Question: Cytosolic proteins bind to iron-responsive elements on mRNAs coding for DMT1. Cytosolic proteins bind to iron-responsive elements on mRNAs coding for proteins involved in iron uptake.

Title: Multiple RNA surveillance pathways limit aberrant expression of iron uptake mRNAs and prevent iron toxicity in S. cerevisiae. Content: Tight regulation of the expression of mRNAs encoding iron uptake proteins is essential to control iron homeostasis and avoid intracellular iron toxicity. We show that many mRNAs encoding iron uptake or iron mobilization proteins are expressed in iron-replete conditions in the absence of the S. cerevisiae RNase III ortholog Rnt1p or of the nuclear exosome component Rrp6p. Extended forms of these mRNAs accumulate in the absence of Rnt1p or of the 5'-->3' exonucleases Xrn1p and Rat1p, showing that multiple degradative pathways contribute to the surveillance of aberrant forms of these transcripts. RNase III-deficient cells are hypersensitive to high iron concentrations, suggesting that Rnt1p-mediated RNA surveillance is required to prevent iron toxicity. These results show that RNA surveillance through multiple ribonucleolytic pathways plays a role in iron homeostasis in yeast to avoid the potentially toxic effects of the expression of the iron starvation response in iron-replete conditions.

Title: Mitochondrial iron chelation ameliorates cigarette-smoke induced bronchitis and emphysema in mice Content: Chronic obstructive pulmonary disease (COPD) is linked to both cigarette smoking and genetic determinants. We have previously identified iron-responsive element-binding protein 2 (IRP2) as an important COPD susceptibility gene and have shown that IRP2 protein is increased in the lungs of individuals with COPD. Here we demonstrate that mice deficient in Irp2 were protected from cigarette smoke (CS)-induced experimental COPD. By integrating RNA immunoprecipitation followed by sequencing (RIP-seq), RNA sequencing (RNA-seq), and gene expression and functional enrichment clustering analysis, we identified Irp2 as a regulator of mitochondrial function in the lungs of mice. Irp2 increased mitochondrial iron loading and levels of cytochrome c oxidase (COX), which led to mitochondrial dysfunction and subsequent experimental COPD. Frataxin-deficient mice, which had higher mitochondrial iron loading, showed impaired airway mucociliary clearance (MCC) and higher pulmonary inflammation at baseline, whereas mice deficient in the synthesis of cytochrome c oxidase, which have reduced COX, were protected from CS-induced pulmonary inflammation and impairment of MCC. Mice treated with a mitochondrial iron chelator or mice fed a low-iron diet were protected from CS-induced COPD. Mitochondrial iron chelation also alleviated CS-induced impairment of MCC, CS-induced pulmonary inflammation and CS-associated lung injury in mice with established COPD, suggesting a critical functional role and potential therapeutic intervention for the mitochondrial-iron axis in COPD.

Title: GRSF1 Regulates RNA Processing in Mitochondrial RNA Granules Content: Various specialized domains have been described in the cytosol and the nucleus; however, little is known about compartmentalization within the mitochondrial matrix. GRSF1 (G-rich sequence factor 1) is an RNA binding protein that was previously reported to localize in the cytosol. We found that an isoform of GRSF1 accumulates in discrete foci in the mitochondrial matrix. These foci are composed of nascent mitochondrial RNA and also contain RNase P, an enzyme that participates in mitochondrial RNA processing. GRSF1 was found to interact with RNase P and to be required for processing of both classical and tRNA-less RNA precursors. In its absence, cleavage of primary RNA transcripts is abnormal, leading to decreased expression of mitochondrially encoded proteins and mitochondrial dysfunction. Our findings suggest that the foci containing GRSF1 and RNase P correspond to sites where primary RNA transcripts converge to be processed. We have termed these large ribonucleoprotein structures "mitochondrial RNA granules. "

Title: The mitochondrial RNA-binding protein GRSF1 localizes to RNA granules and is required for posttranscriptional mitochondrial gene expression. Content: RNA-binding proteins are at the heart of posttranscriptional gene regulation, coordinating the processing, storage, and handling of cellular RNAs. We show here that GRSF1, previously implicated in the binding and selective translation of influenza mRNAs, is targeted to mitochondria where it forms granules that colocalize with foci of newly synthesized mtRNA next to mitochondrial nucleoids. GRSF1 preferentially binds RNAs transcribed from three contiguous genes on the light strand of mtDNA, the ND6 mRNA, and the long noncoding RNAs for cytb and ND5, each of which contains multiple consensus binding sequences. RNAi-mediated knockdown of GRSF1 leads to alterations in mitochondrial RNA stability, abnormal loading of mRNAs and lncRNAs on the mitochondrial ribosome, and impaired ribosome assembly. This results in a specific protein synthesis defect and a failure to assemble normal amounts of the oxidative phosphorylation complexes. These data implicate GRSF1 as a key regulator of posttranscriptional mitochondrial gene expression.

Title: Dnmt3a and Dnmt3b Associate with Enhancers to Regulate Human Epidermal Stem Cell Homeostasis. Content: The genome-wide localization and function of endogenous Dnmt3a and Dnmt3b in adult stem cells are unknown. Here, we show that in human epidermal stem cells, the two proteins bind in a histone H3K36me3-dependent manner to the most active enhancers and are required to produce their associated enhancer RNAs. Both proteins prefer super-enhancers associated to genes that either define the ectodermal lineage or establish the stem cell and differentiated states. However, Dnmt3a and Dnmt3b differ in their mechanisms of enhancer regulation: Dnmt3a associates with p63 to maintain high levels of DNA hydroxymethylation at the center of enhancers in a Tet2-dependent manner, whereas Dnmt3b promotes DNA methylation along the body of the enhancer. Depletion of either protein inactivates their target enhancers and profoundly affects epidermal stem cell function. Altogether, we reveal novel functions for Dnmt3a and Dnmt3b at enhancers that could contribute to their roles in disease and tumorigenesis.

Title: Structural features of the interaction of the 3'-untranslated region of mRNA containing exosomal RNA-specific motifs with YB-1, a potential mediator of mRNA sorting. Content: We have previously shown that YB-1 is the only protein of the HEK293 cell cytoplasmic (S100) extract that specifically interacts with RNA hairpins each containing one of the motifs ACCAGCCU (1), CAGUGAGC (2) and UAAUCCCA (3), which had been identified as often found in exosomal RNA and proposed as potential cis-acting elements targeting RNAs into exosomes. Here we explored the interactions of YB-1 with a fragment of the 3'-untranslated region (UTR) of septin 14 mRNA (SEPT14 RNA), which contains all three motifs. We demonstrated the occurrence of YB-1 among proteins pulled down from the HEK293 S100 extract using biotinylated SEPT14 RNA. With recombinant YB-1, it was found that SEPT14 RNA can bind up to 5 moles of protein per mole of RNA in a cooperative manner, which was shown to be mainly facilitated by the presence of the above motifs. RNA hairpins with motifs 1 and 2 competed with SEPT14 RNA for binding to the protein, whereas that with motif 3 was less competitive, in accordance with the affinity of YB-1 for these RNA hairpins. With YB-1-bound RNA, nucleotides protected from attack by hydroxyl radicals were revealed in all three motifs, although hairpins with motif 2 and especially with motif 1 contained many protected nucleotides outside the motifs, suggesting that the specific environments of these motifs contribute significantly to the YB-1 binding. An analysis of the environments of motifs 1-3 in the HEK293 cell mRNA 3' UTRs gained from RNA-seq data led us to conclude that the primary binding sites of YB-1 in the 3' UTRs are hairpins containing some part of the motif along with its specific surroundings; the consensus sequences of these hairpins were derived. Thus, our findings provide a new understanding of the structural basis of the interactions between YB-1 and mRNAs carrying the aforementioned motifs.

Title: Cyclin A2 is an RNA binding protein that controls Mre11 mRNA translation Content: Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding–dependent role that ensures adequate repair of common replication errors.

Title: A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N1-2'O-Methylated Self RNA. Content: The cytosolic helicase retinoic acid-inducible gene-I (RIG-I) initiates immune responses to most RNA viruses by detecting viral 5'-triphosphorylated RNA (pppRNA). Although endogenous mRNA is also 5'-triphosphorylated, backbone modifications and the 5'-ppp-linked methylguanosine ((m7)G) cap prevent immunorecognition. Here we show that the methylation status of endogenous capped mRNA at the 5'-terminal nucleotide (N1) was crucial to prevent RIG-I activation. Moreover, we identified a single conserved amino acid (H830) in the RIG-I RNA binding pocket as the mediator of steric exclusion of N1-2'O-methylated RNA. H830A alteration (RIG-I(H830A)) restored binding of N1-2'O-methylated pppRNA. Consequently, endogenous mRNA activated the RIG-I(H830A) mutant but not wild-type RIG-I. Similarly, knockdown of the endogenous N1-2'O-methyltransferase led to considerable RIG-I stimulation in the absence of exogenous stimuli. Studies involving yellow-fever-virus-encoded 2'O-methyltransferase and RIG-I(H830A) revealed that viruses exploit this mechanism to escape RIG-I. Our data reveal a new role for cap N1-2'O-methylation in RIG-I tolerance of self-RNA.

Title: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology Content: The canonical role of messenger RNA (mRNA) is to deliver protein-coding information to sites of protein synthesis. However, given that microRNAs bind to RNAs, we hypothesized that RNAs could possess a regulatory role that relies on their ability to compete for microRNA binding, independently of their protein-coding function. As a model for the protein-coding-independent role of RNAs, we describe the functional relationship between the mRNAs produced by the PTEN tumour suppressor gene and its pseudogene PTENP1 and the critical consequences of this interaction. We find that PTENP1 is biologically active as it can regulate cellular levels of PTEN and exert a growth-suppressive role. We also show that the PTENP1 locus is selectively lost in human cancer. We extended our analysis to other cancer-related genes that possess pseudogenes, such as oncogenic KRAS. We also demonstrate that the transcripts of protein-coding genes such as PTEN are biologically active. These findings attribute a novel biological role to expressed pseudogenes, as they can regulate coding gene expression, and reveal a non-coding function for mRNAs.

Title: miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA. Content: MicroRNAs (miRNAs) are ∼22 nt non-coding RNAs that typically bind to the 3' UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non-coding antisense transcripts in human cells. Specifically, we show that miR-671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration-Related protein 1 (CDR1) locus in an Ago2-slicer-dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non-coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels.

Title: Cyclin D1 activation in B-cell malignancy: association with changes in histone acetylation, DNA methylation, and RNA polymerase II binding to both promoter and distal sequences. Content: Cyclin D1 expression is deregulated by chromosome translocation in mantle cell lymphoma and a subset of multiple myeloma. The molecular mechanisms involved in long-distance gene deregulation remain obscure, although changes in acetylated histones and methylated CpG dinucleotides may be important. The patterns of DNA methylation and histone acetylation were determined at the cyclin D1 locus on chromosome 11q13 in B-cell malignancies. The cyclin D1 promoter was hypomethylated and hyperacetylated in expressing cell lines and patient samples, and methylated and hypoacetylated in nonexpressing cell lines. Domains of hyperacetylated histones and hypomethylated DNA extended over 120 kb upstream of the cyclin D1 gene. Interestingly, hypomethylated DNA and hyperacetylated histones were also located at the cyclin D1 promoter but not the upstream major translocation cluster region in cyclin D1-nonexpressing, nontumorigenic B and T cells. RNA polymerase II binding was demonstrated both at the cyclin D1 promoter and 3' immunoglobulin heavy-chain regulatory regions only in malignant B-cell lines with deregulated cyclin D1 expression. Our results suggest a model where RNA polymerase II bound at IgH regulatory sequences can activate the cyclin D1 promoter by either long-range polymerase transfer or tracking.

Title: Cytosolic viral sensor RIG-I is a 5'-triphosphate-dependent translocase on double-stranded RNA. Content: Retinoic acid inducible-gene I (RIG-I) is a cytosolic multidomain protein that detects viral RNA and elicits an antiviral immune response. Two N-terminal caspase activation and recruitment domains (CARDs) transmit the signal, and the regulatory domain prevents signaling in the absence of viral RNA. 5'-triphosphate and double-stranded RNA (dsRNA) are two molecular patterns that enable RIG-I to discriminate pathogenic from self-RNA. However, the function of the DExH box helicase domain that is also required for activity is less clear. Using single-molecule protein-induced fluorescence enhancement, we discovered a robust adenosine 5'-triphosphate-powered dsRNA translocation activity of RIG-I. The CARDs dramatically suppress translocation in the absence of 5'-triphosphate, and the activation by 5'-triphosphate triggers RIG-I to translocate preferentially on dsRNA in cis. This functional integration of two RNA molecular patterns may provide a means to specifically sense and counteract replicating viruses.

Title: Key role of Ubc5 and lysine-63 polyubiquitination in viral activation of IRF3. Content: The mitochondrial antiviral signaling protein (MAVS; also known as IPS-1, VISA, and CARDIF) is essential for innate immune response against RNA viruses. MAVS transduces signals from the cytosolic RIG-I-like receptors, which bind to viral RNAs. But how MAVS activates downstream transcription factors such as IRF3 to induce type-I interferons is not well understood. We have established a cell-free system in which mitochondria derived from virus-infected cells activate IRF3 in the cytosol. Fractionation of the cytosol led to the identification of Ubc5 as a ubiquitin-conjugating enzyme (E2) required for IRF3 activation. Using an inducible RNAi strategy, we demonstrate that catalytically active Ubc5 is required for IRF3 activation by viral infection. The activation of IRF3 also requires two ubiquitin-binding domains of NEMO. Furthermore, we show that replacement of endogenous ubiquitin with its K63R mutant abolishes viral activation of IRF3, demonstrating that K63 polyubiquitination plays a key role in IRF3 activation.

Title: Nuclear HuR accumulation through phosphorylation by Cdk1. Content: A predominantly nuclear RNA-binding protein, HuR translocates to the cytoplasm in response to stress and proliferative signals, where it stabilizes or modulates the translation of target mRNAs. Here, we present evidence that HuR phosphorylation at S202 by the G2-phase kinase Cdk1 influences its subcellular distribution. HuR was specifically phosphorylated in synchronous G2-phase cultures; its cytoplasmic levels increased by Cdk1-inhibitory interventions and declined in response to Cdk1-activating interventions. In keeping with the prominently cytoplasmic location of the nonphosphorylatable point mutant HuR(S202A), phospho-HuR(S202) was shown to be predominantly nuclear using a novel anti-phospho-HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR was linked to its decreased association with 14-3-3 and to its heightened binding to target mRNAs. Our findings suggest that Cdk1 phosphorylates HuR during G2, thereby helping to retain it in the nucleus in association with 14-3-3 and hindering its post-transcriptional function and anti-apoptotic influence.

Title: The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Content: The cyclin-dependent kinase Cdk2 associates with cyclins A, D, and E and has been implicated in the control of the G1 to S phase transition in mammals. To identify potential Cdk2 regulators, we have employed an improved two-hybrid system to isolate human genes encoding Cdk-interacting proteins (Cips). CIP1 encodes a novel 21 kd protein that is found in cyclin A, cyclin D1, cyclin E, and Cdk2 immunoprecipitates. p21CIP1 is a potent, tight-binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by cyclin A-Cdk2, cyclin E-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes. Cotransfection experiments indicate that CIP1 and SV40 T antigen function in a mutually antagonistic manner to control cell cycle progression.

Title: Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein. Content: Cytoplasmic dynein is a microtubule-based motor protein that is responsible for most intracellular retrograde transports along microtubule filaments. The motor domain of dynein contains six tandemly linked AAA (ATPases associated with diverse cellular activities) modules, with the first four containing predicted nucleotide-binding/hydrolysis sites (P1-P4). To dissect the functions of these multiple nucleotide-binding/hydrolysis sites, we expressed and purified Dictyostelium dynein motor domains in which mutations were introduced to block nucleotide binding at each of the four AAA modules, and then examined their detailed biochemical properties. The P1 mutant was trapped in a strong-binding state even in the presence of ATP and lost its motile activity. The P3 mutant also showed a high affinity for microtubules in the presence of ATP and lost most of the microtubule-activated ATPase activity, but retained microtubule sliding activity, although the sliding velocity of the mutant was more than 20-fold slower than that of the wild type. In contrast, mutation in the P2 or P4 site did not affect the apparent binding affinity of the mutant for microtubules in the presence of ATP, but reduced ATPase and microtubule sliding activities. These results indicate that ATP binding and its hydrolysis only at the P1 site are essential for the motor activities of cytoplasmic dynein, and suggest that the other nucleotide-binding/hydrolysis sites regulate the motor activities. Among them, nucleotide binding at the P3 site is not essential but is critical for microtubule-activated ATPase and motile activities of cytoplasmic dynein.

Title: The Inhibition of TDP-43 Mitochondrial Localization Blocks Its Neuronal Toxicity Content: Genetic mutations in TAR DNA-binding protein 43 (TARDBP, also known as TDP-43) cause amyotrophic lateral sclerosis (ALS), and an increase in the presence of TDP-43 (encoded by TARDBP) in the cytoplasm is a prominent histopathological feature of degenerating neurons in various neurodegenerative diseases. However, the molecular mechanisms by which TDP-43 contributes to ALS pathophysiology remain elusive. Here we have found that TDP-43 accumulates in the mitochondria of neurons in subjects with ALS or frontotemporal dementia (FTD). Disease-associated mutations increase TDP-43 mitochondrial localization. In mitochondria, wild-type (WT) and mutant TDP-43 preferentially bind mitochondria-transcribed messenger RNAs (mRNAs) encoding respiratory complex I subunits ND3 and ND6, impair their expression and specifically cause complex I disassembly. The suppression of TDP-43 mitochondrial localization abolishes WT and mutant TDP-43-induced mitochondrial dysfunction and neuronal loss, and improves phenotypes of transgenic mutant TDP-43 mice. Thus, our studies link TDP-43 toxicity directly to mitochondrial bioenergetics and propose the targeting of TDP-43 mitochondrial localization as a promising therapeutic approach for neurodegeneration.

Title: Mitochondrial RNA Granules Are Centers for Posttranscriptional RNA Processing and Ribosome Biogenesis. Content: Cytoplasmic RNA granules play a central role in mRNA metabolism, but the importance of mitochondrial RNA granules remains relatively unexplored. We characterized their proteome and found that they contain a large toolbox of proteins dedicated to RNA metabolism. Investigation of four uncharacterized putative RNA-binding proteins-two RNA helicases, DHX30 and DDX28, and two proteins of the Fas-activated serine-threonine kinase (FASTKD) family, FASTKD2 and FASTKD5-demonstrated that both helicases and FASTKD2 are required for mitochondrial ribosome biogenesis. RNA-sequencing (RNA-seq) analysis showed that DDX28 and FASTKD2 bound the 16S rRNA. FASTKD5 is required for maturing precursor mRNAs that are not flanked by tRNAs and that therefore cannot be processed by the canonical mRNA maturation pathway. Silencing FASTKD5 rendered mature COX I mRNA almost undetectable, which severely reduced the synthesis of COX I, resulting in a complex IV assembly defect. These data demonstrate that mitochondrial RNA granules are centers for posttranscriptional RNA processing and the biogenesis of mitochondrial ribosomes.

Title: Specific and cooperative binding of E. coli single-stranded DNA binding protein to mRNA. Content: Fluorometric titration of E. coli single-stranded DNA binding protein with various RNAs showed that the protein specifically and cooperatively binds to its own mRNA. The binding inhibited in vitro expression of ssb and bla but not nusA. This inhibition takes place at a physiological concentration of SSB. The function of the protein in gene regulation is discussed.

Title: Dnmt1-Independent CG Methylation Contributes to Nucleosome Positioning in Diverse Eukaryotes Content: Dnmt1 epigenetically propagates symmetrical CG methylation in many eukaryotes. Their genomes are typically depleted of CG dinucleotides because of imperfect repair of deaminated methylcytosines. Here, we extensively survey diverse species lacking Dnmt1 and show that, surprisingly, symmetrical CG methylation is nonetheless frequently present and catalyzed by a different DNA methyltransferase family, Dnmt5. Numerous Dnmt5-containing organisms that diverged more than a billion years ago exhibit clustered methylation, specifically in nucleosome linkers. Clustered methylation occurs at unprecedented densities and directly disfavors nucleosomes, contributing to nucleosome positioning between clusters. Dense methylation is enabled by a regime of genomic sequence evolution that enriches CG dinucleotides and drives the highest CG frequencies known. Species with linker methylation have small, transcriptionally active nuclei that approach the physical limits of chromatin compaction. These features constitute a previously unappreciated genome architecture, in which dense methylation influences nucleosome positions, likely facilitating nuclear processes under extreme spatial constraints.

Title: Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Content: The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

Title: The Wilms tumour suppressor protein WT1 (+KTS isoform) binds alpha-actinin 1 mRNA via its zinc-finger domain. Content: Mutations in WT1 are associated with developmental syndromes that affect the urogenital system and neoplasms, including Wilms tumour, acute myeloid leukemia, and breast and prostate cancers. The WT1 protein belongs to the early growth response family of zinc-finger transcription factors. Uniquely to WT1, an evolutionarily conserved alternative splice event inserts the tripeptide KTS, between zinc fingers 3 and 4. Whereas -KTS isoforms bind DNA and activate or repress transcription, +KTS isoforms bind DNA less efficiently and interact with splice factors and RNA in vitro and in vivo. Although candidate DNA targets have been found, physiological mRNA targets are yet to be defined. We examined the distribution of WT1 in ribonucleoprotein (RNP) complexes in nuclear extract prepared from M15 cells, a mouse mesonephric fetal kidney cell line. WT1 cofractionated with the splice factor PSF in large RNP particles >or=2 MDa. We also found that PSF co-immunoprecipitated with WT1, suggesting a functional interaction between these 2 multifunctional proteins. Using yeast three-hybrid library constructed from the co-immunoprecipitated RNA we found that WT1 (+KTS) binds close to or at the start codon of alpha-actinin 1 (ACTN1) mRNA. A band shift assay confirmed the ability of the WT1 zinc-finger domain (+KTS) to bind this sequence in vitro. ACTN1 is the first likely physiological mRNA target of WT1.

Title: Long non-coding RNAs with low expression levels in cells are enriched in secreted exosomes. Content: Long non-coding RNAs (lncRNAs) are involved in regulating chromatin modifications, gene transcription, mRNA translation, and protein function. We recently reported a high variation in the basal expression levels of a panel of lncRNAs in HeLa and MCF-7 cells and their differential response to DNA damage induction. Here, we hypothesized that lncRNA molecules with different cellular expression may have a differential abundance in secreted exosomes, and their exosome levels would reflect cellular response to DNA damage. MALAT1, HOTAIR, lincRNA-p21, GAS5, TUG1, CCND1-ncRNA in exosomes secreted from cultured cells were characterized. A different expression pattern of lncRNAs in exosomes was seen compared to cells. RNA molecules with relative low expression levels (lincRNA-p21, HOTAIR, ncRNA-CCND1) were highly enriched in exosomes. TUG1 and GAS5 levels were moderately elevated in exosomes, whereas MALAT1--which was the most abundant molecule in cells--was present at levels comparable to its cellular levels. lincRNA-p21 and ncRNA-CCND1 were the main molecules; exosome levels of them best reflect the change of their cellular levels upon exposure of the cells to bleomycin-induced DNA damage. In conclusion, we provide evidence that lncRNAs have a differential abundance in exosomes, indicating a selective loading.

Title: CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms. Content: CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150(Glued) subunit. We find that CLIP-170 mutants alter p150(Glued) localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin.

Title: Identification of short-lived long non-coding RNAs as surrogate indicators for chemical stress response. Content: Abiotic and biotic stressors in human cells are often a result of sudden and/or frequent changes in environmental factors. The molecular response to stress involves elaborate modulation of gene expression and is of homeostatic, ecological, and evolutionary importance. Although attention has primarily focused on signaling pathways and protein networks, long non-coding RNAs (ncRNAs) are increasingly involved in the molecular mechanisms associated with responses to cellular stresses. We identified six novel short-lived long ncRNAs (MIR22HG, GABPB-AS1, LINC00152, IDI2-AS1, SNHG15, and FLJ33630) that responded to chemical stressors (cisplatin, cycloheximide, and mercury (II) oxide) in HeLa Tet-off cells. Our results indicate that short-lived long ncRNAs respond to general and specific chemical stressors. The expression levels of the short-lived long ncRNAs were elevated because of prolonged decay rates in response to chemical stressors and interruption of RNA degradation pathways. We propose that these long ncRNAs have the potential to be surrogate indicators of cellular stress responses.

Title: Nicotinic Acid Adenine Dinucleotide 2'-Phosphate (NAADP) Binding Proteins in T-Lymphocytes. Content: Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Although several channels, including two-pore channels (TPC), ryanodine receptor (RYR) and mucolipin (TRP-ML1) have been implicated in NAADP regulation of calcium signaling, the NAADP receptor has not been identified. In this study, the photoaffinity probe, [32P]-5-azido-NAADP ([32P]-5-N3-NAADP), was used to study NAADP binding proteins in extracts from NAADP responsive Jurkat T-lymphocytes. [32P]-5-N3-NAADP photolabeling of Jurkat S100 cytosolic fractions resulted in the labeling of at least ten distinct proteins. Several of these S100 proteins, including a doublet at 22/23 kDa and small protein at 15 kDa displayed selectivity for NAADP as the labeling was protected by inclusion of unlabeled NAADP, whereas the structurally similar NADP required much higher concentrations for protection. Interestingly, the labeling of several S100 proteins (60, 45, 33 and 28 kDa) was stimulated by low concentrations of unlabeled NAADP, but not by NADP. The effect of NAADP on the labeling of the 60 kDa protein was biphasic, peaking at 100 nM with a five-fold increase and displaying no change at 1 µM NAADP. Several proteins were also photolabeled when the P100 membrane fraction from Jurkat cells was examined. Similar to the results with S100, a 22/23 kDa doublet and a 15 kDa protein appeared to be selectively labeled. NAADP did not increase the labeling of any P100 proteins as it did in the S100 fraction. The photolabeled S100 and P100 proteins were successfully resolved by two-dimensional gel electrophoresis. [32P]-5-N3-NAADP photolabeling and two-dimensional electrophoresis should represent a suitable strategy in which to identify and characterize NAADP binding proteins.

Title: Molecular Mechanisms of Vitamin D Action Content: The hormonal metabolite of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D), initiates biological responses via binding to the vitamin D receptor (VDR). When occupied by 1,25D, VDR interacts with the retinoid X receptor (RXR) to form a heterodimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1,25D. By recruiting complexes of either coactivators or corepressors, ligand-activated VDR-RXR modulates the transcription of genes encoding proteins that promulgate the traditional functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. Thus, vitamin D action in a particular cell depends upon the metabolic production or delivery of sufficient concentrations of the 1,25D ligand, expression of adequate VDR and RXR coreceptor proteins, and cell-specific programming of transcriptional responses to regulate select genes that encode proteins that function in mediating the effects of vitamin D. For example, 1,25D induces RANKL, SPP1 (osteopontin), and BGP (osteocalcin) to govern bone mineral remodeling; TRPV6, CaBP9k, and claudin 2 to promote intestinal calcium absorption; and TRPV5, klotho, and Npt2c to regulate renal calcium and phosphate reabsorption. VDR appears to function unliganded by 1,25D in keratinocytes to drive mammalian hair cycling via regulation of genes such as CASP14, S100A8, SOSTDC1, and others affecting Wnt signaling. Finally, alternative, low-affinity, non-vitamin D VDR ligands, e.g., lithocholic acid, docosahexaenoic acid, and curcumin, have been reported. Combined alternative VDR ligand(s) and 1,25D/VDR control of gene expression may delay chronic disorders of aging such as osteoporosis, type 2 diabetes, cardiovascular disease, and cancer.

Title: A Microtubule Interactome: Complexes with Roles in Cell Cycle and Mitosis Content: The microtubule (MT) cytoskeleton is required for many aspects of cell function, including the transport of intracellular materials, the maintenance of cell polarity, and the regulation of mitosis. These functions are coordinated by MT-associated proteins (MAPs), which work in concert with each other, binding MTs and altering their properties. We have used a MT cosedimentation assay, combined with 1D and 2D PAGE and mass spectrometry, to identify over 250 MAPs from early Drosophila embryos. We have taken two complementary approaches to analyse the cellular function of novel MAPs isolated using this approach. First, we have carried out an RNA interference (RNAi) screen, identifying 21 previously uncharacterised genes involved in MT organisation. Second, we have undertaken a bioinformatics analysis based on binary protein interaction data to produce putative interaction networks of MAPs. By combining both approaches, we have identified and validated MAP complexes with potentially important roles in cell cycle regulation and mitosis. This study therefore demonstrates that biologically relevant data can be harvested using such a multidisciplinary approach, and identifies new MAPs, many of which appear to be important in cell division.

Title: Oxidative damage targets complexes containing DNA methyltransferases, SIRT1, and polycomb members to promoter CpG Islands. Content: Cancer cells simultaneously harbor global losses and gains in DNA methylation. We demonstrate that inducing cellular oxidative stress by hydrogen peroxide treatment recruits DNA methyltransferase 1 (DNMT1) to damaged chromatin. DNMT1 becomes part of a complex(es) containing DNMT3B and members of the polycomb repressive complex 4. Hydrogen peroxide treatment causes relocalization of these proteins from non-GC-rich to GC-rich areas. Key components are similarly enriched at gene promoters in an in vivo colitis model. Although high-expression genes enriched for members of the complex have histone mark and nascent transcription changes, CpG island-containing low-expression genes gain promoter DNA methylation. Thus, oxidative damage induces formation and relocalization of a silencing complex that may explain cancer-specific aberrant DNA methylation and transcriptional silencing.

Title: The Arabidopsis Chromatin-Modifying Nuclear siRNA Pathway Involves a Nucleolar RNA Processing Center Content: In Arabidopsis thaliana, small interfering RNAs (siRNAs) direct cytosine methylation at endogenous DNA repeats in a pathway involving two forms of nuclear RNA polymerase IV (Pol IVa and Pol IVb), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3), ARGONAUTE4 (AGO4), the chromatin remodeler DRD1, and the de novo cytosine methyltransferase DRM2. We show that RDR2, DCL3, AGO4, and NRPD1b (the largest subunit of Pol IVb) colocalize with siRNAs within the nucleolus. By contrast, Pol IVa and DRD1 are external to the nucleolus and colocalize with endogenous repeat loci. Mutation-induced loss of pathway proteins causes downstream proteins to mislocalize, revealing their order of action. Pol IVa acts first, and its localization is RNA dependent, suggesting an RNA template. We hypothesize that maintenance of the heterochromatic state involves locus-specific Pol IVa transcription followed by siRNA production and assembly of AGO4- and NRPD1b-containing silencing complexes within nucleolar processing centers.

Title: A genome-wide short hairpin RNA screening of jurkat T-cells for human proteins contributing to productive HIV-1 replication. Content: Short interfering RNAs (siRNAs) have been used to inhibit HIV-1 replication. The durable inhibition of HIV-1 replication by RNA interference has been impeded, however, by a high mutation rate when viral sequences are targeted and by cytotoxicity when cellular genes are knocked down. To identify cellular proteins that contribute to HIV-1 replication that can be chronically silenced without significant cytotoxicity, we employed a shRNA library that targets 54,509 human transcripts. We used this library to select a comprehensive population of Jurkat T-cell clones, each expressing a single discrete shRNA. The Jurkat clones were then infected with HIV-1. Clones that survived viral infection represent moieties silenced for a human mRNA needed for virus replication, but whose chronic knockdown did not cause cytotoxicity. Overall, 252 individual Jurkat mRNAs were identified. Twenty-two of these mRNAs were secondarily verified for their contributions to HIV-1 replication. Five mRNAs, NRF1, STXBP2, NCOA3, PRDM2, and EXOSC5, were studied for their effect on steps of the HIV-1 life cycle. We discuss the similarities and differences between our shRNA findings for HIV-1 using a spreading infection assay in human Jurkat T-cells and results from other investigators who used siRNA-based screenings in HeLa or 293T cells.

Title: STING and the innate immune response to nucleic acids in the cytosol Content: Cytosolic detection of pathogen-derived nucleic acids is critical for the initiation of innate immune defense against diverse bacterial, viral and eukaryotic pathogens. Conversely, inappropriate responses to cytosolic nucleic acids can produce severe autoimmune pathology. The host protein STING has been identified as a central signaling molecule in the innate immune response to cytosolic nucleic acids. STING seems to be especially critical for responses to cytosolic DNA and the unique bacterial nucleic acids called 'cyclic dinucleotides'. Here we discuss advances in the understanding of STING and highlight the many unresolved issues in the field.

Title: Human DNA methylomes at base resolution show widespread epigenomic differences Content: DNA cytosine methylation is a central epigenetic modification that has essential roles in cellular processes including genome regulation, development and disease. Here we present the first genome-wide, single-base-resolution maps of methylated cytosines in a mammalian genome, from both human embryonic stem cells and fetal fibroblasts, along with comparative analysis of messenger RNA and small RNA components of the transcriptome, several histone modifications, and sites of DNA–protein interaction for several key regulatory factors. Widespread differences were identified in the composition and patterning of cytosine methylation between the two genomes. Nearly one-quarter of all methylation identified in embryonic stem cells was in a non-CG context, suggesting that embryonic stem cells may use different methylation mechanisms to affect gene regulation. Methylation in non-CG contexts showed enrichment in gene bodies and depletion in protein binding sites and enhancers. Non-CG methylation disappeared upon induced differentiation of the embryonic stem cells, and was restored in induced pluripotent stem cells. We identified hundreds of differentially methylated regions proximal to genes involved in pluripotency and differentiation, and widespread reduced methylation levels in fibroblasts associated with lower transcriptional activity. These reference epigenomes provide a foundation for future studies exploring this key epigenetic modification in human disease and development.

Title: Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns. Content: A protein of molecular size 62,000 daltons (p62) was detected in HeLa cell nuclear extracts by UV cross-linking to mRNA precursors. p62 binds specifically to the polypyrimidine tract of the 3' splice site region of introns. p62 purified to homogeneity binds the polypyrimidine tract of pre-mRNAs. This binding does not require the AG dinucleotide at the 3' splice site. Alterations in the polypyrimidine tract that reduce the binding of p62 yield a corresponding reduction in the efficiency of formation of a U2 snRNP/pre-mRNA complex and splicing. The p62 protein is retained in the spliceosome, where it remains bound to the pre-mRNA. This polypyrimidine tract binding protein (pPTB) is proposed to be a critical component in recognition of the 3' splice site during splicing.

Title: LIN28 Regulates Stem Cell Metabolism and Conversion to Primed Pluripotency. Content: The RNA-binding proteins LIN28A and LIN28B play critical roles in embryonic development, tumorigenesis, and pluripotency, but their exact functions are poorly understood. Here, we show that, like LIN28A, LIN28B can function effectively with NANOG, OCT4, and SOX2 in reprogramming to pluripotency and that reactivation of both endogenous LIN28A and LIN28B loci are required for maximal reprogramming efficiency. In human fibroblasts, LIN28B is activated early during reprogramming, while LIN28A is activated later during the transition to bona fide induced pluripotent stem cells (iPSCs). In murine cells, LIN28A and LIN28B facilitate conversion from naive to primed pluripotency. Proteomic and metabolomic analysis highlighted roles for LIN28 in maintaining the low mitochondrial function associated with primed pluripotency and in regulating one-carbon metabolism, nucleotide metabolism, and histone methylation. LIN28 binds to mRNAs of proteins important for oxidative phosphorylation and modulates protein abundance. Thus, LIN28A and LIN28B play cooperative roles in regulating reprogramming, naive/primed pluripotency, and stem cell metabolism.

Title: EB virus-encoded RNAs are recognized by RIG-I and activate signaling to induce type I IFN. Content: Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) are nonpolyadenylated, untranslated RNAs, exist most abundantly in latently EBV-infected cells, and are expected to show secondary structures with many short stem-loops. Retinoic acid-inducible gene I (RIG-I) is a cytosolic protein that detects viral double-stranded RNA (dsRNA) inside the cell and initiates signaling pathways leading to the induction of protective cellular genes, including type I interferons (IFNs). We investigated whether EBERs were recognized by RIG-I as dsRNA. Transfection of RIG-I plasmid induced IFNs and IFN-stimulated genes (ISGs) in EBV-positive Burkitt's lymphoma (BL) cells, but not in their EBV-negative counterparts or EBER-knockout EBV-infected BL cells. Transfection of EBER plasmid or in vitro-synthesized EBERs induced expression of type I IFNs and ISGs in RIG-I-expressing, EBV-negative BL cells, but not in RIG-I-minus counterparts. EBERs activated RIG-I's substrates, NF-kappaB and IFN regulatory factor 3, which were necessary for type I IFN activation. It was also shown that EBERs co-precipitated with RIG-I. These results indicate that EBERs are recognized by RIG-I and activate signaling to induce type I IFN in EBV-infected cells.

Title: Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus Content: The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Title: An Argonaute transports siRNAs from the cytoplasm to the nucleus. Content: Ribonucleoprotein complexes consisting of Argonaute-like proteins and small regulatory RNAs function in a wide range of biological processes. Many of these small regulatory RNAs are predicted to act, at least in part, within the nucleus. We conducted a genetic screen to identify factors essential for RNA interference (RNAi) in nuclei of Caenorhabditis elegans and identified the Argonaute protein NRDE-3. In the absence of small interfering RNAs (siRNAs), NRDE-3 resides in the cytoplasm. NRDE-3 binds siRNAs generated by RNA-dependent RNA polymerases acting on messenger RNA templates in the cytoplasm and redistributes to the nucleus. Nuclear redistribution of NRDE-3 requires a functional nuclear localization signal, is required for nuclear RNAi, and results in NRDE-3 association with nuclear-localized nascent transcripts. Thus, specific Argonaute proteins can transport specific classes of small regulatory RNAs to distinct cellular compartments to regulate gene expression.

Title: Regulation of Bcl-2 expression by HuR in HL60 leukemia cells and A431 carcinoma cells. Content: Overexpression of the proto-oncogene bcl-2 promotes abnormal cell survival by inhibiting apoptosis. Expression of bcl-2 is determined, in part, by regulatory mechanisms that control the stability of bcl-2 mRNA. Elements in the 3'-untranslated region of bcl-2 mRNA have been shown to play a role in regulating the stability of the message. Previously, it was found that the RNA binding proteins nucleolin and Ebp1 have a role in stabilizing bcl-2 mRNA in HL60 cells. Here, we have identified HuR as a component of bcl-2 messenger ribonucleoprotein (mRNP) complexes. RNA coimmunoprecipitation assays showed that HuR binds to bcl-2 mRNA in vivo. We also observed an RNA-dependent coprecipitation of HuR and nucleolin, suggesting that the two proteins are present in common mRNP complexes. Moreover, nucleolin and HuR bind concurrently to bcl-2 AU-rich element (ARE) RNA in vitro, suggesting separate binding sites for these proteins on bcl-2 mRNA. Knockdown of HuR in A431 cells leads to down-regulation of bcl-2 mRNA and protein levels. Observation of a decreased ratio of bcl-2 mRNA to heterogeneous nuclear RNA in HuR knockdown cells confirmed a positive role for HuR in regulating bcl-2 stability. Recombinant HuR retards exosome-mediated decay of bcl-2 ARE RNA in extracts of HL60 cells. This supports a role for HuR in the regulation of bcl-2 mRNA stability in HL60 cells, as well as in A431 cells. Addition of nucleolin and HuR to HL60 cell extracts produced a synergistic protective effect on decay of bcl-2 ARE RNA. HuR knockdown also leads to redistribution of bcl-2 mRNA from polysomes to monosomes. Thus, HuR seems to play a positive role in both regulation of bcl-2 mRNA translation and mRNA stability.

Title: GEMC1 is a TopBP1 interacting protein required for chromosomal DNA replication Content: Many of the factors required for chromosomal DNA replication have been identified in unicellular eukaryotes. However, DNA replication is poorly understood in multicellular organisms. Here, we report the identification of GEMC1 (geminin coiled-coil containing protein 1), a novel vertebrate protein required for chromosomal DNA replication. GEMC1 is highly conserved in vertebrates and is preferentially expressed in proliferating cells. Using Xenopus laevis egg extract we show that Xenopus GEMC1 (xGEMC1) binds to the checkpoint and replication factor TopBP1, which promotes binding of xGEMC1 to chromatin during pre-replication complex (pre-RC) formation. We demonstrate that xGEMC1 interacts directly with replication factors such as Cdc45 and the kinase Cdk2-CyclinE, through which it is heavily phosphorylated. Phosphorylated xGEMC1 stimulates initiation of DNA replication, whereas depletion of xGEMC1 prevents the onset of DNA replication owing to the impairment of Cdc45 loading onto chromatin. Similarly, inhibition of GEMC1 expression with morpholino and siRNA oligos prevents DNA replication in embryonic and somatic vertebrate cells. These data suggest that GEMC1 promotes initiation of chromosomal DNA replication in multicellular organisms by mediating TopBP1- and Cdk2-dependent recruitment of Cdc45 onto replication origins.

Title: Analysis of SiO2 nanoparticles binding proteins in rat blood and brain homogenate Content: A multitude of nanoparticles, such as titanium oxide (TiO2), zinc oxide, aluminum oxide, gold oxide, silver oxide, iron oxide, and silica oxide, are found in many chemical, cosmetic, pharmaceutical, and electronic products. Recently, SiO2 nanoparticles were shown to have an inert toxicity profile and no association with an irreversible toxicological change in animal models. Hence, exposure to SiO2 nanoparticles is on the increase. SiO2 nanoparticles are routinely used in numerous materials, from strengthening filler for concrete and other construction composites, to nontoxic platforms for biomedical application, such as drug delivery and theragnostics. On the other hand, recent in vitro experiments indicated that SiO2 nanoparticles were cytotoxic. Therefore, we investigated these nanoparticles to identify potentially toxic pathways by analyzing the adsorbed protein corona on the surface of SiO2 nanoparticles in the blood and brain of the rat. Four types of SiO2 nanoparticles were chosen for investigation, and the protein corona of each type was analyzed using liquid chromatography-tandem mass spectrometry technology. In total, 115 and 48 plasma proteins from the rat were identified as being bound to negatively charged 20 nm and 100 nm SiO2 nanoparticles, respectively, and 50 and 36 proteins were found for 20 nm and 100 nm arginine-coated SiO2 nanoparticles, respectively. Higher numbers of proteins were adsorbed onto the 20 nm sized SiO2 nanoparticles than onto the 100 nm sized nanoparticles regardless of charge. When proteins were compared between the two charges, higher numbers of proteins were found for arginine-coated positively charged SiO2 nanoparticles than for the negatively charged nanoparticles. The proteins identified as bound in the corona from SiO2 nanoparticles were further analyzed with ClueGO, a Cytoscape plugin used in protein ontology and for identifying biological interaction pathways. Proteins bound on the surface of nanoparticles may affect functional and conformational properties and distributions in complicated biological processes.

Title: E2F-6: a novel member of the E2F family is an inhibitor of E2F-dependent transcription Content: The E2F family of transcription factors are essential for the regulation of genes required for appropriate progression through the cell cycle. Five members of the E2F family have been previously reported, namely E2F1-5. All five are key elements in transcriptional regulation of essential genes, and they can be divided into two functional groups, those that induce S-phase progression when overexpressed in quiescent cells (E2Fs 1–3), and those that do not (E2Fs 4–5). Here, we describe the identification of a novel member of this family, which we refer to as E2F-6. E2F-6 shares significant homology with E2Fs 1–5, especially within the DNA binding, heterodimerization and marked box domains. Unlike E2Fs 1–5, E2F-6 lacks a transactivation and a pocket protein binding domain, hence, forms a unique third group within the E2F family. E2F-6 is a nuclear protein that can form heterodimers with the DP proteins (both DP-1 and DP-2) in vitro and in vivo. Our results show that the complex formed between E2F-6 and the DP proteins, possesses high DNA binding activity, displaying a preference for a TTTCCCGC E2F recognition site, which is slightly different to the E2F consensus site derived from the E2 promoter (TTTCGCGC). In contrast to the other members of the E2F family, ectopic expression of E2F-6 inhibits transcription from promoters possessing E2F recognition sites rather than activating transcription. In addition, overexpression of E2F-6 suppresses the transactivational effects of co-expression of E2F-1 and DP-1. The inhibitory effect of E2F-6 is dependent on its DNA binding activity and its ability to form heterodimers with the DPs. Interestingly, ectopic expression of E2F-6 leads to accumulation of cells in S-phase. Our data suggest that E2F-6 expression delays the exit from S-phase rather than inducing S-phase, which further emphasizes the functional difference between E2F-6 and the previously known E2F family members.

Title: Telomere-bound TRF1 and TRF2 stall the replication fork at telomeric repeats. Content: Vertebrate telomeres consist of tandem repeats of T2AG3 and associated proteins including the telomeric DNA-binding proteins, TRF1 and TRF2. It has been proposed that telomeres assume two interswitchable states, the open state that is accessible to various trans-acting factors and the closed state that excludes those factors. TRF1 and TRF2 are believed to promote the formation of the closed state. However, little is known about how those two states influence DNA replication. We analyzed the effects of TRF1 and TRF2 on telomeric replication both in vitro and in vivo. By exploiting the in vitro replication system of linear SV40 DNA, we found that telomeric repeats are a poor replication template. Moreover, the addition of recombinant TRF1 and TRF2 significantly stalled the replication fork progression at telomeric repeats. When TRF1 was overexpressed in HeLa cells, cells with 4N DNA content were accumulated. Furthermore, cytological analyses revealed that the replication focus overlapped with telomere signals at a significantly higher frequency in TRF1-overexpressing cells than in control cells. The results suggest that TRF1 and TRF2 exert inhibitory effects on replication fork progression.

Title: Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing. Content: Immune sensing of DNA is critical for antiviral immunity but can also trigger autoimmune diseases such as lupus erythematosus (LE). Here we have provided evidence for the involvement of a damage-associated DNA modification in the detection of cytosolic DNA. The oxidized base 8-hydroxyguanosine (8-OHG), a marker of oxidative damage in DNA, potentiated cytosolic immune recognition by decreasing its susceptibility to 3' repair exonuclease 1 (TREX1)-mediated degradation. Oxidizative modifications arose physiologically in pathogen DNA during lysosomal reactive oxygen species (ROS) exposure, as well as in neutrophil extracellular trap (NET) DNA during the oxidative burst. 8-OHG was also abundant in UV-exposed skin lesions of LE patients and colocalized with type I interferon (IFN). Injection of oxidized DNA in the skin of lupus-prone mice induced lesions that closely matched respective lesions in patients. Thus, oxidized DNA represents a prototypic damage-associated molecular pattern (DAMP) with important implications for infection, sterile inflammation, and autoimmunity.

Title: Arginine Starvation Impairs Mitochondrial Respiratory Function in ASS1-Deficient Breast Cancer Cells Content: Autophagy is the principal catabolic response to nutrient starvation and is necessary to clear dysfunctional or damaged organelles, but excessive autophagy can be cytotoxic or cytostatic and contributes to cell death. Depending on the abundance of enzymes involved in molecule biosynthesis, cells can be dependent on uptake of exogenous nutrients to provide these molecules. Argininosuccinate synthetase 1 (ASS1) is a key enzyme in arginine biosynthesis, and its abundance is reduced in many solid tumors, making them sensitive to external arginine depletion. We demonstrated that prolonged arginine starvation by exposure to ADI-PEG20 (pegylated arginine deiminase) induced autophagy-dependent death of ASS1-deficient breast cancer cells, because these cells are arginine auxotrophs (dependent on uptake of extracellular arginine). Indeed, these breast cancer cells died in culture when exposed to ADI-PEG20 or cultured in the absence of arginine. Arginine starvation induced mitochondrial oxidative stress, which impaired mitochondrial bioenergetics and integrity. Furthermore, arginine starvation killed breast cancer cells in vivo and in vitro only if they were autophagy-competent. Thus, a key mechanism underlying the lethality induced by prolonged arginine starvation was the cytotoxic autophagy that occurred in response to mitochondrial damage. Last, ASS1 was either low in abundance or absent in more than 60% of 149 random breast cancer biosamples, suggesting that patients with such tumors could be candidates for arginine starvation therapy.

Title: Pituitary adenylate cyclase-activating polypeptide (PACAP(1-38)) enhances N-methyl-D-aspartate receptor function and brain-derived neurotrophic factor expression via RACK1. Content: We recently identified a novel mechanism for modulation of the phosphorylation state and function of the N-methyl-d-aspartate (NMDA) receptor via the scaffolding protein RACK1. We found that RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein-tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). Here, we identified the signaling cascade by which RACK1 is released from the NMDA receptor complex and identified the consequences of the dissociation. We found that activation of the cAMP/protein kinase A pathway in hippocampal slices induced the release of RACK1 from NR2B and Fyn. This resulted in the induction of NR2B phosphorylation and the enhancement of NMDA receptor-mediated activity via Fyn. We identified the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP(1-38)), as a ligand that induced phosphorylation of NR2B and enhanced NMDA receptor potentials. Finally, we found that activation of the cAMP/protein kinase A pathway induced the movement of RACK1 to the nuclear compartment in dissociated hippocampal neurons. Nuclear RACK1 in turn was found to regulate the expression of brain-derived neurotrophic factor induced by PACAP(1-38). Taken together our results suggest that activation of adenylate cyclase by PACAP(1-38) results in the release of RACK1 from the NMDA receptor and Fyn. This in turn leads to NMDA receptor phosphorylation, enhanced activity mediated by Fyn, and to the induction of brain-derived neurotrophic factor expression by RACK1.

Title: The role of iron in Mycobacterium smegmatis biofilm formation: the exochelin siderophore is essential in limiting iron conditions for biofilm formation but not for planktonic growth Content: Many species of mycobacteria form structured biofilm communities at liquid–air interfaces and on solid surfaces. Full development of Mycobacterium smegmatis biofilms requires addition of supplemental iron above 1 μM ferrous sulphate, although addition of iron is not needed for planktonic growth. Microarray analysis of the M. smegmatis transcriptome shows that iron-responsive genes – especially those involved in siderophore synthesis and iron uptake – are strongly induced during biofilm formation reflecting a response to iron deprivation, even when 2 μM iron is present. The acquisition of iron under these conditions is specifically dependent on the exochelin synthesis and uptake pathways, and the strong defect of an iron–exochelin uptake mutant suggests a regulatory role of iron in the transition to biofilm growth. In contrast, although the expression of mycobactin and iron ABC transport operons is highly upregulated during biofilm formation, mutants in these systems form normal biofilms in low-iron (2 μM) conditions. A close correlation between iron availability and matrix-associated fatty acids implies a possible metabolic role in the late stages of biofilm maturation, in addition to the early regulatory role. M. smegmatis surface motility is similarly dependent on iron availability, requiring both supplemental iron and the exochelin pathway to acquire it.

Title: Comparative gene expression profiling of in vitro differentiated megakaryocytes and erythroblasts identifies novel activatory and inhibitory platelet membrane proteins. Content: To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes, a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts, including G6b, G6f, LRRC32, LAT2, and the G protein-coupled receptor SUCNR1, encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins, and flow cytometric analysis confirmed the expression of G6b, G6f, and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b, G6f, and LRRC32 are restricted to the platelet lineage, whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest, because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists.

Title: The Cytosolic Chaperonin CCT/TRiC and Cancer Cell Proliferation Content: The molecular chaperone CCT/TRiC plays a central role in maintaining cellular proteostasis as it mediates the folding of the major cytoskeletal proteins tubulins and actins. CCT/TRiC is also involved in the oncoprotein cyclin E, the Von Hippel-Lindau tumour suppressor protein, cyclin B and p21(ras) folding which strongly suggests that it is involved in cell proliferation and tumor genesis. To assess the involvement of CCT/TRiC in tumor genesis, we quantified its expression levels and activity in 18 cancer, one non-cancer human cell lines and a non-cancer human liver. We show that the expression levels of CCT/TRiC in cancer cell lines are higher than that in normal cells. However, CCT/TRiC activity does not always correlate with its expression levels. We therefore documented the expression levels of CCT/TRiC modulators and partners PhLP3, Hop/P60, prefoldin and Hsc/Hsp70. Our analysis reveals a functional interplay between molecular chaperones that might account for a precise modulation of CCT/TRiC activity in cell proliferation through changes in the cellular levels of prefoldin and/or Hsc/p70 and CCT/TRiC client protein availability. Our observation and approaches bring novel insights in the role of CCT/TRiC-mediated protein folding machinery in cancer cell development.

Title: The dystonia-associated protein torsinA modulates synaptic vesicle recycling. Content: The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with diverse cellular activities) torsinA is responsible for most cases of early onset autosomal dominant primary dystonia. In this study, we found that snapin, which binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the association of the SNARE complex with synaptotagmin, is an interacting partner for both wild type and mutant torsinA. Snapin co-localized with endogenous torsinA on dense core granules in PC12 cells and was recruited to perinuclear inclusions containing mutant DeltaE-torsinA in neuroblastoma SH-SY5Y cells. In view of these observations, synaptic vesicle recycling was analyzed using the lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope of synaptotagmin I. We found that overexpression of wild type torsinA negatively affects synaptic vesicle endocytosis. Conversely, overexpression of DeltaE-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of snapin and/or torsinA using small interfering RNAs had a similar inhibitory effect on the exo-endocytic process. In addition, down-regulation of torsinA causes the persistence of synaptotagmin I on the plasma membrane, which closely resembles the effect observed by the overexpression of the DeltaE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that DeltaE-torsinA exerts its pathological effects through a loss of function mechanism. This may affect neuronal uptake of neurotransmitters, such as dopamine, playing a role in the development of dystonic movements.

Title: Increased expression of CYP1A1 and CYP1B1 in ovarian/peritoneal endometriotic lesions. Content: Endometriosis is an estrogen-dependent disease affecting up to 10% of all premenopausal women. There is evidence that different endometriosis sites show distinct local estrogen concentration, which, in turn, might be due to a unique local estrogen metabolism. We aimed to investigate whether there was a site-specific regulation of selected enzymes responsible for the oxidative metabolism of estrogens in biopsy samples and endometrial and endometriotic stromal cells. Cytochrome P450 (CYP) 1A1 and CYP1B1 mRNA and protein expressions in deep-infiltrating (rectal, retossigmoidal, and uterossacral) lesions, superficial (ovarian and peritoneal) lesions, and eutopic and healthy (control) endometrium were evaluated by real-time PCR and western blot. Using a cross-sectional study design with 58 premenopausal women who were not under hormonal treatment, we were able to identify an overall increased CYP1A1 and CYP1B1 mRNA expression in superficial lesions compared with the healthy endometrium. CYP1A1 mRNA expression in superficial lesions was also greater than in the eutopic endometrium. Interestingly, we found a similar pattern of CYP1A1 and CYP1B1 expression in in vitro stromal cells isolated from ovarian lesions (n=3) when compared with stromal cells isolated from either rectum lesions or eutopic endometrium. In contradiction, there was an increased half-life of estradiol (measured by HPLC-MS-MS) in ovarian endometriotic stromal cells compared with paired eutopic stromal endometrial cells. Our results indicate that there is a site-dependent regulation of CYP1A1 and CYP1B1 in ovarian/peritoneal lesions and ovarian endometriotic stromal cells, whereas a slower metabolism is taking place in these cells.

Title: Mobilization of intracellular copper stores by the ctr2 vacuolar copper transporter. Content: Copper plays an essential role in processes including signaling to the transcription and protein trafficking machinery, oxidative phosphorylation, iron mobilization, neuropeptide maturation, and normal development. Whereas much is known about intracellular mobilization of ions such as calcium, little information is available on how eukaryotic cells mobilize intracellular copper stores. We describe a mechanism by which the Saccharomyces cerevisiae Ctr2 protein provides bioavailable copper via mobilization of intracellular copper stores. Whereas Ctr2 exhibits structural similarity to the Ctr1 plasma membrane copper importer, microscopic and biochemical fractionation studies localize Ctr2 to the vacuole membrane. We demonstrate that Ctr2 mobilizes vacuolar copper stores in a manner dependent on amino acid residues conserved between the Ctr1 and Ctr2 copper transport family and that ctr2 Delta mutants hyper-accumulate vacuolar copper. Furthermore, a Ctr2 mutant that is mislocalized to the plasma membrane stimulates extracellular copper uptake, supporting a direct role for Ctr2 in copper transport across membranes. These studies identify a novel mechanism for copper mobilization and suggest that organisms cope with copper deprivation via the use of intracellular vesicular stores.

Title: The Sp1-like protein BTEB3 inhibits transcription via the basic transcription element box by interacting with mSin3A and HDAC-1 co-repressors and competing with Sp1. Content: Sp1-like proteins are characterized by three conserved C-terminal zinc finger motifs that bind GC-rich sequences found in promoters of numerous genes essential for mammalian cell homeostasis. These proteins behave as transcriptional activators or repressors. Although significant information has been reported on the molecular mechanisms by which Sp1-like activators function, relatively little is known about mechanisms for repressor proteins. Here we report the functional characterization of BTEB3, a ubiquitously expressed Sp1-like transcriptional repressor. GAL4 assays show that the N terminus of BTEB3 contains regions that can act as direct repressor domains. Immunoprecipitation assays reveal that BTEB3 interacts with the co-repressor mSin3A and the histone deacetylase protein HDAC-1. Gel shift assays demonstrate that BTEB3 specifically binds the BTE site, a well characterized GC-rich DNA element, with an affinity similar to that of Sp1. Reporter and gel shift assays in Chinese hamster ovary cells show that BTEB3 can also mediate repression by competing with Sp1 for BTE binding. Thus, the characterization of this protein expands the repertoire of BTEB-like members of the Sp1 family involved in transcriptional repression. Furthermore, our results suggest a mechanism of repression for BTEB3 involving direct repression by the N terminus via interaction with mSin3A and HDAC-1 and competition with Sp1 via the DNA-binding domain.

Title: Foxk proteins repress the initiation of starvation-induced atrophy and autophagy programs Content: Autophagy is the primary catabolic process triggered in response to starvation. Although autophagic regulation within the cytosolic compartment is well established, it is becoming clear that nuclear events also regulate the induction or repression of autophagy. Nevertheless, a thorough understanding of the mechanisms by which sequence-specific transcription factors modulate expression of genes required for autophagy is lacking. Here, we identify Foxk proteins (Foxk1 and Foxk2) as transcriptional repressors of autophagy in muscle cells and fibroblasts. Interestingly, Foxk1/2 serve to counter-balance another forkhead transcription factor, Foxo3, which induces an overlapping set of autophagic and atrophic targets in muscle. Foxk1/2 specifically recruits Sin3A-HDAC complexes to restrict acetylation of histone H4 and expression of critical autophagy genes. Remarkably, mTOR promotes the transcriptional activity of Foxk1 by facilitating nuclear entry to specifically limit basal levels of autophagy in nutrient-rich conditions. Our study highlights an ancient, conserved mechanism whereby nutritional status is interpreted by mTOR to restrict autophagy by repressing essential autophagy genes through Foxk-Sin3-mediated transcriptional control.

Title: LincRNA-p21 suppresses target mRNA translation. Content: Mammalian long intergenic noncoding RNAs (lincRNAs) are best known for modulating transcription. Here we report a posttranscriptional function for lincRNA-p21 as a modulator of translation. Association of the RNA-binding protein HuR with lincRNA-p21 favored the recruitment of let-7/Ago2 to lincRNA-p21, leading to lower lincRNA-p21 stability. Under reduced HuR levels, lincRNA-p21 accumulated in human cervical carcinoma HeLa cells, increasing its association with JUNB and CTNNB1 mRNAs and selectively lowering their translation. With elevated HuR, lincRNA-p21 levels declined, which in turn derepressed JunB and β-catenin translation and increased the levels of these proteins. We propose that HuR controls translation of a subset of target mRNAs by influencing lincRNA-p21 levels. Our findings uncover a role for lincRNA as a posttranscriptional inhibitor of translation.

Title: METHODS AND RESULTS Content: Cytoscape is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework. Although applicable to any system of molecular components and interactions, Cytoscape is most powerful when used in conjunction with large databases of protein-protein, protein-DNA, and genetic interactions that are increasingly available for humans and model organisms. Cytoscape's software Core provides basic functionality to layout and query the network; to visually integrate the network with expression profiles, phenotypes, and other molecular states; and to link the network to databases of functional annotations. The Core is extensible through a straightforward plug-in architecture, allowing rapid development of additional computational analyses and features. Several case studies of Cytoscape plug-ins are surveyed, including a search for interaction pathways correlating with changes in gene expression, a study of protein complexes involved in cellular recovery to DNA damage, inference of a combined physical/functional interaction network for Halobacterium, and an interface to detailed stochastic/kinetic gene regulatory models.

Title: Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Content: 5-methylcytosine (5mC) in DNA plays an important role in gene expression, genomic imprinting, and suppression of transposable elements. 5mC can be converted to 5-hydroxymethylcytosine (5hmC) by the Tet (ten eleven translocation) proteins. Here, we show that, in addition to 5hmC, the Tet proteins can generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) from 5mC in an enzymatic activity-dependent manner. Furthermore, we reveal the presence of 5fC and 5caC in genomic DNA of mouse embryonic stem cells and mouse organs. The genomic content of 5hmC, 5fC, and 5caC can be increased or reduced through overexpression or depletion of Tet proteins. Thus, we identify two previously unknown cytosine derivatives in genomic DNA as the products of Tet proteins. Our study raises the possibility that DNA demethylation may occur through Tet-catalyzed oxidation followed by decarboxylation.

Title: Cloning of three novel neuronal Cdk5 activator binding proteins. Content: Neuronal Cdc2-like kinase (Nclk) is involved in the regulation of neuronal differentiation and neuro-cytoskeleton dynamics. The active kinase consists of a catalytic subunit, Cdk5, and a 25 kDa activator protein (p25nck5a) derived from a 35 kDa neuronal-specific protein (p35nck5a). As an extension of our previous study (Qi, Z., Tang, D., Zhu, X., Fujita, D.J., Wang, J.H., 1998. Association of neurofilament proteins with neuronal Cdk5 activator. J. Biol. Chem. 270, 2329-2335), which showed that neurofilament is one of the p35nck5a-associated proteins, we now report the isolation of three other novel p35nck5a-associated proteins using the yeast two-hybrid screen. The full-length forms of these three novel proteins, designated C42, C48 and C53, have a molecular mass of 66, 24, and 57 kDa, respectively. Northern analysis indicates that these novel proteins are widely expressed in human tissues, including the heart, brain, skeletal muscle, placenta, lung, liver, kidney and pancreas. The bacterially expressed glutathione S-transferase (GST)-fusion forms of these three proteins were able to co-precipitate p35nck5a complexed with Cdk5 from insect cell lysate. Among these three proteins, only C48 and C53 can be phosphorylated by Nclk, suggesting that they may be the substrates of Nclk. Sequence homology searches have suggested that the C48 protein is marginally related to restin protein, whereas the C42 protein has homologues of unknown function in Caenorhabditis elegans and Arabidopsis thaliana.

Title: p53 binding to nucleosomal DNA depends on the rotational positioning of DNA response element. Content: The sequence-specific binding to DNA is crucial for the p53 tumor suppressor function. To investigate the constraints imposed on p53-DNA recognition by nucleosomal organization, we studied binding of the p53 DNA binding domain (p53DBD) and full-length wild-type p53 protein to a single p53 response element (p53RE) placed near the nucleosomal dyad in six rotational settings. We demonstrate that the strongest p53 binding occurs when the p53RE in the nucleosome is bent in the same direction as observed for the p53-DNA complexes in solution and in co-crystals. The p53RE becomes inaccessible, however, if its orientation in the core particle is changed by approximately 180 degrees. Our observations indicate that the orientation of the binding sites on a nucleosome may play a significant role in the initial p53-DNA recognition and subsequent cofactor recruitment.

Title: Competition between target sites of regulators shapes post-transcriptional gene regulation Content: Post-transcriptional gene regulation (PTGR) of mRNA turnover, localization and translation is mediated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). These regulators exert their effects by binding to specific sequences within their target mRNAs. Increasing evidence suggests that competition for binding is a fundamental principle of PTGR. Not only can miRNAs be sequestered and neutralized by the targets with which they interact through a process termed 'sponging', but competition between binding sites on different RNAs may also lead to regulatory crosstalk between transcripts. Here, we quantitatively model competition effects under physiological conditions and review the role of endogenous sponges for PTGR in light of the key features that emerge.

Title: Drosophila CPEB Orb2A Mediates Memory Independent of Its RNA-Binding Domain Content: Long-term memory and synaptic plasticity are thought to require the synthesis of new proteins at activated synapses. The CPEB family of RNA binding proteins, including Drosophila Orb2, has been implicated in this process. The precise mechanism by which these molecules regulate memory formation is however poorly understood. We used gene targeting and site-specific transgenesis to specifically modify the endogenous orb2 gene in order to investigate its role in long-term memory formation. We show that the Orb2A and Orb2B isoforms, while both essential, have distinct functions in memory formation. These two isoforms have common glutamine-rich and RNA-binding domains, yet Orb2A uniquely requires the former and Orb2B the latter. We further show that Orb2A induces Orb2 complexes in a manner dependent upon both its glutamine-rich region and neuronal activity. We propose that Orb2B acts as a conventional CPEB to regulate transport and/or translation of specific mRNAs, whereas Orb2A acts in an unconventional manner to form stable Orb2 complexes that are essential for memory to persist.

Title: Mutations in G protein beta subunits promote transformation and kinase inhibitor resistance Content: Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Title: Broad antiretroviral defence by human APOBEC3G through lethal editing of nascent reverse transcripts Content: Viral replication usually requires that innate intracellular lines of defence be overcome, a task usually accomplished by specialized viral gene products. The virion infectivity factor (Vif) protein of human immunodeficiency virus (HIV) is required during the late stages of viral production to counter the antiviral activity of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; also known as CEM15), a protein expressed notably in human T lymphocytes. When produced in the presence of APOBEC3G, vif-defective virus is non-infectious. APOBEC3G is closely related to APOBEC1, the central component of an RNA-editing complex that deaminates a cytosine residue in apoB messenger RNA. APOBEC family members also have potent DNA mutator activity through dC deamination; however, whether the editing potential of APOBEC3G has any relevance to HIV inhibition is unknown. Here, we demonstrate that it does, as APOBEC3G exerts its antiviral effect during reverse transcription to trigger G-to-A hypermutation in the nascent retroviral DNA. We also find that APOBEC3G can act on a broad range of retroviruses in addition to HIV, suggesting that hypermutation by editing is a general innate defence mechanism against this important group of pathogens.

Title: A comprehensive genomic binding map of gene and chromatin regulatory proteins in Saccharomyces. Content: Hundreds of different proteins regulate and implement transcription in Saccharomyces. Yet their interrelationships have not been investigated on a comprehensive scale. Here we determined the genome-wide binding locations of 200 transcription-related proteins, under normal and acute heat-shock conditions. This study distinguishes binding between distal versus proximal promoter regions as well as the 3' ends of genes for nearly all mRNA and tRNA genes. This study reveals (1) a greater diversity and specialization of regulation associated with the SAGA transcription pathway compared to the TFIID pathway, (2) new regulators enriched at tRNA genes, (3) a global co-occupancy network of >20,000 unique regulator combinations that show a high degree of regulatory interconnections among lowly expressed genes, (4) regulators of the SAGA pathway located largely distal to the core promoter and regulators of the TFIID pathway located proximally, and (5) distinct mobilization of SAGA- versus TFIID-linked regulators during acute heat shock.

Title: Dual Regulation of miRNA Biogenesis Generates Target Specificity in Neurotrophin-Induced Protein Synthesis Content: Control of translation is a fundamental source of regulation in gene expression. The induction of protein synthesis by brain-derived neurotrophic factor (BDNF) critically contributes to enduring modifications of synaptic function, but how BDNF selectively affects only a minority of expressed mRNAs is poorly understood. We report that BDNF rapidly elevates Dicer, increasing mature miRNA levels and inducing RNA processing bodies in neurons. BDNF also rapidly induces Lin28, causing selective loss of Lin28-regulated miRNAs and a corresponding upregulation in translation of their target mRNAs. Binding sites for Lin28-regulated miRNAs are necessary and sufficient to confer BDNF responsiveness to a transcript. Lin28 deficiency, or expression of a Lin28-resistant Let-7 precursor miRNA, inhibits BDNF translation specificity and BDNF-dependent dendrite arborization. Our data establish that specificity in BDNF-regulated translation depends upon a two-part posttranscriptional control of miRNA biogenesis that generally enhances mRNA repression in association with GW182 while selectively derepressing and increasing translation of specific mRNAs.

Title: Transcription initiation complex structures elucidate DNA opening Content: Transcription of eukaryotic protein-coding genes begins with assembly of the RNA polymerase (Pol) II initiation complex and promoter DNA opening. Here we report cryo-electron microscopy (cryo-EM) structures of yeast initiation complexes containing closed and open DNA at resolutions of 8.8 Å and 3.6 Å, respectively. DNA is positioned and retained over the Pol II cleft by a network of interactions between the TATA-box-binding protein TBP and transcription factors TFIIA, TFIIB, TFIIE, and TFIIF. DNA opening occurs around the tip of the Pol II clamp and the TFIIE ‘extended winged helix’ domain, and can occur in the absence of TFIIH. Loading of the DNA template strand into the active centre may be facilitated by movements of obstructing protein elements triggered by allosteric binding of the TFIIE ‘E-ribbon’ domain. The results suggest a unified model for transcription initiation with a key event, the trapping of open promoter DNA by extended protein–protein and protein–DNA contacts.

Title: Multiple degradation pathways regulate versatile CIP/KIP CDK inhibitors. Content: The mammalian CIP/KIP family of cyclin-dependent kinase (CDK) inhibitors (CKIs) comprises three proteins--p21(Cip1/WAF1), p27(Kip1), and p57(Kip2)--that bind and inhibit cyclin-CDK complexes, which are key regulators of the cell cycle. CIP/KIP CKIs have additional independent functions in regulating transcription, apoptosis and actin cytoskeletal dynamics. These divergent functions are performed in distinct cellular compartments and contribute to the seemingly contradictory observation that the CKIs can both suppress and promote cancer. Multiple ubiquitin ligases (E3s) direct the proteasome-mediated degradation of p21, p27 and p57. This review analyzes recent data highlighting our current understanding of how distinct E3 pathways regulate subpopulations of the CKIs to control their diverse functions.

Title: Functional elements within the dynein microtubule-binding domain Content: Dynein interacts with microtubules through an ATP-sensitive linkage mapped to a structurally complex region of the heavy chain following the fourth P-loop motif. Virtually nothing is known regarding how binding affinity is achieved and modulated during ATP hydrolysis. We have performed a detailed dissection of the microtubule contact site, using fragment expression, alanine substitution, and peptide competition. Our work identifies three clusters of amino acids important for the physical contact with microtubules; two of these fall within a region sharing sequence homology with MAP1B, the third in a region just downstream. Amino acid substitutions within any one of these regions can eliminate or weaken microtubule binding (KK3379, 80, E3385, K3387, K3397, KK3410,11, W3414, RKK3418-20, F3426, R3464, S3466, and K3467), suggesting that their activities are highly coordinated. A peptide that actively displaces MAP1B from microtubules perturbs dynein binding, supporting previous evidence for similar sites of interaction. We have also identified four amino acids whose substitutions affect release of the motor from the microtubule (E3413, R3444, E3460, and C3469). These suggest that nucleotide-sensitive affinity may be locally controlled at the site of contact. Our work is the first detailed description of dynein-tubulin interactions and provides a framework for understanding how affinity is achieved and modulated.

Title: Oncofetal long noncoding RNA PVT1 promotes proliferation and stem cell-like property of hepatocellular carcinoma cells by stabilizing NOP2. Content: UNLABELLED Many protein-coding oncofetal genes are highly expressed in murine and human fetal liver and silenced in adult liver. The protein products of these hepatic oncofetal genes have been used as clinical markers for the recurrence of hepatocellular carcinoma (HCC) and as therapeutic targets for HCC. Herein we examined the expression profiles of long noncoding RNAs (lncRNAs) found in fetal and adult liver in mice. Many fetal hepatic lncRNAs were identified; one of these, lncRNA-mPvt1, is an oncofetal RNA that was found to promote cell proliferation, cell cycling, and the expression of stem cell-like properties of murine cells. Interestingly, we found that human lncRNA-hPVT1 was up-regulated in HCC tissues and that patients with higher lncRNA-hPVT1 expression had a poor clinical prognosis. The protumorigenic effects of lncRNA-hPVT1 on cell proliferation, cell cycling, and stem cell-like properties of HCC cells were confirmed both in vitro and in vivo by gain-of-function and loss-of-function experiments. Moreover, mRNA expression profile data showed that lncRNA-hPVT1 up-regulated a series of cell cycle genes in SMMC-7721 cells. By RNA pulldown and mass spectrum experiments, we identified NOP2 as an RNA-binding protein that binds to lncRNA-hPVT1. We confirmed that lncRNA-hPVT1 up-regulated NOP2 by enhancing the stability of NOP2 proteins and that lncRNA-hPVT1 function depends on the presence of NOP2. CONCLUSION Our study demonstrates that the expression of many lncRNAs is up-regulated in early liver development and that the fetal liver can be used to search for new diagnostic markers for HCC. LncRNA-hPVT1 promotes cell proliferation, cell cycling, and the acquisition of stem cell-like properties in HCC cells by stabilizing NOP2 protein. Regulation of the lncRNA-hPVT1/NOP2 pathway may have beneficial effects on the treatment of HCC.

Title: Post-prenylation-processing enzymes as new targets in oncogenesis Content: RAS and many other oncogenic proteins undergo a complex series of post-translational modifications that are initiated by the addition of an isoprenoid lipid through a process known as prenylation. Following prenylation, these proteins usually undergo endoproteolytic processing by the RCE1 protease and then carboxyl methylation by a unique methyltransferase known as isoprenylcysteine carboxyl methyltransferase (ICMT). Although inhibitors that have been designed to target the prenylation step are now in advanced-stage clinical trials, their utility and efficacy seem to be limited. Recent findings, however, indicate that the inhibition of these post-prenylation-processing steps — particularly that of ICMT-catalysed methylation — might provide a better approach to the control of cancer-cell proliferation.

Title: Genome-wide analysis reveals SR protein cooperation and competition in regulated splicing. Content: SR proteins are well-characterized RNA binding proteins that promote exon inclusion by binding to exonic splicing enhancers (ESEs). However, it has been unclear whether regulatory rules deduced on model genes apply generally to activities of SR proteins in the cell. Here, we report global analyses of two prototypical SR proteins, SRSF1 (SF2/ASF) and SRSF2 (SC35), using splicing-sensitive arrays and CLIP-seq on mouse embryo fibroblasts (MEFs). Unexpectedly, we find that these SR proteins promote both inclusion and skipping of exons in vivo, but their binding patterns do not explain such opposite responses. Further analyses reveal that loss of one SR protein is accompanied by coordinated loss or compensatory gain in the interaction of other SR proteins at the affected exons. Therefore, specific effects on regulated splicing by one SR protein actually depend on a complex set of relationships with multiple other SR proteins in mammalian genomes.

Title: Genetic and Functional Diversification of Small RNA Pathways in Plants Content: Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense.

Title: Long non-coding RNAs as novel players in β cell function and type 1 diabetes Content: BACKGROUND Long non-coding RNAs (lncRNAs) are a sub-class within non-coding RNA repertoire that have emerged as crucial regulators of the gene expression in various pathophysiological conditions. lncRNAs display remarkable versatility and wield their functions through interactions with RNA, DNA, or proteins. Accumulating body of evidence based on multitude studies has highlighted the role of lncRNAs in many autoimmune and inflammatory diseases, including type 1 diabetes (T1D). This review highlights emerging roles of lncRNAs in immune and islet β cell function as well as some of the challenges and opportunities in understanding the pathogenesis of T1D and its complications. CONCLUSION We accentuate that the lncRNAs within T1D-loci regions in consort with regulatory variants and enhancer clusters orchestrate the chromatin remodeling in β cells and thereby act as cis/trans-regulatory determinants of islet cell transcriptional programs.

Title: Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene. Content: One type of RNA editing involves the conversion of adenosine residues into inosine in double-stranded RNA through the action of adenosine deaminases acting on RNA (ADAR). A-to-I RNA editing of the coding sequence could result in synthesis of proteins not directly encoded in the genome. ADAR edits also non-coding sequences of target RNAs, such as introns and 3'-untranslated regions, which may affect splicing, translation, and mRNA stability. Three mammalian ADAR gene family members (ADAR1-3) have been identified. Here we investigated phenotypes of mice homozygous for ADAR1 null mutation. Although live ADAR1-/- embryos with normal gross appearance could be recovered up to E11.5, widespread apoptosis was detected in many tissues. Fibroblasts derived from ADAR1-/- embryos were also prone to apoptosis induced by serum deprivation. Our results demonstrate an essential requirement for ADAR1 in embryogenesis and suggest that it functions to promote survival of numerous tissues by editing one or more double-stranded RNAs required for protection against stress-induced apoptosis.

Title: NuA4 lysine acetyltransferase Esa1 is targeted to coding regions and stimulates transcription elongation with Gcn5. Content: NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.

Title: Pseudoknots: RNA Structures with Diverse Functions Content: RNA molecules fulfill a diverse set of biological functions within cells, from the transfer of genetic information from DNA to protein, to enzymatic catalysis. Reflecting this range of roles, simple linear strings of RNA—made up of uracil, guanine, cytosine, and adenine—form a variety of complex three-dimensional structures. Just as proteins form distinct structural motifs such as zinc fingers and beta barrels, certain structures are also commonly adopted by RNA molecules. Among the most prevalent RNA structures is a motif known as the pseudoknot. First recognized in the turnip yellow mosaic virus [1], a pseudoknot is an RNA structure that is minimally composed of two helical segments connected by single-stranded regions or loops (Figure 1). Although several distinct folding topologies of pseudoknots exist, the best characterized is the H type. In the H-type fold, the bases in the loop of a hairpin form intramolecular pairs with bases outside of the stem (Figure 1A and and1B).1B). This causes the formation of a second stem and loop, resulting in a pseudoknot with two stems and two loops (Figure 1C). The two stems are able to stack on top of each other to form a quasi-continuous helix with one continuous and one discontinuous strand. The single-stranded loop regions often interact with the adjacent stems (loop 1–stem 2 or loop 2–stem 1) to form hydrogen bonds and to participate in the overall structure of the molecule. Hence, this relatively simple fold can yield very complex and stable RNA structures. Due to variation of the lengths of the loops and stems, as well as the types of interactions between them, pseudoknots represent a structurally diverse group. It is fitting that they play a variety of diverse roles in biology. These roles include forming the catalytic core of various ribozymes [2,3], self-splicing introns [4], and telomerase [5]. Additionally, pseudoknots play critical roles in altering gene expression by inducing ribosomal frameshifting in many viruses [6–9].

Title: The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells Content: The recognition of pathogenic DNA is important to the initiation of antiviral responses. Here we report the identification of DDX41, a member of the DEXDc family of helicases, as an intracellular DNA sensor in myeloid dendritic cells (mDCs). Knockdown of DDX41 expression by short hairpin RNA blocked the ability of mDCs to mount type I interferon and cytokine responses to DNA and DNA viruses. Overexpression of both DDX41 and the membrane-associated adaptor STING together had a synergistic effect in promoting Ifnb promoter activity. DDX41 bound both DNA and STING and localized together with STING in the cytosol. Knockdown of DDX41 expression blocked activation of the mitogen-activated protein kinase TBK1 and the transcription factors NF-κB and IRF3 by B-form DNA. Our results suggest that DDX41 is an additional DNA sensor that depends on STING to sense pathogenic DNA.

Title: Brahma-related gene 1-dependent STAT3 recruitment at IL-6-inducible genes. Content: IL-6 is an immunoregulatory cytokine with multiple functions in hemopoiesis, proliferation, and tumorigenesis. IL-6 triggers phosphorylation, dimerization, and nuclear translocation of STAT3, which binds to target promoters and activates transcription. Brahma-related gene 1 (BRG1), the enzymatic engine of the yeast-mating type-switching and sucrose-nonfermenting chromatin-remodeling complex, is essential for recruitment of STAT1 or STAT1/STAT2-containing complexes to IFN targets. We hypothesized that BRG1 might also be required for STAT3 recruitment. In this study, we show that induction of a subset of human IL-6-responsive genes is BRG1 dependent. BRG1 is constitutively present at these targets and is required for STAT3 recruitment, downstream histone modifications, and IL-6-induced chromatin remodeling. IL-6-induced recruitment of STAT3 to the IFN regulatory factor 1 promoter and subsequent mRNA synthesis is BRG1 dependent, even though IFN-gamma-mediated STAT1 recruitment to this locus is BRG1 independent. BRG1 also increased basal expression of IFN-induced transmembrane protein 3 and IFN-gamma-induced protein 16, and the basal chromatin accessibility at the promoter of IFN regulatory factor 1. The effect on basal expression was STAT3 independent, as revealed by small interfering RNA knockdown. Together with prior observations, these data reveal that BRG1 has a broad role in mediating STAT accessibility at multiple cytokine-responsive promoters and exposes promoter specific differences in both the effect of BRG1 on basal chromatin accessibility and on access of different STAT proteins to the same target.

Title: Primate cytomegaloviruses encode and express an IL-10-like protein. Content: An open reading frame (ORF) with homology to interleukin-10 (IL-10) has been identified in rhesus cytomegalovirus (RhCMV). The IL-10-like protein is generated from a multispliced, polyadenylated early gene transcript encompassing part of the corresponding UL111A ORF of human CMV (HCMV). Immunological analyses confirm expression of the IL-10-like protein both in tissue culture and in RhCMV-infected rhesus macaques. Conserved ORFs were subsequently identified in human, baboon, and African green monkey CMV, and a fully processed transcript has been mapped in fibroblasts infected with the Towne strain of HCMV. The conservation of this previously unrecognized ORF suggests that the protein may play an essential role in primate CMV persistence and pathogenesis.

Title: A coactivator of pre-mRNA splicing. Content: The nuclear matrix antigen recognized by the monoclonal antibody (mAb) B1C8 is a novel serine (S) and arginine (R)-rich protein associated with splicing complexes and is named here SRm160 (SR-related matrix protein of 160 kD). SRm160 contains multiple SR repeats, but unlike proteins of the SR family of splicing factors, lacks an RNA recognition motif. SRm160 and a related protein SRm300 (the 300-kD nuclear matrix antigen recognized by mAb B4A11) form a complex that is required for the splicing of specific pre-mRNAs. The SRm160/300 complex associates with splicing complexes and promotes splicing through interactions with SR family proteins. Binding of SRm160/300 to pre-mRNA is normally also dependent on U1 snRNP and is stabilized by U2 snRNP. Thus, SRm160/300 forms multiple interactions with components bound directly to important sites within pre-mRNA. The results suggest that a complex of the nuclear matrix proteins SRm160 and SRm300 functions as a coactivator of pre-mRNA splicing.

Title: Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome. Content: The transcription factor HNF3 and linker histones H1 and H5 possess winged-helix DNA-binding domains, yet HNF3 and other fork head-related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild-type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site-directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome-binding activities that are not predicted from studies of factor interactions with free DNA.

Title: Piezo1 Is as a Novel Trefoil Factor Family 1 Binding Protein that Promotes Gastric Cancer Cell Mobility In Vitro Content: Trefoil factor family 1 (TFF1) is a member of the TFF-domain peptide family involved in epithelial restitution and cell motility. Recently, we screened Piezo1 as a candidate TFF1-binding protein. We aimed to confirm Piezo1 as a novel TFF1 binding protein and to assess the role of this interaction in mediating gastric cancer cell mobility. This interaction was confirmed by co-immunoprecipitation and co-localisation of TFF1 and Piezo1 in GES-1 cells. We used stable RNA interference to knockdown Piezo1 protein expression and restored the expression of TFF1 in the gastric cancer cell lines SGC-7901 and BGC-823. Cell motility was evaluated using invasion assay and migration assay in vitro. The expression levels of the integrin subunits β1, β5, α1 as well as the expression of β-catenin and E-cadherin were detected by Western blot. We demonstrate that TFF1, but not TFF2 or TFF3, bind to and co-localize with Piezo1 in the cytoplasm in vitro. TFF1 interacts with the C-terminal portion of the Piezo1 protein. Wound healing and trans-well assays demonstrated that the restored expression of TFF1 promoted cell mobility in gastric cancer cells, and this effect was attenuated by the knockdown of Piezo1. Western blots demonstrated the decreased expression of integrin β1 in Piezo1-knockdown cells. Our data demonstrate that Piezo1 is a novel TFF1 binding protein that is important for TFF1-mediated cell migration and suggest that this interaction may be a therapeutic target in the invasion and metastasis of gastric cancer.

Title: The evolution of RNAs with multiple functions. Content: Increasing numbers of transcripts have been reported to transmit both protein-coding and regulatory information. Apart from challenging our conception of the gene, this observation raises the question as to what extent this phenomenon occurs across the genome and how and why such dual encoding of function has evolved in the eukaryotic genome. To address this question, we consider the evolutionary path of genes in the earliest forms of life on Earth, where it is generally regarded that proteins evolved from a cellular machinery based entirely within RNA. This led to the domination of protein-coding genes in the genomes of microorganisms, although it is likely that RNA never lost its other capacities and functionalities, as evidenced by cis-acting riboswitches and UTRs. On the basis that the subsequent evolution of a more sophisticated regulatory architecture to provide higher levels of epigenetic control and accurate spatiotemporal expression in developmentally complex organisms is a complicated task, we hypothesize: (i) that mRNAs have been and remain subject to secondary selection to provide trans-acting regulatory capability in parallel with protein-coding functions; (ii) that some and perhaps many protein-coding loci, possibly as a consequence of gene duplication, have lost protein-coding functions en route to acquiring more sophisticated trans-regulatory functions; (iii) that many transcripts have become subject to secondary processing to release different products; and (iv) that novel proteins have emerged within loci that previously evolved functionality as regulatory RNAs. In support of the idea that there is a dynamic flux between different types of informational RNAs in both evolutionary and real time, we review recent observations that have arisen from transcriptomic surveys of complex eukaryotes and reconsider how these observations impact on the notion that apparently discrete loci may express transcripts with more than one function. In conclusion, we posit that many eukaryotic loci have evolved the capacity to transact a multitude of overlapping and potentially independent functions as both regulatory and protein-coding RNAs.

Title: The NBS1–Treacle complex controls ribosomal RNA transcription in response to DNA damage Content: Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.

Title: Characterization of a single-stranded DNA-binding protein from Pseudomonas aeruginosa PAO1. Content: Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as in DNA replication, repair, and recombination, and is essential for cell survival. We characterized the single-stranded DNA (ssDNA)-binding properties of Pseudomonas aeruginosa PAO1 SSB (PaSSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified PaSSB by gel filtration chromatography revealed a stable tetramer in solution. In fluorescence titrations, PaSSB bound 22-32 nucleotides (nt) per tetramer depending on salt concentration. Using EMSA, we characterized the stoichiometry of PaSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 29 ± 1 nt. Furthermore, EMSA results indicated that the dissociation constants of PaSSB for the first tetramer were less than those for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding mode of PaSSB is expected to be noncooperative.

Title: Archaeal chromatin and transcription. Content: Archaea contain a variety of sequence-independent DNA binding proteins consistent with the evolution of several different, sometimes overlapping and exchangeable solutions to the problem of genome compaction. Some of these proteins undergo residue-specific post-translational lysine acetylation or methylation, hinting at analogues of the histone modifications that regulate eukaryotic chromatin structure and transcription. Archaeal transcription initiation most closely resembles the eukaryotic RNA polymerase II (RNAPII) system, but Archaea do not appear to have homologues of the multisubunit complexes that remodel eukaryotic chromatin and activate RNAPII initiation. In contrast, they have sequence-specific regulators that repress and perhaps activate archaeal transcription by mechanisms superficially similar to the bacterial paradigm of regulating promoter binding by RNAP. Repressors compete with archaeal TATA-box binding protein (TBP) and TFB for the TATA-box and TFB-recognition elements (BRE) of the archaeal promoter, or with archaeal RNAP for the site of transcription initiation. Transcript-specific regulation by repressors binding to sites of transcript initiation is consistent with such sites having very little sequence conservation. However, most Archaea have only one TBP and/or TFB that presumably must therefore bind to similar TATA-box and BRE sequences upstream of most genes. Repressors that function by competing with TBP and/or TFB binding must therefore also make additional contacts with transcript-specific regulatory sites adjacent or remote from the TATA-box/BRE region. The fate of the archaeal TBP and TFB following transcription initiation remains to be determined. Based on functional homology with their eukaryotic RNAPII-system counterparts, archaeal TBP and possibly also TFB should remain bound to the TATA-box/BRE region after transcription initiation. However, this seems unlikely as it might limit repressor competition at this site to only the first round of transcription initiation.

Title: Structural basis for the activation of microtubule assembly by the EB1 and p150Glued complex. Content: Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. Here we report the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued. Each molecule of the EB1 dimer contains two helices forming a conserved four-helix bundle, while also providing p150Glued binding sites in its flexible tail region. Combining crystallography, NMR, and mutational analyses, our studies reveal the critical interacting elements of both EB1 and p150Glued, whose mutation alters microtubule polymerization activity. Moreover, removal of the key flexible tail from EB1 activates microtubule assembly by EB1 alone, suggesting that the flexible tail negatively regulates EB1 activity. We, therefore, propose that EB1 possesses an auto-inhibited conformation, which is relieved by p150Glued as an allosteric activator.

Title: Coupling cAMP signaling to transcription in the liver: pivotal role of CREB and CREM. Content: Transcriptional factors binding to cAMP-responsive elements (CREs) in the promoters of various genes belong to the basic domain-leucine zipper superfamily and are composed of three genes in mammals, CREB, CREM, and ATF-1. A large number of CREB, CREM, and ATF-1 proteins are generated by posttranscriptional events, mostly alternative splicing, and regulate gene expression by acting as activators or repressors. Activation is classically brought about by signaling-dependent phosphorylation of a key acceptor site (Ser133 in CREB) by a number of possible kinases, including PKA, CamKIV, and Rsk-2. Phosphorylation is the prerequisite for the interaction of CBP (CREB-binding protein), a co-activator that has also histone acetyltransferase activity. Repression may involve dynamic dephosphorylation of the activators and thus decreased association with CBP. Another pathway of transcriptional repression on CRE sites implicates the inducible repressor ICER (inducible cAMP early repressor), a product of the CREM gene. Being an inducible repressor, ICER is involved in autoregulatory feedback loops of transcription that govern the down-regulation of early response genes, such as the proto-oncogene c-fos. The liver represents a remarkable physiological setting where cAMP-responsive signaling plays a major role. Indeed, a finely tuned program of gene expression is triggered by partial hepatectomy, so that through specific checkpoints a coordinated regeneration of the tissue is obtained. Temporal kinetics of transcriptional activation after hepatectomy reveals a pattern of early induction for several genes, some of them controlled by the CREB/CREM transcription factors. An important role of CREM in liver physiology was suggested by the robust induction of ICER after partial hepatectomy. The delay in tissue regeneration in CREM-deficient mice confirmed the important function of this factor in regulating hepatocyte proliferation. As gene induction is accompanied by critical changes in chromatin organization, the deciphering of the specific modification codes that histones display during liver regeneration and physiology will provide exciting new insights into the dynamics of chromatin architecture.

Title: Ribosome Profiling Provides Evidence that Large Noncoding RNAs Do Not Encode Proteins Content: Large noncoding RNAs are emerging as an important component in cellular regulation. Considerable evidence indicates that these transcripts act directly as functional RNAs rather than through an encoded protein product. However, a recent study of ribosome occupancy reported that many large intergenic ncRNAs (lincRNAs) are bound by ribosomes, raising the possibility that they are translated into proteins. Here, we show that classical noncoding RNAs and 5' UTRs show the same ribosome occupancy as lincRNAs, demonstrating that ribosome occupancy alone is not sufficient to classify transcripts as coding or noncoding. Instead, we define a metric based on the known property of translation whereby translating ribosomes are released upon encountering a bona fide stop codon. We show that this metric accurately discriminates between protein-coding transcripts and all classes of known noncoding transcripts, including lincRNAs. Taken together, these results argue that the large majority of lincRNAs do not function through encoded proteins.

Title: Parathyroid hormone induces E4bp4 messenger ribonucleic acid expression primarily through cyclic adenosine 3',5'-monophosphate signaling in osteoblasts. Content: PTH binding to its receptor activates protein kinase A (PKA), protein kinase C (PKC), and calcium signaling to induce transcription of primary response genes in osteoblasts. Adenovirus E4 promoter-binding protein/nuclear factor regulated by IL-3 (E4BP4/NFIL3), a transcriptional repressor, is a PTH-induced primary response gene in primary mouse osteoblasts (MOBs). Here we investigate the signaling pathway(s) that lead to PTH induction of E4bp4 mRNA expression. Ten and 100 nm PTH induced maximum E4bp4 expression in MOBs. Forskolin (FSK), an adenylate cyclase inducer, 8-bromo-cAMP, a cAMP analog, and phorbol myristate acetate, a PKC activator, increased E4bp4 mRNA levels, whereas ionomycin, a calcium ionophore, had no effect. Pretreatment of cells with 30 microm H89, a PKA inhibitor, strongly inhibited PTH- and FSK-induced E4bp4 expression. In contrast, overnight pretreatment with 1 microm phorbol myristate acetate to down-regulate PKC signaling did not alter PTH and FSK effects. Moreover, PTH (3-34) that does not activate cAMP signaling did not increase E4bp4 expression. Prostaglandin E(2), which signals through cAMP, increased E4bp4 mRNA at all doses, whereas prostaglandin F(2alpha) that primarily activates PKC and calcium signaling, induced E4bp4 only at high doses and fluprostenol that only activates PKC and calcium signaling, had no effect. Finally, 80 microg/kg PTH (1-34) ip injection induced E4bp4 mRNA expression at 1 h in mice. In contrast, 80 microg/kg PTH (3-34) had no effect. Our data suggest that PTH-induced E4bp4 mRNA expression is mediated primarily through cAMP-PKA signaling in vitro and in vivo. In conjunction with our previous report, we hypothesize that E4bp4 attenuates transcription of osteoblastic genes possessing E4bp4 promoter binding sites.

Title: ADAM13 disintegrin and cysteine-rich domains bind to the second heparin-binding domain of fibronectin. Content: ADAM13 is a member of the disintegrin and metalloprotease protein family that is expressed on cranial neural crest cells surface and is essential for their migration. ADAM13 is an active protease that can cleave fibronectin in vitro and remodel a fibronectin substrate in vivo. Using a recombinant secreted protein containing both disintegrin and cysteine-rich domains of ADAM13, we show that this "adhesive" region of the protein binds directly to fibronectin. Fibronectin fusion proteins corresponding to the various functional domains were used to define the second heparin-binding domain as the ADAM13 binding site. Mutation of the syndecan-binding site (PPRR --> PPTM) within this domain abolishes binding of the recombinant disintegrin and cysteine-rich domains of ADAM13. We further show that the adhesive disintegrin and cysteine-rich domain of ADAM13 can promote cell adhesion via beta(1) integrins. This adhesion requires integrin activation and can be prevented by antibodies to the cysteine-rich domain of ADAM13 and beta(1) integrin. Finally, wild type, but not the E/A mutant of ADAM13 metalloprotease domain, can be shed from the cell surface, releasing the metalloprotease domain associated with the disintegrin and cysteine-rich domains. This suggests that ADAM13 shedding may involve its own metalloprotease activity and that the released protease may interact with both integrins and extracellular matrix proteins.

Title: The mechanosensitive ion channel Piezo1 is inhibited by the peptide GsMTx4. Content: Cells can respond to mechanical stress by gating mechanosensitive ion channels (MSCs). The cloning of Piezo1, a eukaryotic cation selective MSC, defines a new system for studying mechanical transduction at the cellular level. Because Piezo1 has electrophysiological properties similar to those of endogenous cationic MSCs that are selectively inhibited by the peptide GsMTx4, we tested whether the peptide targets Piezo1 activity. Extracellular GsMTx4 at micromolar concentrations reversibly inhibited ∼80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibition was voltage insensitive, and as seen with endogenous MSCs, the mirror image d enantiomer inhibited like the l. The rate constants for binding and unbinding based on Piezo1 current kinetics provided association and dissociation rates of 7.0 × 10(5) M(-1) s(-1) and 0.11 s(-1), respectively, and a K(D) of ∼155 nM, similar to values previously reported for endogenous MSCs. Consistent with predicted gating modifier behavior, GsMTx4 produced an ∼30 mmHg rightward shift in the pressure-gating curve and was active on closed channels. In contrast, streptomycin, a nonspecific inhibitor of cationic MSCs, showed the use-dependent inhibition characteristic of open channel block. The peptide did not block currents of the mechanical channel TREK-1 on outside-out patches. Whole-cell Piezo1 currents were also reversibly inhibited by GsMTx4, and although the off rate was nearly identical to that of outside-out patches, differences were observed for the on rate. The ability of GsMTx4 to target the mechanosensitivity of Piezo1 supports the use of this channel in high-throughput screens for pharmacological agents and diagnostic assays.

Title: Regulation of pluripotency by RNA binding proteins. Content: Establishment, maintenance, and exit from pluripotency require precise coordination of a cell's molecular machinery. Substantial headway has been made in deciphering many aspects of this elaborate system, particularly with respect to epigenetics, transcription, and noncoding RNAs. Less attention has been paid to posttranscriptional regulatory processes such as alternative splicing, RNA processing and modification, nuclear export, regulation of transcript stability, and translation. Here, we introduce the RNA binding proteins that enable the posttranscriptional regulation of gene expression, summarizing current and ongoing research on their roles at different regulatory points and discussing how they help script the fate of pluripotent stem cells.

Title: A Screen for Endocytic Motifs Content: Sorting signals for cargo selection into coated vesicles are usually in the form of short linear motifs. Three motifs for clathrin-mediated endocytosis have been identified: YXXPhi, [D/E]XXXL[L/I] and FXNPXY. To search for new endocytic motifs, we made a library of CD8 chimeras with random sequences in their cytoplasmic tails, and used a novel fluorescence-activated cell sorting (FACS)-based assay to select for endocytosed constructs. Out of the five tails that were most efficiently internalized, only one was found to contain a conventional motif. Two contain dileucine-like sequences that appear to be variations on the [D/E]XXXL[L/I] motif. Another contains a novel internalization signal, YXXXPhiN, which is able to function in cells expressing a mutant mu2 that cannot bind YXXPhi, indicating that it is not a variation on the YXXPhi motif. Similar sequences are present in endogenous proteins, including a functional YXXXPhiN (in addition to a classical YXXPhi) in cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Thus, the repertoire of endocytic motifs is more extensive than the three well-characterized sorting signals.

Title: CLIP identifies Nova-regulated RNA networks in the brain. Content: Nova proteins are neuron-specific antigens targeted in paraneoplastic opsoclonus myoclonus ataxia (POMA), an autoimmune neurologic disease characterized by abnormal motor inhibition. Nova proteins regulate neuronal pre-messenger RNA splicing by directly binding to RNA. To identify Nova RNA targets, we developed a method to purify protein-RNA complexes from mouse brain with the use of ultraviolet cross-linking and immunoprecipitation (CLIP).Thirty-four transcripts were identified multiple times by Nova CLIP.Three-quarters of these encode proteins that function at the neuronal synapse, and one-third are involved in neuronal inhibition. Splicing targets confirmed in Nova-/- mice include c-Jun N-terminal kinase 2, neogenin, and gephyrin; the latter encodes a protein that clusters inhibitory gamma-aminobutyric acid and glycine receptors, two previously identified Nova splicing targets. Thus, CLIP reveals that Nova coordinately regulates a biologically coherent set of RNAs encoding multiple components of the inhibitory synapse, an observation that may relate to the cause of abnormal motor inhibition in POMA.

Title: Decoding Mammalian Ribosome-mRNA States by Translational GTPase Complexes Content: In eukaryotes, accurate protein synthesis relies on a family of translational GTPases that pair with specific decoding factors to decipher the mRNA code on ribosomes. We present structures of the mammalian ribosome engaged with decoding factor⋅GTPase complexes representing intermediates of translation elongation (aminoacyl-tRNA⋅eEF1A), termination (eRF1⋅eRF3), and ribosome rescue (Pelota⋅Hbs1l). Comparative analyses reveal that each decoding factor exploits the plasticity of the ribosomal decoding center to differentially remodel ribosomal proteins and rRNA. This leads to varying degrees of large-scale ribosome movements and implies distinct mechanisms for communicating information from the decoding center to each GTPase. Additional structural snapshots of the translation termination pathway reveal the conformational changes that choreograph the accommodation of decoding factors into the peptidyl transferase center. Our results provide a structural framework for how different states of the mammalian ribosome are selectively recognized by the appropriate decoding factor⋅GTPase complex to ensure translational fidelity.

Title: Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1. Content: Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.

Title: Transposable elements have rewired the core regulatory network of human embryonic stem cells Content: Detection of new genomic control elements is critical in understanding transcriptional regulatory networks in their entirety. We studied the genome-wide binding locations of three key regulatory proteins (POU5F1, also known as OCT4; NANOG; and CTCF) in human and mouse embryonic stem cells. In contrast to CTCF, we found that the binding profiles of OCT4 and NANOG are markedly different, with only ∼5% of the regions being homologously occupied. We show that transposable elements contributed up to 25% of the bound sites in humans and mice and have wired new genes into the core regulatory network of embryonic stem cells. These data indicate that species-specific transposable elements have substantially altered the transcriptional circuitry of pluripotent stem cells.

Title: Identification and characterization of a widely expressed form of adenylyl cyclase. Content: A novel mammalian adenylyl cyclase was identified by reverse transcription-polymerase chain reaction amplification using degenerate primers based on a conserved region of previously described adenylyl cyclases (Premont, R. T. (1994) Methods Enzymol. 238, 116-127). The full-length cDNA sequence obtained from mouse brain predicts a 1353-amino acid protein possessing a 12-membrane span topology, and containing two regions of high similarity with the catalytic domains of adenylyl cyclases. Comparison of this novel adenylyl cyclase with the eight previously described mammalian enzymes indicates that this type 9 adenylyl cyclase sequence is the most divergent, defining a sixth distinct subclass of mammalian adenylyl cyclases. The AC9 gene has been localized to human chromosome band 16p13.3-13.2. The 8.5-kb mRNA encoding the type 9 adenylyl cyclase is widely distributed, being readily detected in all tissues tested, and is found at very high levels in skeletal muscle and brain. AC9 mRNA is found throughout rat brain but is particularly abundant in hippocampus, cerebellum, and neocortex. An antiserum directed against the carboxyl terminus of the type 9 adenylyl cyclase detects native and expressed recombinant AC9 protein in tissue and cell membranes. Levels of the AC9 protein are highest in mouse brain membranes. Characterization of expressed recombinant AC9 reveals that the protein is a functional adenylyl cyclase that is stimulated by Mg2+, forskolin, and mutationally activated Gsalpha. AC9 activity is not affected by Ca2+/calmodulin or by G protein betagamma-subunits. Thus AC9 represents a functional G protein-regulated adenylyl cyclase found in brain and in most somatic tissues.

Title: Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Content: Replication protein A [RPA; also known as replication factor A (RFA) and human single-stranded DNA-binding protein] is a single-stranded DNA-binding protein that is required for multiple processes in eukaryotic DNA metabolism, including DNA replication, DNA repair, and recombination. RPA homologues have been identified in all eukaryotic organisms examined and are all abundant heterotrimeric proteins composed of subunits of approximately 70, 30, and 14 kDa. Members of this family bind nonspecifically to single-stranded DNA and interact with and/or modify the activities of multiple proteins. In cells, RPA is phosphorylated by DNA-dependent protein kinase when RPA is bound to single-stranded DNA (during S phase and after DNA damage). Phosphorylation of RPA may play a role in coordinating DNA metabolism in the cell. RPA may also have a role in modulating gene expression.