Genome-Wide Analysis of Histidine Repeats Reveals Their Role in the Localization of Human Proteins to the Nuclear Speckles Compartment
[['Single amino acid repeats ( SARs ) , also known as homopolymeric tracts , are very common in eukaryotes [1] and between 18–20% of proteins in the human genome contain such repetitive sequences [2] .', 'Although most of them are thought to be functionally neutral , recent evidence suggests they may play important functional or structural roles .', 'Indeed , there is an overrepresentation of SARs-containing proteins ( SARPs ) among transcription factors , kinases and proteins required for development [2]–[5] .', 'The intrinsic disorder of such repeats converts them into flexible spacer elements between individual folded domains , allowing SARPs to associate in large , multiprotein complexes [5] , [6] .', 'In addition , it is thought that disordered regions can bind to multiple targets with weak affinity , an ideal property for elements involved in transcriptional and signal transduction processes [7] .', 'Homopolymeric tracts are often encoded by trinucleotide repeats , a class of microsatellites .', 'Their repetitive nature facilitates DNA replication slippage , and the expansion or contraction of the repeats ( for review , see [8] ) .', 'Although genetic variability of these repeats provides a substrate for adaptive evolution [9] , [10] , uncontrolled expansion of such unstable regions within coding sequences has been associated with a number of developmental and inherited neurodegenerative disorders [2] , [11] , as well as with several types of cancer [12] .', "For example , polyglutamine expansions have been associated with Huntington's disease and certain types of spinocerebellar ataxia ( for review , see [11] ) .", 'In addition , alanine repeats are related to several developmental disorders ( for review , see [13] ) , and aspartate hyperexpansions with two types of dysplasia and osteoarthritis [14] , [15] .', 'Some of the mechanisms thought to underlie the pathogenic effects of expanded tracts involve the deregulation of transcriptional activity and the formation of toxic protein aggregates ( for review , see [11] , [16] ) .', 'Nevertheless , the functions of many homopeptidic segments found in proteins have not yet been elucidated .', 'Among homopolymeric tracts , histidine ( His ) repeats are relatively rare [5] .', 'However , their frequency increases from about 1 . 4% to 4 . 3% when we consider repeats of at least 8 instead of 5 residues , indicating that they are generally longer than other types of SARs [4] .', 'The physicochemical properties of His make it a versatile amino acid that can fulfill different roles , influencing protein conformation and enzymatic activity .', 'For instance , His-repeats are found in Zn-finger domains that are implicated in interactions between nucleic acids and proteins ( for review , see [17] ) , and a His-stretch has been described as a protein interacting surface of the transcriptional regulator cyclin T1 [18] , [19] .', 'Nevertheless , there is still no clear function associated to His homopeptides .', 'We previously described the His-repeat in the DYRK1A protein kinase as both necessary and sufficient to target this protein to nuclear speckles [20] .', 'A protein segment containing a His-tract is also involved in the accumulation of cyclin T1 in these nuclear structures [20] , [21] .', 'These results provided the first evidence that His-repeats may act as nuclear speckle-targeting signals , although the extent to which this was true in other proteins remained to be determined .', 'Nuclear speckles ( also known as the splicing factor compartment -SFC- or as interchromatin granule clusters -IGCs- ) are subnuclear structures defined as compartments in which components of the RNA splicing machinery are stored and assembled ( for review , see [22] ) .', 'They mainly contain splicing factors ( snRNPs and serine/arginine-rich ( SR ) proteins ) , as well as transcription factors , 3′-RNA processing factors , translation factors , ribosomal proteins , a subpopulation of the RNA polymerase II and some kinases and phosphatases [23] , [24] .', 'Like other nuclear bodies , nuclear speckles are highly dynamic structures that change in number , shape and size depending on the transcriptional state and the phase of the cell cycle [22] .', 'Here , we have performed an in-depth analysis of polyHis-containing proteins in the human genome .', 'A significant fraction of the proteins identified are transcription factors and developmental proteins with a nuclear phase .', 'The subcellular localization of several of these proteins shows that most of them accumulate in nuclear speckles through their His-repeat .', 'The presence of DNA-binding or protein-protein interaction domains , and the transcriptional state of the cell , are factors that affect the retention of transcription factors with His-repeats in nuclear speckles , illustrating the dynamic behavior of these proteins .', 'Together , these results define the His-repeat as a novel and general targeting signal for nuclear speckles .'], ['For a typical protein of 400 amino acids and of average composition , a run of any individual amino acid is significant if there are 5 or more consecutive residues [25] .', 'Following this premise , we established a threshold of 5 His residues to determine the minimum number of His necessary for a His-containing protein to accumulate in nuclear speckles .', 'We generated plasmids to express green fluorescent protein ( GFP ) fusion proteins with 5 , 6 , 7 , 8 or 9 His , and we analyzed the subcellular localization of these fusion proteins by direct fluorescence in transfected HeLa cells .', 'Nuclear speckles were identified by indirect immunofluorescence with an antibody against the splicing factor SC35 , an endogenous marker of the nuclear speckles compartment [26] .', 'No significant differences in the staining pattern were observed when GFP and GFP-5xHis were compared ( Figure S1 ) .', 'However , from the 6xHis constructs onwards , a positive relationship was detected between the accumulation in nuclear speckles and the length of the His-tract .', 'While GFP-6xHis only weakly concentrated in SC35-positive speckles , this association became stronger as the number of His residues increased , and it was clearly evident with a fusion protein containing 9 His ( Figure 1A and S1 ) .', 'To confirm that the GFP-His fusions almost completely co-localized with SC35 positive speckles , we carried out an immunofluorescence analysis with protein markers of other subnuclear compartments that are compatible with such staining , including promyelocytic leukemia ( PML ) bodies ( for review , see [27] ) , Sumo-bodies ( for review , see [28] ) or paraspeckles [29] .', 'No co-localization between the GFP-9xHis fusion protein and any of the protein markers ( PML , Sumo1 , PSP1 ) was detected ( Figure S2 ) .', 'Finally , the subnuclear localization of GFP fusion proteins with polyproline or polyglutamine tracts , which are particularly enriched in transcription factors [4] and that have been shown to be functional as transcriptional activators [30] , was also analyzed .', 'These fusion proteins showed nucleoplasmic staining and no colocalization with SC35 ( Figure 1B ) , in agreement with previous results with longer amino acid tracts [31] .', 'Therefore , His homopolymeric tracts seem to specifically accumulate in the nuclear speckles compartment .', 'To extrapolate these results to real proteins , we performed a bioinformatics screen of the Ensembl database [32] to identify all the human proteins containing at least one His-repeat of 5 or more residues .', 'The lower-limit of 5 His residues was set to cover all possible functionally significant repeats [25] .', 'Our search identified 86 Ensembl genes ( Table S1 ) .', 'As some of the proteins encoded by these genes contained more than one repeat , there was a total of 99 repeats with 5 residues or more .', 'The average size of the His-repeats was 7 . 5 , with the longest repeat containing 15 residues ( LOC730417 ) .', 'The majority of the repeats were well conserved in the corresponding mouse orthologous proteins; 54% showed exactly the same length and 30% differed in only one or two repeat units .', 'When more than one His-repeat was present in a protein , they were generally very close to each other such that they could be considered as “extended” His-repeat tracts ( for instance , H4GNSSH13 in DYRK1A ) .', 'Thus , we defined “extended” tracts as regions that contained at least one pure His-repeat of 5 residues or more , that had His residues at the start and/or end of the tract , and that contained other “interrupting” residues ( often P , Q , G , S , A ) which covered <50% of the tract .', 'Such extended tracts were present in half of the proteins containing pure His-repeats ( 43 out of 86 ) .', 'Significantly , none of the His-repeats were situated within characterized protein domains and unlike other repeats [4] , we did not find them preferentially located at the amino- , carboxy- , or central part of the proteins .', 'We compared the length distribution of His-repeats in coding sequences to that of equivalent sequences in non-coding regions , the latter defined as sequences containing at least five tandem CAY ( CAC or CAT: His encoding triplets ) .', 'Accordingly , we identified 7815 such repeats in non-coding genomic regions .', 'Interestingly , although much longer repeats existed in the non-coding regions ( the longest was 154 trinucleotides ) , their average size ( 7 . 24 ) was smaller than in coding regions .', 'Indeed , the distribution of the repeat size was significantly different between coding and non-coding sequences ( p-value\u200a=\u200a0 . 003 , non-parametric Kolmogorov-Smirnov test ) .', 'In coding sequences , there was an under-representation of short repeats ( size 5 ) with respect to longer repeats ( around 7 ) when compared to non-coding sequences ( Figure 2A and 2B , respectively ) .', 'As the length distribution of non-coding repeats is likely to reflect neutral mutational processes , this difference points to selective retention of relatively long His-repeats in protein sequences .', 'The population of proteins containing other types of amino acid repeats , such as polyglutamine , polyalanine , polyglycine , polyserine and polyproline , is enriched in transcription factors [4] .', 'We examined whether any such bias in Gene Ontology terms ( GO; [33] ) existed in the gene dataset encoding His-repeats .', 'Among proteins containing His-repeats there was a strong over-representation of nuclear proteins ( 72% with respect to 26% in the complete protein dataset , p-value<10−5 , Figure 3A ) .', 'In addition , 75% of the His repeat-containing nuclear proteins were also annotated with the GO term ‘regulation of transcription’ , in comparison with 49% of those in the complete nuclear protein dataset .', 'Even more striking was the strong over-representation of developmental factors among nuclear proteins with His-repeats , especially those involved in the development of the nervous system ( 22% with respect to 3% in the complete gene dataset , p-value<10−5 , Figure 3B ) .', 'This finding is in agreement with previous work [34] and it might be linked to the fact that increased formation of homopolymeric runs in human proteins may be a recent evolutionary event , concomitant with complex brain development [2] .', 'The GO terms analysis indicated that most of the polyHis-containing proteins are nuclear proteins , and therefore they might be targeted to nuclear speckles .', 'Thus , we analyzed the distribution of a group of the nuclear-annotated proteins with pure His-repeats of different lengths ( longer than 5 residues ) and several proteins with extended repeats .', 'The subcellular localization of the His-containing proteins was analyzed by generating GFP fusion proteins with the open reading frames of candidate proteins in a mammalian expression vector .', 'The subcellular distribution of the fusion proteins was analyzed by direct fluorescence in transient transfected cells and nuclear speckles were identified by anti-SC35 staining .', 'As previously described for cyclin T1 and DYRK1A [20] , other polyHis-containing proteins also showed punctate nuclear staining that co-localized with SC35 , such as the transcription factors POU4F2 or YY1 , or the protein kinase NLK ( Figure 4A ) .', 'Fluorescence images revealed differences in the staining patterns for the His-repeats-containing proteins , with some of them showing more nucleoplasmic staining than others ( Figure 4A; see other examples in Figures 5–8 ) .', 'The His-repeat seemed to be necessary for this subnuclear localization since deletion of the polyHis segment alone from POU4F2 or DYRK1A ( the extended His-repeat ) completely abrogated the accumulation of these proteins in SC35-labelled nuclear speckles ( Figure 4B ) .', 'These results indicate that the His-repeat can act as a nuclear speckle localization signal .', 'Moreover , deletion of the His-repeat did not interfere with the biochemical function of the protein , that is “kinase” for DYRK1A or “transcriptional activator” for POU4F2 ( Figure 4C and 4D , respectively ) .', 'Similar results were obtained when the His-repeat was deleted in NLK ( Figure S3 ) .', 'These data indicate that the deletion has not induced a general alteration of protein structure , and further suggest that the His-tract conveys a novel behavior to the host protein without affecting its basic activity .', 'Interestingly , a significant fraction ( 64% ) of the genes encoding proteins with His-repeats had closely-related paralogues in the human genome .', 'According to Ensembl annotations , 74% of them had been presumably formed by gene duplication at the dawn of vertebrate evolution ( Table S2 ) .', 'However , in most cases none of the paralogues contained a similar His-repeat in their primary sequence .', 'This indicates that the repeat had only later appeared in one of the duplicate copies , probably by duplication slippage .', 'To approximately date their appearance , we inspected all the orthologous and paralogous vertebrate proteins in Ensembl for the presence of similar His-repeats .', 'In 11 out of 39 cases , the repeat was found in all vertebrate orthologues but in none of the paralogues , indicating that they arose soon after the duplication event .', 'However , the dominant class was repeats formed at the base of the placental mammals ( 14 cases ) .', 'Notably , a large number of alanine and glycine repeats are also proposed to be specific to mammals [35]–[37] .', 'Indeed , the increased repeat expansion in this clade may be related to the increased GC content [38] , [39] .', 'Finally , the His-repeats in the BMP2K and PBXIP1 genes were restricted to primates , suggesting they arose relatively recently .', 'Given the significant number of polyHis-containing proteins with paralogous proteins without His-repeats , we reasoned that if the His-repeat were responsible for their accumulation in speckles then the paralogous copy without the repeat should not be found in this subnuclear compartment .', 'To confirm this hypothesis , we examined the FAM76A and FAM76B pair of paralogues .', 'A sequence alignment of these two proteins highlighted their high degree of conservation , except in the region containing the His-repeat ( Figure 5A ) .', 'As hypothesized , the paralogue without the His-tract , FAM76A , presented a diffuse nucleoplasmic staining , while the protein with the polyHis segment , FAM76B , accumulated in nuclear speckles ( Figure 5B ) .', 'Similar results were obtained for other pairs of paralogous proteins such as DYRK1A/DYRK1B or POU4F2/POU4F3 ( Figure S4 ) .', 'Thus , these findings further indicate that the His-repeats in these proteins are necessary for their localization to nuclear speckles .', 'The initial analysis of the nuclear localization of polyHis-containing candidates revealed that some proteins did not apparently localize to nuclear speckles .', 'These proteins contained other protein domains such as DNA binding domains or protein-protein interacting regions .', 'For instance , the transcription factors MEOX2 and OTX1 harbor a homeobox DNA-binding domain in their C- and N-terminal regions , respectively ( Figure 6A and Figure S5 ) .', 'In the case of the Sumo E3 ligase CBX4 , its C-terminal region includes domains that interact with the polycomb protein CtBP2 and the transcriptional repressor RING1 ( Figure 6B ) .', 'These domains mediate the localization of CBX4 to subnuclear foci , that are compatible with polycomb bodies [40] .', 'Therefore , we hypothesized that the accumulation of proteins to nuclear speckles may be influenced by other interactions .', 'To confirm this hypothesis , we deleted the DNA binding domain in MEOX2 and assessed its nuclear distribution .', 'Accordingly , while the wild type protein presented the dispersed distribution typical of most transcription factors ( Figure 6A ) , compatible with active transcription sites [41] , the mutant protein in which the homeobox was eliminated ( MEOX2ΔHB ) fully co-localized with SC35 ( Figure 6A ) .', 'Similar results were obtained with the OTX1 transcription factor ( Figure S5 ) .', 'In the case of CBX4 , we assessed whether deleting the C-terminal fragment spanning the CtBP2 and RING1-interacting domains ( CBX4ΔPB ) similarly affected its distribution .', 'While the wild type CBX4 protein was present in nuclear foci that were not positive for SC35 , the mutant CBX4ΔPB co-localized with SC35 in the nucleus ( Figure 6B ) .', 'These results confirmed that the accumulation of some of the polyHis-containing proteins in nuclear speckles was influenced by their binding to other nuclear components , such as DNA or diverse subnuclear structures .', 'Moreover , they suggest that competition between distinct protein regions dictates the steady state subnuclear localization of the protein .', 'In mammalian cells , the structure and function of nuclear speckles is sensitive to the transcriptional state of the cell ( for review , see [22] ) .', 'When cells are treated with RNA polymerase II transcription inhibitors , there is a decrease in the splicing activity and a redistribution of the components of speckles , which are recruited to larger and rounder nuclear speckles [42] .', 'Most of the His-containing proteins were transcription factors and since our results showed that DNA binding activity influenced their accumulation in speckles , we wondered whether their failure to localize to this subnuclear compartment might be reverted by inhibiting RNA polymerase II activity .', 'Two proteins , FOXG1B and HOXA1 , that did not produce speckled staining at the steady state , co-localized with SC35 in fewer but larger speckles after α-amanitin treatment ( Figure 7A and 7B ) .', 'Interestingly , the diffuse nucleoplasmic distribution of several other transcription factors became punctate in cells treated with α-amanitin , and it overlapped with SC35 staining ( Table 1 and Figure S6A ) .', 'These dynamic changes in distribution could be observed by in vivo imaging ( Videos S1 and S2 ) .', 'For HOXA1 , we noticed that the staining not only overlapped with SC35 foci but it also adopted a “capped structure” , as described for the recently reported S1-1 nuclear domains [43] .', 'We therefore analyzed co-localization with an anti-S1-1 antibody as a marker of this nuclear domain , and we found that the HOXA1 signal co-localized with both the SC35 and the S1-1 staining ( Figure S6B ) .', 'Since nuclear speckles and S1-1 domains have been suggested to be functionally connected [43] , it is possible that HOXA1 could traffic between these two subnuclear domains .', 'The dependence on the polyHis segment for this dynamic behavior was analyzed using a HOXA1 mutant protein in which the His-repeat was eliminated .', 'Accordingly , there was no change in the subcellular distribution of this mutant protein when cells were exposed to α-amanitin ( Figure 7C ) .', 'HOXA1-dependent reporter assays confirmed that deletion of the His-repeat did not abolish the transcriptional activity of this transcription factor ( Figure 7D ) , suggesting that the mutation affected specifically the subnuclear localization of the protein .', 'We also analyzed the effect of RNA polymerase II inhibition on three polyHis-containing proteins considered to be cytosolic: the negative regulator of the Wnt-canonical pathway NKD2; the mitotic kinase PLK2; and the PRICKLE family member PRICKLE3 ( also known as LMO6 ) .', 'Both NKD2 and PLK2 remained in the cytoplasm under basal conditions and upon exposure to α-amanitin ( not shown ) .', 'However , exposure to this inhibitor produced the translocation of a proportion of PRICKLE3 to the nucleus , where it co-localized with SC35 ( Figure 8A ) .', 'Incubation with leptomycin B , an inhibitor of CRM1-dependent nuclear export , caused the relocalization of PRICKLE3 to the nucleus ( Figure 8B ) , indicating that it is a shuttling protein and further suggesting that its targeting to nuclear speckles may be linked to the yet unknown role of PRICKLE3 within the nucleus .', 'Leptomycin B treatment induced accumulation of PRICKLE 3 in PML bodies ( Figure S7 ) .', 'The results of the analysis of the subcellular localization of several polyHis-containing nuclear proteins are summarized in Table 1 and notably , 15 out of 22 of these proteins displayed nuclear staining compatible with their accumulation in nuclear speckles .', 'Thus , proteins with His-repeats seem to localize dynamically in the splicing factor compartment .'], ['The mammalian nucleus is a highly complex organelle that is both physically and functionally compartmentalized ( for review , see [22] , [47] ) .', 'The subnuclear structures are associated with specific biological activities related to the synthesis , processing and modification of RNA , and they can be distinguished by morphological criteria and the presence of specific protein markers .', 'One such compartment is that of the nuclear speckles .', 'The mechanisms responsible for the formation and regulation of these structures are not yet known and as for many other nuclear bodies , it has been proposed that they are highly dynamic self-organizing entities [48] .', 'A rapid exchange of protein components between subnuclear compartments has been reported , which can be explained by a reaction-diffusion model [49] .', 'However , the kinetics associated to a particular protein can be affected by its binding to other molecules , either proteins or nucleic acids , which in turn can aid its recruitment to a specific compartment .', 'Accordingly , a few protein domains have been described that direct proteins to nuclear speckles , such as the arginine/serine-rich ( RS ) -domain in SR proteins [50] or the RNA recognition motif [51] .', 'Other regions in specific proteins have also been reported to act as speckle-localizing sequences , like the threonine-proline repeats in SF3B1/SF3b155 [52] and the “Forkhead Associated” domain in PPP1R8/NIPP1 [53] .', 'We previously showed that the His-tract in the DYRK1A protein kinase and the regulator of transcription cyclin T1 [20] , [21] is responsible for the accumulation of these proteins in nuclear speckles .', 'Given that the functions of many of the polyHis-containing proteins were related to DNA and RNA metabolism , it was plausible that this role as a subnuclear targeting signal could be more general in other proteins .', 'Indeed , a significant proportion of the polyHis-containing proteins analyzed have the ability to accumulate in nuclear speckles either at the steady-state or upon transcription inhibition .', 'This targeting may respond to the nature of nuclear speckles as sites of storage , recycling and degradation of factors involved in DNA and RNA metabolism [22] , [54] .', 'The uneven distribution found among different speckle-positive His-repeats-containing proteins is also observed among splicing factors that accumulate in speckles for instance [22] , [26] , and could reflect differential binding affinities for distinct targets within the nucleus .', 'Importantly , accumulation in nuclear speckles is dependent on the presence of the His-tract , as confirmed by both deletion analysis in some candidate proteins and by the behavior of paralogous proteins lacking the His-repeat .', 'Apart from the previously mentioned DYRK1A and cyclin T1 [20] , [21] , only HOXA9 had already been reported to accumulate in nuclear speckles of unknown nature [55] .', 'Given that our analysis was performed by transient transfection of plasmids expressing the candidate proteins fused to GFP , we tried to rule out non-physiological effects due to overexpression .', 'This is particularly relevant since expanded homopolymeric tracts , including polyHis expansions , have been associated with protein aggregation [16] , [31] , [56] .', 'As a cellular defense mechanism against protein misfolding and aggregation , protein aggregates are thought to be sequestered in inclusions that also contain molecular chaperones and components of the ubiquitin proteasome system [57] .', 'We did not detect any co-localization of candidate proteins with an anti-ubiquitin antibody ( Figure S8 ) , suggesting that the speckled staining was not produced by the formation of intranuclear protein aggregates .', 'In addition , no cytosolic granules were detected ( Figure S1 and S2 ) , in contrast with results published with longer His tracts ( 26 His residues; [31] ) .', 'We also analyzed the behavior of a stably expressed polyHis-containing protein ( DYRK1A ) fused to GFP during the cell cycle .', 'Nuclear speckles disassemble when cells enter mitosis and the proteins associated with them become diffusely distributed throughout the cytoplasm [58] .', 'As shown in Figure S9 , the fusion protein totally recapitulated these changes during the cell cycle indicating that poly-His expression does not interfere with the intrinsic dynamics of the compartment .', 'As additional support for the specificity of the subcellular localization , we did not detect an accumulation of the GFP-9xHis chimera in other subnuclear compartments and there was no colocalization with different marker proteins or any specific accumulation in the cytoplasm of the transfected cells , suggesting that the fusion protein is not recruited to a specific cytosolic organelle .', 'It seems most likely that the His-repeat acts as a nuclear speckle-targeting signal by serving as an interaction surface for resident molecules in the speckle .', 'The features of His make it a versatile amino acid , strongly represented in enzymatic and binding activities .', "Histidine's imidazole side-chain allows it to shift from a neutral to positive charge in a pH-dependent fashion , a property that may have an impact on the binding capabilities of a His-stretch .", 'Moreover , the presence of His in a β-strand provides a charge gradient that could mediate protein-protein or protein-DNA via electrostatic interactions .', 'His is also known as an excellent ligand to coordinate metal ions [17] , which can also participate in organizing interacting domains .', 'All these mechanisms may contribute to finely regulate the binding properties of His-repeats .', 'Examples of His-stretches as protein-protein interacting domains can be found in cyclin T1 when interacting with RNA polymerase II and granulin [18] , [19] , and DYRK1A interacting with Sprouty2 [59] .', 'The ability of His-tracts to target proteins to the nuclear speckles compartment seems to be specific to His since other homopolymeric amino acid tracts do not display such activity according to our results ( 9xGln and 9xPro as GFP fusions; 13xAla in NLKΔHis , 16xGly and 7xSer in POU4F2ΔHis ) and those published for longer amino acid tracts [31] .', 'Speckle-positive His-repeats vary from simple amino acid runs ( for instance , H10 in HOXA1 ) to complex repeats ( HPSNH5NH2SHKHSH in cyclin T1 ) , suggesting that the number of His residues is not decisive for its functional role but rather , the spacing between residues may be important .', 'We failed to find a specific code underlying targeting to nuclear speckles , except that a minimum of 6 His residues is required for this effect , which indicates a high degree of flexibility in this functional signal .', 'Considering that His-repeats are widely used as tags for affinity-purification and immunodetection of expressed proteins , we would like to stress the fact that more than 6 His residues may alter the original localization of a tagged protein .', 'Only 22% of SARPs have paralogous proteins [60] , whereas a large fraction of the genes encoding proteins with His-repeats have closely-related paralogues .', 'We found that many of them were derived from gene duplications at the base of vertebrate evolution , when two rounds of whole-genome duplication took place [61] .', 'Interestingly , most of the paralogues lacked the His-repeat , suggesting that this repeat had been gained after the duplication of the gene .', 'Further analysis of the distribution of these repeats revealed that they were gained during two periods of vertebrate evolution: soon after gene duplication or before placental mammal radiation .', 'The comparison of the subcellular distribution of three pairs of paralogous proteins , FAM76B/FAM76A , DYRK1A/DYRK1B and POU4F2/POU4F3 , confirmed that only those members containing His-repeats localized to nuclear speckles .', 'Notably , in approximately 30% of the duplicate gene pairs derived from the S . cerevisiae whole-genome duplication event , the two protein members of the pair localize to distinct subcellular compartments [62] .', 'This and other evidence led to the proposal that protein subcellular relocalization might be an important evolutionary mechanism for the functional diversification of duplicate genes [63] .', 'Therefore , the appearance of a new repeat , or variations in the length and composition of an existing one , may have been an important mechanism for functional diversification .', 'The acquisition of a new His-repeat might have contributed to the reorganization of protein-protein interaction networks and more specifically , to nuclear speckle targeting as a novel cell property associated to the paralogous protein .', 'This might be relatively rapid on an evolutionary time scale because of the high mutation rates associated with microsatellites [64] .', 'In fact , the expansion and contraction of repeats within transcription factors has been linked to major morphological changes in vertebrates [65] , [66] .', 'Given that a high proportion of the polyHis-containing proteins have roles in developmental processes , mutations involving His-repeats may have played a significant part in diversification and adaptation .', 'Several of the His-containing proteins that did not accumulate in nuclear speckles were transcription factors .', 'The fact that these proteins contain domains that may control their specific localization within the nucleus , such as DNA binding regions or protein-protein interaction domains , led us to think that competition between His-repeats and other protein regions might regulate their intranuclear distribution .', 'Our results show a direct correlation between loss of DNA binding activity and accumulation in nuclear speckles .', 'Similar behavior was recently described for the transcription factor GATA-4 , although the subnuclear compartment to which it localized was not identified [67] .', 'Although we cannot ignore that the elimination of the DNA binding domains may result in a conformational change that exposes the His-repeat , we favor a loss of retention in the chromosomal compartment as being responsible for the enrichment in nuclear speckles .', 'This assumption is supported by the results with inhibitors of RNA polymerase II-dependent transcription , since treatment with α-amanitin caused re-localization to nuclear speckles of many of the proteins with a dispersed nuclear distribution under basal conditions .', 'In this regard , we noted that the subgroup of proteins unable to accumulate in nuclear speckles was enriched in proteins with more than one DNA binding domain , a feature that may confer a more immobile character to these proteins .', 'Thus , we propose that the intranuclear localization of some transcription factors with His-repeats is the net result of competition for binding to different recruiting sites within the nucleus , such as DNA , nuclear speckles or other nuclear bodies .', 'Moreover , this dynamic behavior might also explain why among the proteins listed in Table 1 , only OTX1 a
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Single amino acid repeats are prevalent in eukaryote organisms , although the role of many such sequences is still poorly understood .', 'We have performed a comprehensive analysis of the proteins containing homopolymeric histidine tracts in the human genome and identified 86 human proteins that contain stretches of five or more histidines .', 'Most of them are endowed with DNA- and RNA-related functions , and , in addition , there is an overrepresentation of proteins expressed in the brain and/or nervous system development .', 'An analysis of their subcellular localization shows that 15 of the 22 nuclear proteins identified accumulate in the nuclear subcompartment known as nuclear speckles .', 'This localization is lost when the histidine repeat is deleted , and significantly , closely related paralogous proteins without histidine repeats also fail to localize to nuclear speckles .', 'Hence , the histidine tract appears to be directly involved in targeting proteins to this compartment .', 'The removal of DNA-binding domains or treatment with RNA polymerase II inhibitors induces the re-localization of several polyhistidine-containing proteins from the nucleoplasm to nuclear speckles .', 'These findings highlight the dynamic relationship between sites of transcription and nuclear speckles .', 'Therefore , we define the histidine repeats as a novel targeting signal for nuclear speckles , and we suggest that these repeats are a way of generating evolutionary diversification in gene duplicates .', 'These data contribute to our better understanding of the physiological role of single amino acid repeats in proteins .']
['Single amino acid repeats are common in eukaryotic proteins .', 'Some of them are associated with developmental and neurodegenerative disorders in humans , suggesting that they play important functions .', 'However , the role of many of these repeats is unknown .', 'Here , we have studied histidine repeats from a bioinformatics as well as a functional point of view .', 'We found that only 86 proteins in the human genome contain stretches of five or more histidines , and that most of these proteins have functions related with RNA synthesis .', 'When studying where these proteins localize in the cell , we found that a significant proportion accumulate in a subnuclear organelle known as nuclear speckles , via the histidine repeat .', 'This is a structure where proteins related to the synthesis and processing of RNA accumulate .', 'In some cases , the localization is transient and depends on the transcriptional requirements of the cell .', 'Our findings are important because they identify a common cellular function for stretches of histidine residues , and they support the notion that histidine repeats contribute to generate evolutionary diversification .', 'Finally , and considering that some of the proteins with histidine stretches are key elements in essential developmental processes , variation in these repeats would be expected to contribute to human disease .']
['computational biology/sequence motif analysis', 'cell biology/nuclear structure and function', 'genetics and genomics/functional genomics', 'genetics and genomics/nuclear structure and function', 'biochemistry/bioinformatics', 'computational biology/genomics', 'genetics and genomics/bioinformatics']