micrornas ( mirnas ) are short noncoding regulatory molecules , involved in diverse biological processes .
biogenesis of mirnas involve a nuclear phase , where the microprocessor complex , comprising drosha , an rnase iii - like enzyme and its cofactor dgcr8 , process primary mirnas ( pri - mirnas ) into a 70 nt pre - mirna ( han et  al . , 2004 ;
this occurs cotranscriptionally from both independently transcribed and intron - encoded mirnas ( ballarino et  al . , 2009 ;
following drosha - mediated rna cleavage and pre - mirna release from the nascent rna , 5 and 3 nascent rna ends are trimmed by 5-3 xrn2 and 3-5 exosome ( morlando et  al . , 2008 ) , and
the pre - mirna precursor is exported to the cytoplasm ( lund et  al . , 2004 ; yi et  al . , 2003
) . here , a second rnase iii enzyme , dicer , further processes the pre - mirna into the mature mirna duplex ( bernstein et  al . , 2001 ) that targets specific mrnas for degradation or translational inactivation ( reviewed in bartel , 2009 ) .
mirna levels are tightly regulated at the posttranscriptional level by a number of rna - binding proteins ( siomi and siomi , 2010 ) .
furthermore , drosha can directly regulate levels of microprocessor complex by cleaving hairpin structures in dgcr8 mrna , thereby decreasing dgcr8 protein levels ( han et  al .
2009 ) . along the same lines , drosha knockdown in drosophila leads to upregulation of some mrnas containing conserved rna hairpins , potentially recognized by the microprocessor complex ( kadener et  al . , 2009 ) .
several recent studies demonstrated the ability of microprocessor complex to cleave mrnas , thus regulating their expression .
many drosha - dependent mrna cleavage events were identified in mescs , consistent with microprocessor regulation of coding mrnas through direct cleavage ( karginov et  al . , 2010 ) .
drosha can also cleave the tar hairpin of the hiv-1 transcript , resulting in premature termination of rna polymerase ii ( pol ii ) ( wagschal et  al . ,
a recent dgcr8 hits - clip analysis extended these observations and revealed general noncanonical functions of the microprocessor complex ( macias et  al . , 2012 ) .
transcriptome and proteome studies of mice lacking drosha and dicer suggest that both enzymes have nonredundant functions , as their deficiency can induce different phenotypes ( chong et  al . , 2010 ) .
although many rnas were stabilized by drosha depletion , some were downregulated , consistent with drosha possessing independent functions to its role in canonical mirna biogenesis . in human cells
drosha exists in two distinct multiprotein complexes ( gregory et  al . , 2004 ) .
the smaller complex , containing just drosha and dgcr8 , is necessary and sufficient for mirna processing .
the larger complex , displaying only weak pre - mirna processing activity in  vitro , contains dead - box rna helicases , double - stranded rna - binding proteins , hnrnp proteins , members of fus / tls family of proteins , and the snip1 protein , implying additional functions in gene expression .
thus , dead box helicases p68/p72 increase drosha processing efficiency for a subset of mirnas and at gene - specific promoters interact with transcriptional coactivators and pol ii and regulate alternative splicing ( fuller - pace and ali , 2008 ) .
nuclear scaffolding protein hnrnpu and members of fus / tls family are also associated with regulation of transcription ( wang et  al . , 2008 ) .
snip1 , a component of a large snip1/skip - associated complex , involved in transcriptional regulation and cotranscriptional processing , interacts with drosha and plays a role in mirna biogenesis ( fujii  et  al . , 2006 ; yu et  al . , 2008
ars2 is implicated in rna silencing that functions in antiviral defense in flies and cell proliferation in mammals ( gruber et  al . , 2009 ;
it interacts with the nuclear cap - binding complex ( cbp20/cbp80 ) and is involved in mirna biogenesis , suggesting a link  between rna silencing and rna - processing pathways .
cbp20/cbp80 proteins are also implicated in mirna biogenesis in plants ( kim et  al . ,
overall , the existence of this large drosha - complex with only weak mirna - processing activity suggests that drosha  may play multiple roles in mirna - independent gene regulation . using genome - wide and gene - specific approaches
we now show that drosha binds to the promoter - proximal regions of many human genes in a transcription - dependent manner . similarly , dgcr8 binds promoter - proximal regions of many human genes , suggesting that the whole microprocessor is recruited at promoter regions .
we also find that drosha interacts with pol ii and its depletion from human cells causes transcriptional downregulation with a concomitant decrease in nascent and mature mrna levels .
this positive function of drosha in gene expression is mediated through its interaction with the rna - binding protein cbp80 and dependent on the n - terminal protein - interaction domain of drosha .
thus , results presented in this paper demonstrate an mirna- and cleavage - independent function of drosha .
consequently binding of drosha to nascent mirna sequences can be detected using chromatin immunoprecipitation ( chip ) analysis ( morlando et  al . , 2008 ) .
to investigate genome - wide binding of drosha , we employed a chip - on - chip approach using human 5.1.1 encode array .
our results show that , in addition to mirna regions , drosha binds many human genes .
this binding is enriched at the transcriptional start sites ( tsss ) , compared to the ends of transcripts ( txend ) , other gene body , or intergenic regions ( figure  1a ; full gene list in table s1 ) .
in particular , we observed that , out of 160 tss - proximal probes , 55 ( 34% ) had more than 2-fold enrichment of drosha - chip signal over the input signal .
this is significantly more than 1,669 ( 10% ) out of 15,998 total probes or 953 ( 11% ) out of 8,333 probes in the gene bodies ( p  < 0.0001 , fisher s exact test ) .
the same level of enrichment was observed for four ( 40% ) out of ten probes overlapping with annotated mirna regions ( figure  1aii ) . by way of illustration , we present selenpb1 and eef1a1 genes , which scored positively in our genomic analysis , where drosha selectively binds to 5-proximal gene regions ( figure  s1 ) .
genes that are not expressed in hela cells such as il4 and tarm1 demonstrated only background drosha signal ( figure  s1 ) .
we also identified genes , such as g6pd , znf687 , and fundc2 , which are expressed in hela cells but did not demonstrate any  significant drosha enrichment ( figure  s3a ) .
we further confirmed these array data using gene - specific primers in chip  experiments .
in particular , we observed an enrichment of drosha binding to promoter - proximal region of selenbp1 gene , colocalizing with pol ii peak ( figure  s2a ) .
we did not detect any significant binding of drosha or pol ii to il4 and tarm1 genes , further confirming chip - on - chip results ( figures s2b and s2c ) .
we also did not detect any significant drosha binding over promoter - proximal region , enriched for pol ii , on g6pd gene ( figure  s3c ) . to investigate the function of drosha at the beginning of human genes in more detail , we compared pol ii and drosha binding profiles for specific human genes . both pol ii and drosha
were enriched over the promoter - proximal regions of human -actin , gapdh , ptb , -actin , and intronless taf7 genes ( figures 1b , 1c , and s4a  s4c ) .
the enrichment of drosha observed over these genes is higher than over the mir-330 locus within the eml2 gene , used as a positive control for drosha binding ( morlando et  al . , 2008 )
, we used chip for histone h3 , which was found to be depleted from promoter - proximal regions and enriched in the body of these genes ( figure  1eiii ) . to test if drosha binding to these genes is transcription dependent , we treated hela cells with pol ii transcriptional inhibitor actinomycin d. following this treatment , we observed 80% reduction in pre - mrna levels and a substantial reduction in drosha binding to mir-330 locus , -actin , and -actin genes ( figures 1e and s4c ) .
in contrast , we saw no effect on histone h3 binding ( figure  1eiii ) .
these results indicate that , in addition to mirna sequences , drosha binds 5 end regions of human protein - coding genes in a transcription - dependent manner .
furthermore , we also searched for potential correlation between drosha recruitment using our drosha chip - array data and level of pol ii signal determined from available chromatin immunoprecipitation sequencing ( chip - seq ) experiments in ucsc depository .
we have calculated pol ii signal in 1 kb region around the tss for refseq transcripts covered by probes in the drosha array .
following this analysis , we identified a weak ( pearson r  = 0.41 ) but significant ( p  = 1e-6 ) correlation between the drosha probe signal at tss and pol ii chip density ( figure  s5 ) , indicating that drosha is preferentially recruited to the pol ii - rich regions .
overall , these results suggest that drosha binding may be required for enhanced gene expression .
drosha is known to bind mirna sequences as part of the microprocessor complex , which also contains dgcr8 , a double - stranded rna - binding protein that is deleted in the digeorge syndrome ( landthaler et  al . , 2004 ) . to investigate whether drosha binds 5 regions of human genes together with dgcr8 , we carried out chip experiments in hela cells ( figure  2a ) .
dgcr8 was found to be enriched over the mir-330 locus , -actin , gapdh , and selenbp1 promoter - proximal gene regions ( figure  2a ) .
these regions overlap drosha binding regions ( figures 1 , s2 , and s4 ) , suggesting that drosha and dgcr8 may form a complex at promoter - proximal regions of human genes . to substantiate these findings in a genome - wide fashion
, we analyzed a dgcr8 hits - clip experiment that was performed with endogenous and overexpressed dgcr8 protein in hek293 t cells to identify dgcr8 endogenous rna targets ( macias et  al . , 2012 ) .
in particular , we observed 1,101 dgcr8 clusters , corresponding to 967 genes , in the sense orientation ( see supplemental material in macias et  al . , 2012 ) and 196 dgcr8 clusters , corresponding to 174 genes , in antisense orientation ( figure  2c ; table s2 for gene identity ) .
interestingly ,  significant clusters of dgcr8 binding were seen at  1,000/+200  bp from the tsss and just upstream of the transcription termination regions ( ttss ) ( false discovery rate [ fdr ]  < 0.01 ) ( figure  2b ) . in particular , we observed significant enrichment of dgcr8 binding in the region within 200 nt downstream of the tss ( sense versus antisense ks test p  < 2.2e-16 ; figure  2b ) .
dgcr8 binding in this region is most pronounced in genes expressed at medium and high levels when compared to genes expressed at lower levels ( ks test p  < 2.2e-16 in both cases ) ( figure  s7a ) .
these results clearly indicate that dgcr8 , similar to drosha , binds to promoter - proximal regions of human genes .
however , it should be noted that when we analyzed dgcr8 binding to spliced mrna transcripts ( cdnas ) ( figure  s6 ) , we also see an enrichment over exonic sequences immediately downstream of the tss .
again , this is seen in genes with medium and high expression ( ks test p  < 2.2e-16 in both cases ) ( figure  s7b ) .
this may be related to the reported binding of dgcr8 to multiple exonic regions ( macias et  al . , 2012 ) , potentially reflecting an additional function .
binding of drosha to promoter - proximal regions of human genes may be related to its potential role in processing mirnas from these genomic regions , even though such elements are not annotated in the human genome databases . to test
if drosha binds and cleaves mirnas present in promoter - proximal regions , we performed in  vitro pri - mirna cleavage assays in hela nuclear extracts ( guil and cceres , 2007 ) . using in  vitro transcription reactions , we generated radiolabeled rnas from gene regions , identified by drosha chip .
we employed mir-17 - 92 cluster within the c13orf25 human genomic region as a positive control for mirna production .
following in  vitro cleavage assay in hela extracts , mirnas were generated from the control mir-17 - 19a sequences but not from the promoter - proximal region of the -actin gene ( figure  2d ) .
we also failed to detect pre - mirnas or rna cleavage products in  vitro for promoter - proximal regions of other genes , identified in drosha chip assays ( data not shown ) .
these findings suggest that drosha binding over promoter - proximal regions is not associated with mirna synthesis and rna cleavage . to investigate the promoter - associated function of drosha , we performed rnai - induced drosha knockdown in hela cells ( figure  3a ) .
following drosha depletion , we observed a small increase in pol ii binding over the promoters of -actin and gapdh genes , using chip analysis ( figure  3a ) .
we also observed an 20%40% decrease in pre - mrna levels and 30%60% decrease in poly(a)+ rna levels in hela cells depleted for drosha ( figure  3b ) .
in contrast , we did not detect any significant change in the levels of poly(a)+ and pre - mrna corresponding to g6pd , znf687 , and fundc2 genes , for which we observed lack of promoter - proximal drosha binding ( figures s3b and s3c ) .
if drosha has a direct effect on transcription , we employed nuclear run - on ( nro ) using br - utp as the labeled nucleotide ( core and lis , 2008 ; lin et  al . ,
the advantage of br - utp nro over pol ii chip is the ability of br - utp nro to detect actively transcribing polymerases , as opposed to total pol ii levels detected by chip . as demonstrated in figure  3c , we observed a substantial decrease in the level of nascent transcription for taf7 , ptb , and selenbp1 genes in drosha - knocked - down cells . as a negative control for this experiment , we used il4 and tarm1 genes .
these genes are not expressed in hela cells ; hence , we did not observe any detectable transcription signal in br - utp nro reactions .
these results suggest that drosha has a direct positive effect on transcription , consistent with the genome - wide expression data , where highly expressed genes correlated with increased drosha binding on tsss ( figure  1a ) .
furthermore , the antisense transcripts detected over the promoter - proximal region of the -actin gene were also decreased upon drosha depletion ( figure  s4d ) .
these results further indicate that drosha is not involved in the cleavage  of nascent sense or antisense rna transcripts , pointing toward a cleavage - independent function of drosha in human gene expression .
we next employed a -actin / egfp reporter plasmid , where transcription of egfp is driven by the -actin promoter , fused to  -actin exon 1 and intron 1 regions ( qin and gunning , 1997 ) ( figure  3d ) .
as described above , drosha binds the promoter - proximal region of -actin gene ( figure  s4c ) .
interestingly , in hela cells depleted for drosha we observed 75% reduction in expression of egfp mrna from the plasmid ( figure  3d ) , recapitulating downregulation of endogenous -actin mature and pre - mrnas ( figure  3b ) .
these results suggest that drosha plays a positive role in the regulation of -actin / egfp expression .
next , we cotransfected -actin / egfp reporter plasmid with rnai - resistant flag - tagged drosha ( wt ) , a catalytic mutant e110aq ( aq ) , unable to process mirnas , and 390 construct into 293 cells , depleted for drosha , using sirna2 ( han et  al . , 2004 ) .
the 390 drosha construct is active in mirna processing but lacks the n - terminal proline - rich and rs - rich domains , proposed to be important for drosha protein - protein interactions ( han et  al . , 2004 )
both mrnas and proteins corresponding to wt , aq , and 390 drosha were expressed at a high level in 293 t cells , depleted for endogenous drosha ( figure  3ei and 3eii ) . overexpression of both wt and aq catalytic drosha mutant resulted in the increase of egfp mrna ( figure  3eiii ) .
in contrast , overexpression of 390 construct had no significant effect on the expression of egfp mrna .
these results suggest that drosha plays a positive role in the regulation of the -actin promoter .
importantly , this drosha function is independent of its ability to cleave rna but does require the n - terminal protein - protein interaction domain .
we hypothesize that the cleavage - independent stimulatory role of drosha on gene expression may be mediated through its association with protein cofactors .
we therefore performed coimmunoprecipitation experiments and found that transiently overexpressed flag - tagged drosha interacts with both unphosphorylated ( iia ) and phosphorylated ( iio ) forms of pol ii in 293 t cells , supporting its role in transcriptional regulation .
drosha also interacts with cbp80 and ars2 proteins , involved in mirna biogenesis and rna processing pathways , respectively ( gruber et  al . , 2009 ; sabin et  al . , 2009
furthermore , by chip analysis we found that cbp80 protein binds promoter - proximal regions of -actin and gapdh genes , colocalizing with drosha binding ( figures 4b and 4c ) .
we next observed that drosha - cbp80 interaction is not affected by the aq mutation in the catalytic domain of drosha but is significantly reduced when the n - terminal domain of drosha is deleted in 390 construct or when both 390 and aq mutations are combined ( figure  4d , top panel ) .
these results strongly support the view that drosha interacts with cbp80 through its n - terminal rs domain .
we also studied the interaction of drosha with pol ii ( figure 4d , middle ) .
similar to cbp80 , a catalytic mutation in drosha ( aq ) does not affect its interaction with pol ii . however , the drosha 390 construct showed reduced interaction with pol ii , and specifically with its phosphorylated form ( iio ) .
this suggests that the n - terminal domain of drosha is particularly important for drosha - mediated transcriptional effects in human cells because it mediates interaction of drosha with cbp80 and pol ii .
we also observed that interaction of drosha with cbp80 and pol ii is rna independent ( figure  4e ) .
consequently , this result provides a molecular explanation for the ability of drosha to regulate gene expression in a mirna- and cleavage - independent manner .
we demonstrate the existence of a mirna- and cleavage - independent function of drosha in the regulation of human gene expression . combining whole - genome analysis with gene - specific approaches ,
we demonstrate that drosha binds to the 5 ends of human genes in a transcription - dependent manner ( figure  1 ) .
this binding is not associated with mirna biogenesis or rna cleavage but correlates with the level of gene expression ( figures  1 and 2 ) .
depletion of drosha from hela cells led to direct downregulation of transcription based on nuclear run - on and pol ii accumulation at the promoters of these genes , resulting in reduced levels of pre - mrna and poly(a ) mrna ( figure  3 ) .
chip and hits - clip analysis of dgcr8 also revealed binding sites at promoter - proximal regions , enriched at genes expressed at a higher level , suggesting that drosha and dgcr8 may bind to promoter regions as a complex ( figures 2 and s7 ) .
a positive function of drosha in the regulation of gene expression was further confirmed using a heterologous reporter construct , where drosha overexpression influences reporter expression ( figure  3 ) .
this function of drosha is independent of its catalytic activity and mediated through its n - terminal domain , which interacts with the rna - binding protein cbp80 and pol ii , which also bind to the 5 end of human genes ( figures 3 and 4 ) . taken together , these results suggest a model whereby drosha promotes gene expression in a cleavage - independent manner by binding to rna hairpins formed at the 5 ends of the nascent rnas and interacting with cbp80 and pol ii , through its n - terminal rs domain ( figure  4f ) . initially , we hypothesized that this positive function of drosha may be associated with generation / regulation of short promoter - associated transcripts ( prompts ) , originally predicted to regulate transcription through changes in chromatin structure , promoter methylation , or pol ii recycling ( preker et  al . , 2008 ) .
prompts may direct drosha to the beginning of the gene through interaction with its binding factors . in our studies ,
both sense and antisense transcripts , detected over the promoter - proximal regions , were destabilized in drosha - depleted cells ( figure  s4d ) , suggesting that drosha does not cleave these transcripts and its function is not mediated through prompts .
drosha binding at the beginning of human genes may be also related to pol ii release from promoter - proximal pausing events and be required for the recruitment of transcription / rna processing factors .
interestingly , drosha binding coincides with the binding site of the negative elongation factor nelf , which defines the  late  elongation checkpoint , ensuring conversion to productive elongation mediated by phosphorylation of pol ii ctd , nelf , and dsif by the positive transcription elongation factor b ( p - tefb ) ( egloff et  al . , 2009 ) .
as both nucleosome positioning and chromatin modifications play an important role in the elongation functions of p - tefb , we also checked whether drosha knockdown causes a change in chromatin modifications .
gc - rich sequences are known to be enriched in the promoter regions of human genes ( calistri et  al . , 2011 ) .
transcripts for these sequences are likely to form rna hairpin structures , predicted to be suitable for microprocessor binding mediated by dgcr8 ( macias et  al . , 2012 ;
if drosha were to cleave such rnas , the consequences of drosha cleavage across the whole human genome would be detrimental to cellular survival .
we hypothesize that drosha does not cleave promoter - associated rna but is instead utilized as a positive regulator of gene expression .
we further analyzed the structures found in promoter transcripts and compared them to mirna , snornas , and protein - coding region structures , found to be bound by dgcr8 in clip analysis .
our bioinformatic analysis confirmed that structures in promoter regions have shorter stems , higher minimum free energy , and lower base - pairing probability ( figure  s8 ) .
in addition , promoter transcripts also lack motifs important for the processing of the stem , such as tg dinucleotide at the base of the stem and cnnc motif downstream of the stem , as described in auyeung et  al .
, this analysis suggests that promoter structures may be less stable , and their recognition and processing by the microprocessor complex may be less efficient compared to mirnas or other sequences cleaved by drosha .
evidence that components of the mirna machinery may have additional functions comes from several previous studies . in early - stage thymocytes
, drosha recognizes and cleaves mrnas harboring secondary stem - loop structures ( chong et  al . , 2010 ) .
drosha can also regulate viral gene expression of kaposi s sarcoma - associated herpesvirus ( kshv ) ( lin and sullivan , 2011 ) .
dgcr8 together with drosha controls the abundance of many cellular rnas , including noncoding rnas , mrnas , and alternatively spliced isoforms ( macias et  al . , 2012 ) .
finally , microprocessor can regulate transcription of some cellular mrna and hiv provirus , causing pol ii premature termination through the cleavage of the tar stem - loop structure ( wagschal et  al . , 2012 ) .
these regulatory activities of drosha each involve recognition and cleavage of target rnas in an analogous manner to its function in mirna biogenesis in contrast to the positive function of drosha on transcription described here .
possibly this positive function of drosha is related to the action of rna - binding factors , known to modulate the function of drosha in human cells .
such factors are likely to interact with the n - terminal proline - rich and rs domains , proposed to be the main sites of drosha protein - protein interactions .
we confirmed the previously described interaction between drosha and cbp80 and ars2 proteins ( gruber et  al .
, 2009 ; sabin et  al . , 2009 ) . furthermore , our results also show that drosha interacts with the c - terminal domain ( ctd ) of pol ii ( figure  4 ) .
this interaction may be direct or mediated via proteins , containing rs domains and interacting with the ctd , potentially modulating drosha s cleavage - independent function .
interestingly , in drosophila the key rnai components dicer 2 and argonaute 2 associate with chromatin and interact with the core transcription machinery , affecting pol ii dynamics ( cernilogar et  al . , 2011 ) , further supporting the role of rnai machinery in transcriptional regulation . because both drosha and dicer may have additional roles to mirna biogenesis future studies
will be necessary to evaluate the full spectrum of molecular functions that these rnase - iii - like enzymes play in human cells .
pck - flag wt , aq , and 390 drosha constructs ( han et  al . , 2004 ) and -actin / egfp plasmid ( qin and gunning , 1997 ) were described previously .
pflag - cmv-2 ( flag ) was purchased from sigma - aldrich ( e7398 ) .
total rna was harvested using trizol reagent ( invitrogen ) followed by dnase i treatment ( roche ) .
total rna ( 2  g ) was reverse - transcribed using superscript reverse transcriptase ( invitrogen ) and random hexamer primers ( invitrogen ) or gene - specific primers , as described in the supplemental experimental procedures and table s3 , followed by qpcr .
gene - specific primers were used in all figures , apart from figure  3eiii , where random hexamers were used for quantitative rt - pcr ( qrt - pcr ) , following the manufacturer s description ( invitrogen ) .
mir-17 - 19 and -actin gene regions were amplified from genomic dna using t7+-actin ( f / r ) and t7+mir-17 - 19 ( f / r ) primers accordingly . in  vitro processing
the rnai was carried out as described ( wollerton et  al . , 2004 ) .
mrna target sequence for drosha small interfering rna ( sirna ) duplex was 5-cgaguaggcuucgugacuu-3 ( sirna1 ) and 5-gaguauuuacuugcucag uaa-3 ( sirna2 ) .
sirna1 was used in all figures apart from figure  3e , where sirna2 was used .
pull - downs were carried out in rnase a nontreated extracts , except figure  4e where 50  g / ml rnase a was used to treat cell extracts during the pull - down procedure as described in gruber et  al .
western blots were probed with drosha ( abcam ) , actin ( sigma - aldrich ) , gapdh ( sigma - aldrich ) , pol ii ( covance ) , and cbp80 ( sigma ) antibodies .
chip analysis was carried out as previously described ( west et  al . , 2004 ) .
drosha chip - on - chip experiments were carried out as described in de gobbi et  al .
hits - clip for dgcr8 was based on a published protocol ( wang et  al . , 2009 ) with minor modifications , as described in macias et  al .
the br - utp nro was carried out largely as described ( lin et  al . , 2008 ; skourti - stathaki et  al . ,
nuclear pellets were resuspended in transcription buffer ( 40  mm tris - hcl [ ph 7.9 ] , 300  mm kcl , 10  mm mgcl2 , 40% glycerol , 2  mm dtt ) and 10  mm mix of ratp , rctp , rgtp , and br - utp or rutp ( in the control samples ) .
total rna was isolated using trizol reagent ( invitrogen ) according to manufacturer s instructions and treated with rnase - free dnase i ( roche ) .
two microliters of anti - bru antibody ( sigma - aldrich ) was preincubated with 30  l of protein g dynabeads ( upstate ) and 10  g trna per sample for 1  hr at 4c .
the beads were washed three times with rsb-100 buffer ( 10  mm tris - hcl [ ph 7.4 ] , 100  mm nacl , 2.5  mm mgcl2 , 0.4% triton x-100 ) and resuspended in 150  l rsb-100 with 40  u rnase - out ( invitrogen ) and 5  g of glycogen .
total rna was added to the beads and incubated for additional 1  hr at 4c .
rna bound to the beads was extracted with trizol reagent followed by dnase i treatment .
the rt reaction was performed using superscript iii reverse transcriptase ( invitrogen ) following the manufacturers instructions .
the real - time quantitative pcr was performed using a corbett research rotor - gene gg-3000 machine .
the pcr mixture contained quantitest sybr green pcr master mix ( qiagen ) , 2  l of template cdna , and primers from table s3 .
cycling parameters were 95c for 15  min , followed by 45 cycles of 95c for 15 s , 58c for 20 s , and 72c for 20 s. fluorescence intensities were plotted against the number of cycles by using an algorithm provided by the manufacturer .
amount of nascent br - utp rna was calculated by subtracting the background of u - rna produced over a specific gene probe .
unless otherwise stated , results are shown as the average values from at least three independent biological experiments sd .
0.05 ; p  < 0.05 ; p  < 0.05 ) , on the basis of an unpaired , two - tailed distribution determined with a student s t test .
analyzed dgcr8 binding at promoter regions by hits - clip and carried analysis in figures s6s8 .
 summarydrosha is the main rnase iii - like enzyme involved in the process of microrna ( mirna ) biogenesis in the nucleus . using whole - genome chip - on - chip analysis , 
 we demonstrate that , in addition to mirna sequences , drosha specifically binds promoter - proximal regions of many human genes in a transcription - dependent manner . 
 this binding is not associated with mirna production or rna cleavage . 
 drosha knockdown in hela cells downregulated nascent gene transcription , resulting in a reduction of polyadenylated mrna produced from these gene regions . 
 furthermore , we show that this function of drosha is dependent on its n - terminal protein - interaction domain , which associates with the rna - binding protein cbp80 and rna polymerase ii . 
 consequently , we uncover a previously unsuspected rna cleavage - independent function of drosha in the regulation of human gene expression . 
Introduction
Results
Discussion
Experimental Procedures
Author Contributions