InteractiveFly: GeneBrief

drosha: Biological Overview | References

Mature microRNAs (miRNAs) are generated via a two-step processing pathway to yield ~22-nucleotide small RNAs that regulate gene expression at the post-transcriptional level. Initial cleavage is catalysed by Drosha, a nuclease of the RNase III family, which acts on primary miRNA transcripts (pri-miRNAs) in the nucleus (Lee, 2003). The pre-miRNA is then exported by Exportin 5 to the cytoplasm, where it is further cleaved by Dicer into miRNA. Drosha cleaves at the base of pre-miRNA to excise it from pri-miRNA. Dicer cuts again near the loop of pre-miRNA to produce mature miRNA. Drosha exists in a multiprotein complex, the Microprocessor, and begin the process of deconstructing that complex into its constituent components. Along with Drosha, the Microprocessor also contains Pasha (partner of Drosha), a double-stranded RNA binding protein. Suppression of Pasha expression in Drosophila cells or C. elegans interferes with pri-miRNA processing, leading to an accumulation of pri-miRNAs and a reduction in mature miRNAs. Finally, depletion or mutation of pash-1 in C. elegans causes de-repression of a let-7 reporter and the appearance of phenotypic defects overlapping those observed upon examination of worms with lesions in Dicer (dcr-1) or Drosha (drsh-1). Considered together, these results indicate a role for Pasha in miRNA maturation and miRNA-mediated gene regulation (Denli, 2004).

miRNAs are a class of small, non-coding RNAs that enter the RNA interference (RNAi) pathway to regulate the expression of protein-encoding genes at the post-transcriptional level. miRNA production begins with the synthesis of pri-miRNAs, ranging in size from several hundred nucleotides (nt) to several kilobases. These are recognized and cleaved into precursor miRNAs (pre-miRNAs) in the nucleus by an RNase III family nuclease, Drosha (Lee, 2003); the pre-miRNAs are short, hairpin RNAs of approximately 70 nt, bearing the 2-nucleotide 3' overhang that is a signature of RNase III-mediated cleavage. Pre-miRNAs are exported to the cytoplasm by a RanGTP/exportin 5-dependent mechanism (Lund, 2004), with their characteristic overhang contributing to their entry into this export pathway. Once in the cytoplasm, pre-miRNAs are recognized and processed into their mature, ~22-nt form by Dicer, with the 3' overhang again playing a role in specifying cleavage. Mature miRNAs enter RISC (RNA-induced silencing complex) in an asymmetric fashion such that for most miRNAs, only one strand is enabled to recognize and repress the expression of target genes. The outcome of this recognition is either endonucleolytic cleavage of the targeted messenger RNA or interference with protein synthesis by a mechanism that remains unclear. With the ultimate goal of addressing the mechanisms by which pri-miRNAs are tagged for entry into the RNAi pathway and processed at specific sites, a biochemical characterization has been undertaken of Drosha and its associated factors (Denli, 2004).

Lee (2003) has shown that epitope-tagged, human Drosha protein releases pre-miRNAs from pri-miRNAs in vitro and contributes to miRNA maturation in vivo. Processing was dependent upon the presence of a double-stranded region around the cleavage position. It was asked whether a pri-miRNA processing activity also existed in Drosophila cells. Although they are absent from Schizosaccharomyces pombe and Arabidopsis, homologues of mammalian Drosha are present in Drosophila and C. elegans (Wu, 2000). Indeed, S2 cell extracts contain an activity that can recognize a primary-miRNA, in this case primary-bantam, and cleave this into discrete products that can be identified as primary-bantam and the regions of the primary-miRNA that flank that mature sequence; pre-bantam and the 5' and 3' flanks are 60, 104 and 112 nt, respectively. Immunoprecipitates obtained using an affinity-purified Drosha antibody were capable of generating pre-bantam from primary-bantam. Examination of the supernatants showed that a substantial fraction of primary-miRNA processing activity was depleted from the extract by immunoprecipitation. Additional primary-miRNAs from both human and Drosophila were similarly processed by extracts and immunoprecipitates. Considered together, these results implicate Drosha and/or its associated factors as the major source of primary-miRNA processing activity in Drosophila extracts (Denli, 2004).

The accuracy of pri-miRNA processing by immunoaffinity-purified Drosha was assessed in two ways. First, the processing of two pri-miRNA substrates were examined that were labelled at their 5' or 3' termini. Cleavage of primary-bantam or primary-miR30a yielded the expected end fragments. The end of pre-miR30a has been mapped by primer extension of in vivo and in vitro processed transcripts. Upon analysis of primary-miR30a, processed using immunoaffinity-purified Drosophila Drosha, two prominent extension products were found. The smaller is consistent with the expected pre-miR30a product, based both on a labelled DNA marker and on primer extension of a synthetic version of the expected pre-miR30a product. A larger product, differing from the expected product by 2-3 nt, is of unknown origin. However, it should be noted that many nucleases, including Dicer, are known to make multiple cleavages within their substrates. In contrast, Drosha does not operate on fully duplexed substrates (Bernstein, 2001; Denli, 2004).

To investigate how Drosha selects its substrates and how the enzyme determines its specific cleavage sites, attempts were made to characterize native complexes containing the enzyme. Biochemical fractionation indicates that Drosha is present within an ~500 kDa complex, which presumably contains additional protein components. Given its role in microRNA metabolism, this complex has been dubbed the Microprocessor (Denli, 2004).

Recent, genome-wide two-hybrid analysis of Drosophila has generated a substantial list of candidate protein-protein interactions. In examining the list, a potential interaction between Drosha and a double-stranded RNA (dsRNA) binding protein, originally dubbed CG1800 (candidate gene 1800), was noted. This protein consists of two domains, a WW domain near the amino terminus and a dsRNA binding domain near the carboxyl terminus. Notably, WW domains interact with proline-rich domains, such as the one present near the N terminus of Drosha. Along with Drosha, CG1800 is conserved in C. elegans and mammals, where it was called DGCR8. Like Drosha, CG1800 homologues are absent from the Arabidopsis and S. pombe genomes. Results from the published two-hybrid studies, as well as evidence of physical and genetic interaction presented in this study, suggested that CG1800 be designated as Pasha (partner of Drosha) (Denli, 2004).

Consistent with the possibility that they might both be present in a single complex, Drosha and Pasha are predominantly nuclear proteins. Furthermore, Pasha antiserum co-immunoprecipitates Drosha, essentially depleting it from whole cell extracts. This interaction was not disrupted by treatment of extracts or immunoprecipitates with RNase. Analysis of the reciprocal immunoprecipitate required the use of a T7-tagged Pasha, since the antibody heavy chain interferes with detection of this protein in Western blots with the rabbit anti-Pasha antibody. In T7-Pasha-expressing cells, a Drosha antibody co-immunoprecipitates tagged Pasha. Drosha-Pasha complexes are functional since both Drosha and Pasha immunoprecipitates are able to process pri-miRNA into pre-miRNA in vitro with similar efficiencies. To test whether Drosha and Pasha coexist in the 500 kDa Microprocessor, chromatographic profiles from S2 cell extracts were examined. Both proteins along with pri-miRNA processing activity peaked in an ~500 kDa fraction (Denli, 2004).

To examine the functional connection between Pasha and miRNA metabolism, it was asked whether suppression or mutation of Pasha had any affect on primary-miRNA processing or miRNA function. In Drosophila S2 cells, Pasha dsRNAs caused a modest suppression of Pasha mRNA. This resulted in both an accumulation of pri-miR2a and a reduction in levels of mature miR2a. Similar effects on pri-miR30a were seen upon transfection of mammalian cells with two different human Pasha siRNAs. Similarly, treatment of C. elegans with pash-1 dsRNA, by feeding of RNAi-hypersensitive rrf-3 mutant worms, resulted in an accumulation of primary-let-7 with a decrease in the mature species. Notably, similar effects were seen upon suppression of Dicer (dcr-1), although in this case the pre-miRNA also accumulated. A comparison of these two results suggests that Pasha acts in primary-miRNA metabolism upstream of pre-miRNA production, consistent with its placement by biochemistry as a component of the Microprocessor. Precisely why pri-miRNAs also accumulate in worms treated with Dicer dsRNA but not in worms treated with control dsRNAs is at present unclear (Denli, 2004).

For studies of the biological function of Drosha and Pasha in vivo, C. elegans was used. A deletion allele of Drosha, drsh-1(tm0654) causes a sterile phenotype, with no other visible defects. Specifically, the presence of alae and the structure of the vulva were examined, since these structures are often affected by lesions in miRNA pathway genes like dcr-1. Most probably, such defects are not observed in drsh-1(tm0654) animals because of a strong maternal rescue. Defects in the alae and vulva structures are easily detected in pash-1 RNAi knock-down animals and to a lesser degree in a pash-1(pk2083) nonsense mutant. Typical defects include protrusion or bursting of the vulva, and gaps or absence of the alae. In addition, a more sensitive and specific assay was used to directly monitor the activity of the let-7 miRNA. The assay is based on a lacZ reporter that is silenced by let-7 through sequences in the 3' UTR. This results in detectable lacZ staining in all larval stages because let-7 is not yet expressed in these stages, but an absence of lacZ in the let-7 expressing adult. As a control, dcr-1 mutant animals were used. Indeed, in this mutant background lacZ is reactivated in the adult. It was then asked whether the adult-specific silencing of this reporter requires drsh-1 and/or pash-1. Both pash-1(pk2083) and drsh-1(tm0654) lead to re-expression of the reporter in the adult stage, indicating that in vivo, both Drosha and Pasha proteins are required for the function or synthesis of the let-7 miRNA (Denli, 2004).

Considered together, these results indicate that Pasha and Drosha are components of a multiprotein machine, the Microprocessor, which converts pri-miRNAs into pre-miRNAs. Although the experiments presented here do not permit assigning of a definitive function to Pasha within the Microprocessor, it is reasonable to speculate that Pasha might have one of several roles. For example, it could help in identifying primary miRNA transcripts, facilitating delivery of these to Drosha for cleavage. Indeed, Pasha immunoprecipitates contain primary-miRNAs. Alternatively, Pasha could help to orient the pri-miRNA in the Microprocessor, contributing to the specific positioning of the Drosha cleavage site. A parallel study shows an association between DGCR8 and human Drosha, and presents biochemical and genetic evidence that these proteins cooperate to determine the specificity of Drosha cleavage (Gregory, 2004). On the basis of the results presented here, Pasha joins RDE-4, Hyl-1 and R2D2 to form a growing list of dsRNA-binding proteins that play important yet distinct roles in the RNAi pathwa (Denli, 2004).

Genome-wide analysis of mRNAs regulated by Drosha and Argonaute proteins in Drosophila melanogaster

RNA silencing pathways are conserved gene regulation mechanisms that elicit decay and/or translational repression of mRNAs complementary to short interfering RNAs and microRNAs (miRNAs). The fraction of the transcriptome regulated by these pathways is not known, but it is thought that each miRNA may have hundreds of targets. To identify transcripts regulated by silencing pathways at the genomic level, mRNA expression profiles were examined in Drosophila melanogaster cells depleted of four Argonaute paralogs (i.e., AGO1, AGO2, PIWI, or Aubergine) that play essential roles in RNA silencing. Cells depleted of the miRNA-processing enzyme Drosha were also examined. The results reveal that transcripts differentially expressed in Drosha-depleted cells have highly correlated expression in the AGO1 knockdown and are significantly enriched in predicted and validated miRNA targets. The levels of a subset of miRNA targets are also regulated by AGO2. Moreover, AGO1 and AGO2 silence the expression of a common set of mobile genetic elements. Together, these results indicate that the functional overlap between AGO1 and AGO2 in Drosophila is more important than previously thought (Rehwinkel, 2006).

Using microarray analysis of Drosophila cells depleted of Drosha and Argonaute proteins, this study shows that transcripts whose levels are likely to be directly regulated by silencing pathways (up-regulated transcripts) represent less than 20% of the Drosophila S2 cell transcriptome. Computational predictions of miRNA targets indicate that more than 30% of the transcriptome is targeted by miRNAs. There are several possible explanations for these seemingly contradictory observations. (1) It was shown that not all authentic targets change levels in a detectable manner. This indicates that although microarrays are a valuable tool to identify miRNA targets, many targets may escape detection using this approach. (2) Some miRNAs and targets are expressed in a tissue-specific manner, so it is likely that only a subset of miRNA/target pairs is expressed in S2 cells. (3) Current models of miRNA function suggest that miRNAs expressed in a given cell type target transcripts that are already expressed at low levels but avoid housekeeping genes or genes that are expressed in these cells at high levels. These targets may escape detection by microarray analysis. Nevertheless, among transcripts regulated by the Argonaute proteins several were found that are expressed at relatively high levels, suggesting that miRNAs not only silence the expression of undesirable, low-abundance transcripts but may also play a role in fine-tuning the expression of abundant mRNAs (Rehwinkel, 2006).

AGO1 and AGO2 are thought to have nonoverlapping functions in Drosophila. This study shows that these proteins regulate the expression levels of a common set of miRNA targets. The observation that Drosha also regulates these transcripts strongly supports the idea that regulation is mediated by miRNAs. In agreement with this, it was observed that AGO2 can associate with endogenous miRNAs, although less efficiently than does AGO1. In this way, AGO2 may also regulate the expression levels of a subset of miRNA targets. Nonetheless, when miRNA function were assayed by overexpressing miRNAs together with luciferase-based mRNA reporters, it was observed that miRNA-mediated translational repression requires AGO1 but not AGO2. It is therefore possible that in this assay the fraction of miRNAs incorporated into AGO2-containing RISC is too small to observe changes in the expression levels of the reporter. Dicer-1 is involved in miRNA biogenesis and is also required for the assembly of RISC complexes, so these observations suggest that Dicer-1 may load AGO2-containing RISCs with miRNAs, at least to some extent (Rehwinkel, 2006).

A partial functional overlap between AGO1 and AGO2 is also suggested by the observation that these proteins regulate the expression of a common set of transposable elements. It remains, however, to be established whether this regulation occurs via similar mechanisms and whether it happens at the transcriptional or posttranscriptional level (Rehwinkel, 2006).

Apart from the common regulated transcripts, transcripts regulated exclusively by AGO2 but not by Drosha or AGO1 have also been identified, suggesting that AGO2 may regulate the expression of these transcripts by an miRNA-independent mechanism that might involve endogenous siRNAs (Rehwinkel, 2006).

The levels of hid and reaper mRNAs (two experimentally validated miRNA targets increase in cells in which the miRNA pathway is impaired. Moreover, by analyzing changes in mRNA levels, additional miRNA targets have been identified and validated in Drosophila. The observation that miRNA targets change levels following inhibition of the miRNA pathway lends further support to the idea that miRNAs can reduce the levels of the targeted transcripts and not just the expression of the translated protein. Along these lines, it has recently been shown that miRNAs can trigger a strong reduction in target levels in C. elegans. Among the 136 core transcripts, 21% are between 1.5- and 2-fold up-regulated, 73% exhibited changes in the 2- to 5-fold range, and 6% were at least 5-fold up-regulated in AGO1-depleted cells. Thus, although changes in transcript levels can be used to validate miRNA targets, the effects can be modest and, as mentioned above, not all targets can be identified using this approach (Rehwinkel, 2006).

In human cells, the Argonaute proteins localize to P-bodies. These are specialized cytoplasmic foci in which the enzymes involved in mRNA degradation in the 5'-to-3' direction colocalize (e.g., the DCP1:DCP2 decapping complex and the 5'-to-3' exonuclease XRN1. In addition, mRNA decay intermediates, miRNA targets, and miRNAs have been observed in P-bodies, suggesting a functional link between P-bodies and RNA silencing pathways. Consistent with this, it has been shown that P-body components play a crucial role in silencing pathways. In particular, the RNA-binding protein GW182 (a P-body component in metazoa) and the DCP1:DCP2 decapping complex are required for miRNA-mediated gene silencing in Drosophila cells. Likewise, human GW182 plays a role in silencing mediated by miRNAs and siRNAs. Finally, the C. elegans protein AIN-1, which is related to GW182, is also required for regulation of a subset of miRNA targets. Together with the observation that miRNAs inhibit cap-dependent but not cap-independent translation initiation, these observations suggest a model in which miRNA targets are stored in P-bodies after translation inhibition, where they are maintained in a silenced state by associating with proteins that prevent translation or possibly by removal of the cap structure. Decapping or simply the storage of miRNA targets in P-bodies may make these mRNAs susceptible to degradation, providing a possible explanation for the reduction in mRNA levels. In agreement with this, depletion of a 5'-to-3' exonuclease in C. elegans partially restores the levels of miRNA targets (Rehwinkel, 2006).

Nevertheless, not all authentic miRNA targets change expression levels. Thus, it is possible that the extent of the degradation depends on the number of miRNA binding sites and/or the stability of the miRNA:mRNA duplexes. It is also possible that the rate of mRNA decay triggered by miRNAs for some targets does not exceed the rate of transcription and that thus the steady-state levels of these targets remain unchanged. It would therefore be of interest to determine whether miRNAs generally cause a reduction in the half-life of targeted transcripts (Rehwinkel, 2006).

The human DiGeorge syndrome critical region gene 8 and its D. melanogaster homolog are required for miRNA biogenesis

MicroRNAs (miRNAs) represent a family of small noncoding RNAs that are found in plants and animals. miRNAs are expressed in a developmentally and tissue-specific manner and regulate the translational efficiency and stability of partial or fully sequence-complementary mRNAs. miRNAs are excised in a stepwise process from double-stranded RNA precursors that are embedded in long RNA polymerase II primary transcripts (pri-miRNA). Drosha RNase III catalyzes the first excision event, the release in the nucleus of a hairpin RNA (pre-miRNA), which is followed by export of the pre-miRNA to the cytoplasm and further processing by Dicer to mature miRNAs. This study characterized the human DGCR8, the DiGeorge syndrome critical region gene 8, and its Drosophila melanogaster homolog. Viochemical and cell-based readouts demonstrate the requirement of DGCR8 for the maturation of miRNA primary transcripts. RNAi knockdown experiments of fly and human DGCR8 resulted in accumulation of pri-miRNAs and reduction of pre-miRNAs and mature miRNAs. These results suggest that DGCR8 and Drosha interact in human cells and reside in a functional primary-miRNA processing complex (Landthaler, 2004).

A genome-wide, two-hybrid interaction screen for Drosophila proteins has revealed an interaction between Drosha and the WW domain-containing protein dmDGCR8 (CG1800), which is homologous to the human DiGeorge syndrome critical region gene 8 protein, DGCR8. The D. melanogaster homolog is referred to as dmDGCR8. The DGCR8 orthologs contain two dsRNA binding motifs. This observation prompted an investigation to see if these proteins are involved in maturation of primary miRNA transcripts. RNAi-based reverse-genetic methods have been widely applied to study gene function in D. melanogaster S2 suspension cell cultures. This method was adapted for inactivation of dmDGCR8 as well as several other genes known to be involved in nuclear and cytoplasmic miRNA processing. DsRNA of about 500 base pair (bp) in length, directed against the coding region of the respective targets, was added without transfection reagent to the S2 cells growing in suspension culture medium. To examine miRNA processing, mir-2 gene clusters, which have been shown to be expressed in S2 cells, were examined. After 5 days, total RNA was isolated and probed for the presence of the closely sequence-related mature miR-2a and -2b (collectively referred to as miR-2) and their precursors. Depletion of Drosha and dmDGCR8 resulted in a 4.5- and 12-fold reduction of mature miR-2, respectively, and a 10- to 12-fold reduction of the pre-miR-2 hairpin. In contrast, depletion of the cytoplasmic pre-miRNA processing factor Dicer-1 resulted in a 6.5-fold increase of pre-miRNA and no measurable reduction of mature miRNA. Depletion of Dicer-2, which is the enzyme predominantly responsible for generating siRNAs from long dsRNA in D. melanogaster, had no significant effect on miRNA processing (Landthaler, 2004).

In order to characterize the molecular defect in miRNA processing induced by dmDGCR8 depletion, accumulation of unprocessed primary miRNA transcripts (primary-miRNAs) was sought by quantitative RT/PCR (qRT/RCR) analysis. Drosophla expresses members of the miR-2/-13 family from four different loci. The first and third cluster are embedded in sense orientation within the introns of protein-coding genes, whereas the second and fourth genes appear to have their own promoter. Expression for the first, third and fourth miR-2/-13 loci was not detected by qRT/PCR analysis using total RNA from untreated S2 cells. Knockdown of dmDGCR8 or Drosha resulted in a 5- to 23-fold accumulation of pri-miRNAs for intronic miR-2 loci 1 and 4 (Landthaler, 2004).

A reciprocal BLAST search of the protein database using the D. melanogaster dmDGCR8 coding sequence identified a gene in the DiGeorge syndrome critical region, DGCR8, as the likely human homolog. The DGCR8 protein is 40% identical to dmDGCR8 and shares a highly similar domain structure. In order to determine whether DGCR8 functions in miRNA maturation, the protein was depleted in cultured human cells by RNAi and the effect on miRNA biogenesis was examined for a selection of human miRNA genes. Total RNA was isolated from cells 6 days after transfection, with siRNA duplexes targeting either GFP, DGCR8, hDrosha, or Dicer and probed for four different miRNA genes by Northern analysis. Specific knockdown of mRNAs was confirmed by qRT-PCR. Similar to results observed for miR-2 in S2 cells, depletion of hDrosha or DGCR8 in HeLa S3 and HEK 293 cells resulted in a reduction of pre-miR16 and mature miR-16 compared to RNA isolated from untreated cells or cells transfected with GFP control siRNA. Knockdown of Dicer in both human cell types led to the expected increase of the pre-miRNA hairpin. To examine whether the decrease in pre- and mature miRNA levels by knockdowns of DGCR8 or hDrosha is restricted to miR-16, RNA extracted from HeLa cell knockdowns was probed with probes specific for miR-18, miR-21, or miR-27a, which derive from distinct miRNA primary transcripts (pri-miRNAs). As for miR-16, probing for miR-21 revealed a decrease in pre- and mature-miRNA levels in hDrosha- and DGCR8-depleted cells. A reduction of mature miR-18 and miR-27a, for which only the mature miRNA was detectable in the control experiments, was observed in DGCR8, hDrosha, and Dicer knockdowns (Landthaler, 2004).

Because the reduction of pre-miRNAs and mature miRNAs could be a consequence of decreased primary-miRNA transcription in DGCR8 and hDrosha knockdown cells, a qRT/PCR assay was used to monitor the relative expression level of two primary-miRNAs. miRNA-specific reverse primers were used to convert primary-miR-18 and primary-miR-27a into cDNA. For qPCR analysis, PCR primers were positioned such that the resulting amplification product would span the hDrosha-processing site. Knockdown of DGCR8 or hDrosha resulted in a 2- to 5.5-fold increase in the relative amounts of primary-miR-18 and primary-miR-27a compared to the GFP siRNA control. The observed decrease of pre- and mature miRNAs levels in DGCR8 knockdown cells, with a simultaneous increase in primary-miRNAs, indicates that DGCR8 is necessary for miRNA maturation (Landthaler, 2004).

To assess the role of miRNA-processing factors directly in living cells, a HeLa cell-based positive-EGFP readout assay, that senses endogenous miR-21-guided target RNA cleavage, was applied. The EGFP mRNA, which carries a fully complementary miR-21 sequence in its 3' UTR, is constantly cleaved by the endogenously expressed miR-21 but derepressed by transfection of an anti-miR-21 2'-O-methyl oligoribonucleotide that blocks endogenous miR-21 ribonucleoprotein complexes. A control anti-miR-7 2'-O-methyl oligoribonucleotide was unable to derepress EGFP. Similar to the depletion of Ago2, the putative RISC endonuclease, knockdowns of DGCR8 and hDrosha with validated siRNAs resulted in an upregulation of EGFP compared to cells transfected with control siRNA targeting lamin A/C, further supporting that DGCR8, like hDrosha, is required for miR-21 maturation in vivo (Landthaler, 2004).

To determine whether hDrosha also interacts with DGCR8, as might be expected from the D. melanogaster 2-hybrid analysis, coimmunoprecipitations (co-IPs) were performed of transiently expressed, epitope-tagged hDrosha and DGCR8. HEK 293 cells were either transfected with pmyc-hDrosha alone or transfected simultaneously with pmyc-hDrosha and pFLAG/HA (as empty vector control) or pFLAG/HA-DGCR8. Total lysates from the transfected cells were immunoprecipitated with anti-myc or anti-FLAG antibodies, and the precipitated protein complex was analyzed by SDS-PAGE followed by Western blotting. myc-hDrosha and FLAG/HA-DGCR8 coimmunoprecipitated when either antibody was used for IPs. The treatment of the anti-FLAG IPs with a cocktail of RNase A and RNase T1 had no obvious effect on the coprecipitation, suggesting the interaction of myc-hDrosha and FLAG/HA-DGCR8 was RNA independent. No interaction could be detected in cells singly transfected with pmyc-hDrosha or cotransfected with the empty expression vector pFLAG/HA (Landthaler, 2004).

The hDrosha and DGCR8 protein-protein interaction is likely mediated through two conserved domains. The N terminus of Drosha contains a proline-rich region. This domain generally provides a ligand for binding to a WW motif. Such a motif has been identified in the DGCR8 protein sequence (Landthaler, 2004).

The interaction of hDrosha and DGCR8 suggests that both proteins reside in a functional protein complex. hDrosha executes primary-miRNA processing in the nucleus, and immunopurified hDrosha was able to cleave a primary-miRNA, releasing a pre-miRNA hairpin (Lee, 2003). Myc-hDrosha was expressed by transient transfection in HEK 293 cells and the protein was isolated by immunoprecipitation (IP) from total cell lysate by using anti-myc antibody. The immunoprecipitated complex or protein G sepharose beads were incubated with radiolabeled primary-miR-27a. Only the immunoprecipitated complex cleaved the long primary-miR-27a into a smaller RNA of the expected length of about 64 nt, whereas recombinant mouse Dicer generated small RNAs of about 22 nt (Landthaler, 2004).

Interestingly, anti-FLAG-immunoprecipitated complexes from lysates prepared from cells cotransfected with pmyc-hDrosha and pFLAG/HA-DGCR8 also cut the primary-miR-27a and released a major cleavage product of about 64 nt. In summary, these data suggest that FLAG/HA-DGCR8 and myc-hDrosha interact in vivo and are present in a functional primary-miRNA-processing protein complex (Landthaler, 2004).

In plants, miRNA accumulation depends on the activity of the predominantly nuclear proteins DCL1, HEN1, and HYL1. dcl1 null alleles are embryonic lethal, whereas partial loss-of-function dcl1 mutants are viable, but show reduced miRNA accumulation and developmental defects. hen1 and hyl1 null alleles exhibit reduced miRNA levels and developmental defects that overlap with those of partial loss-of-function dcl1 mutants, suggesting that DCL1, HEN1 and HYL1 act together in the nucleus. The HYL1 protein, which also contains a tandem dsRBD, may play a molecular role in miRNA primary transcript recognition in plants, similar to DGCR8 in animals (Landthaler, 2004 and references therein).

Several possible models can be envisioned for explaining the function of DGCR8. DGCR8, containing a tandem dsRNA binding domain (dsRBD), is involved in the recognition of miRNA precursors prior or concomitant to cleavage, and it positions Drosha, containing a single dsRBD, at the miRNA hairpin stem base. Alternatively, DGCR8 functions in stabilizing and handing over the Drosha-cleaved pre-miRNA to the nuclear export complex. For both of these models, one might predict either mutually exclusive/sequential or simultaneous/cooperative miRNA recognition. Structural and biochemical analyses have shown that two dsRNA binding motifs bind to regions of 11 to 16 bp of dsRNA, suggesting the possibility for simultaneous binding of DGCR8 and Drosha to a miRNA stem loop structure. Independent of these proposed models, this study demonstrates that the Drosha-interactor DGCR8 is involved in miRNA biogenesis in D. melanogaster and humans (Landthaler, 2004).

Search PubMed for articles about Drosophila Drosha

Bernstein, E., Caudy, A. A., Hammond, S. M. and Hannon, G. J. (2003). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363-366. Medline abstract: 11201747

Denli, A. M., Tops, B. B., Plasterk, R. H., Ketting, R. F. and Hannon, G. J. (2004). Processing of primary microRNAs by the Microprocessor complex. Nature 432(7014): 231-5. Medline abstract: 15531879

Gregory, R. I., et al. (2004). The Microprocessor complex mediates the genesis of microRNAs. Nature 432(7014): 235-40. Medline abstract: 15531877

Lee, Y. et al. (2003). The nuclear RNase III Drosha initiates microRNA processing. Nature 425: 415-419. Medline abstract: 14508493

Landthaler, M., Yalcin, A. and Tuschl, T. (2004), The human DiGeorge syndrome critical region gene 8 and Its D. melanogaster homolog are required for miRNA biogenesis. Curr. Biol. 14(23): 2162-7. Medline abstract: 15589161

Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E. and Kutay, U. (2004). Nuclear export of microRNA precursors. Science 303: 95-98. Medline abstract: 14631048

Rehwinkel, J., et al. (2006). Genome-wide analysis of mRNAs regulated by Drosha and Argonaute proteins in Drosophila melanogaster. Mol. Cell. Biol. 26: 2965-2975. Medline abstract: 16581772

Wu, H., Xu, H., Miraglia, L. J. and Crooke, S. T. (2000). Human RNase III is a 160-kDa protein involved in preribosomal RNA processing. J. Biol. Chem. 275: 36957-36965. Medline abstract: 10948199

date revised: 30 December 2007

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