InteractiveFly: GeneBrief

drosha: Biological Overview | References

BIOLOGICAL OVERVIEW

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 pathway (Denli, 2004).

Posttranscriptional Crossregulation between Drosha and DGCR8

The Drosha-DGCR8 complex, also known as Microprocessor, is essential for microRNA (miRNA) maturation. Drosha functions as the catalytic subunit, while DGCR8 (also known as Pasha) recognizes the RNA substrate. Although the action mechanism of this complex has been intensively studied, it remains unclear how Drosha and DGCR8 are regulated and if these proteins have any additional role(s) apart from miRNA processing. This study, carried out in both Drosophila and mammalian cell lines, shows that Drosha and DGCR8 (Partner of drosha) regulate each other posttranscriptionally. The Drosha-DGCR8 complex cleaves the hairpin structures embedded in the DGCR8 mRNA and thereby destabilizes the mRNA. DGCR8 stabilizes the Drosha protein via protein-protein interaction. This crossregulation between Drosha and DGCR8 may contribute to the homeostatic control of miRNA biogenesis. Furthermore, microarray analyses suggest that a number of mRNAs may be downregulated in a Microprocessor-dependent, miRNA-independent manner. This study reveals a previously unsuspected function of Microprocessor in mRNA stability control (Han, 2009).

This paper reports two main findings: (1) Drosha can act directly to eliminate a specific mRNA. Thus, Drosha plays an additional role in posttranscriptional control apart from miRNA processing. (2) This direct action of Drosha is part of a feedback control system between Drosha and DGCR8, which also includes a protein stabilization component (Han, 2009).

The Drosha protein cleaves the hairpins on the DGCR8 mRNA and destabilizes the mRNA, while the DGCR8 protein positively regulates the Drosha protein via protein-protein interaction. Drosha may be involved in the stability control of other mRNAs (Han, 2009).

Most eukaryotic RNase III proteins interact with dsRBD-containing proteins. It seems to be a general theme that RNase III proteins are stabilized by their binding partners. For instance, human Dicer binds to PACT, a protein containing three dsRBDs (Lee, 2006). Depletion of PACT results in the reduction of Dicer protein. Similarly, in Drosophila, Dicer-2, and a dsRNA-binding protein, R2D2, interact with each other and depend on each other for stable expression. The present study shows that DGCR8 stabilizes Drosha protein through protein-protein interaction. Although the molecular details of such protein degradation/stabilization remain to be determined, the data from mouse KO cells suggest that protein stability control of RNase III may be significant in vivo (Han, 2009).

Drosha and its cofactor, DGCR8, are engaged in a complex regulatory circuit. If Drosha and DGCR8 levels are elevated in the cell, Microprocessor would cleave and destabilize the DGCR8 mRNA, resulting in the reduction of DGCR8. This will in turn reduces the Drosha protein through protein destabilization, lowering the Microprocessor activity. This autoregulatory feedback circuit may help minimize the potentially harmful fluctuation of Microprocessor activity in the cells. In light of this, it is noteworthy that this study observed an interesting dosage compensation effect in Dgcr8 heterozygous cells that contains only one copy of the Dgcr8 gene. In heterozygous cells, the DGCR8 level is expected to be 50% of that in wild-type cells. However, the DGCR8 protein level in heterozygous cells is over 80% of the normal level. Moreover, the miR-130a level in heterozygous cells is similar to that in wild-type MEF cells. Similarly, it has been shown that miRNA levels in Dgcr8 heterozygous ES cells are over 85% of those in wild-type ES cells (Wang, 2007). Thus, Microprocessor activity in heterozygous cells may be maintained at over 80% of that in wild-type cells. Dgcr8 heterozygous mice have been shown to produce less miRNAs in the brain than does the wild-type (Stark, 2008). However, significant changes were observed only for a small subset of miRNAs, while the majority of miRNAs remained largely unaffected. Taken together, these results indicate that the feedback circuit found in this study may be significant in vivo and contribute to tight control of miRNA production in the cell. The evolutionary evidence also suggests that the regulations between Drosha and DGCR8 may play an important role in controlling miRNA biogenesis in vivo. The hairpins in DGCR8 mRNAs are conserved throughout evolution, and similar regulation occurs not only in mammalian cells but also in Drosophila cultured cells. Further work will be required to confirm the physiological significance of this regulation (Han, 2009).

Both Drosha and DGCR8 are ubiquitously expressed proteins, but the levels of these proteins vary depending on the cell types. It is conceivable that Drosha and DGCR8 are modulated by additional factors in a cell type-specific manner. Previous studies showed that Drosha and DGCR8 are associated with multiple factors (Gregory, 2004; Faller, 2007; Shiohama, 2007]). The functional significance of these interactions remains to be determined. Given the general suppression of miRNAs in cancer and stem cells, it would be particularly interesting to investigate how the miRNA processing activities are modulated during tumorigenesis and cell differentiation.

RNase III family members vary widely and play diverse roles in RNA metabolism. For instance, yeast RNase III protein, Rnt1, functions in the processing of pre-rRNAs, small nuclear RNAs, and small nucleolar RNAs. Rnt1 also cleaves and controls certain specific mRNAs. Dicer functions mainly in small RNA pathways, but it was recently shown that human Dicer can target mRNAs with CNG repeat-containing hairpins. Drosha is known to play a critical role in miRNA maturation. The present study reveals a function of Drosha in mRNA stability control (Han, 2009).

The EvoFold program predicted that vertebrate DGCR8 mRNAs contain two highly conserved hairpin structures, both of which can be cleaved by Drosha in vitro. The cleavage product from the first hairpin was detected by northern blotting. In recent deep sequencing studies, small RNAs from the second hairpin of DGCR8 mRNA were detected and named miR-1306. From the peripheral blood of a dog, miR-1306-5p was sequenced, while miR-1306-3p was identified in human embryonic stem cells. Taken together, both hairpins (A and B) are cleaved by Microprocessor in at least certain cell types. But these hairpins seem to be quite inefficient precursors for miRNA biogenesis because the small RNAs were detected only by massive parallel sequencing approach. So it remains to be determined whether miR-1306 is a functional miRNA, a by-product of regulated mRNA cleavage, or both (Han, 2009).

It is noteworthy that over 10% of the known miRNAs are located in the exonic regions of protein-coding or noncoding transcripts. Because Drosha processing is expected to destabilize the host transcripts, it would be interesting to investigate whether such exonic miRNA hairpins serve dual roles as RNA instability elements as well as miRNA precursors. It would also be of great interest to search for additional mRNAs that are controlled by Drosha. To begin investigating this possibility, a series of knockdown and microarray experiments were performed. It was found that 335 out of 16,309 mRNAs detected in HeLa cells by Affymetrix chip are upregulated over 2-fold in Drosha-depleted cells without significant changes in Dicer-depleted cells. Most mRNAs that are upregulated in Drosha-depleted cells are also upregulated in DGCR8-depleted cells. Out of the 335 mRNAs, 104 mRNAs are elevated over 2-fold in DGCR8-depleted cells without changing significantly in Ago2-depleted cells. These genes do not show significant fold changes in Ago2 knockdown cells. Thus, in HeLa cells, ~100 genes are Microprocessor dependent and RISC independent. Although it is possible that some of these genes are regulated indirectly through unknown mechanism(s), the result implies that there may be a significant number of genes that are directly controlled by Microprocessor (Han, 2009).

A Drosophila genetic screen yields allelic series of core microRNA biogenesis factors and reveals post-developmental roles for microRNAs

Canonical animal microRNAs (miRNAs) are ~22-nt regulatory RNAs generated by stepwise cleavage of primary hairpin transcripts by the Drosha and Dicer RNase III enzymes. A genetic screen was performed using an miRNA-repressed reporter in the Drosophila eye, and the first reported alleles were recovered of fly drosha, an allelic series of its dsRBD partner pasha, and novel alleles of dicer-1. Analysis of drosha mutants provided direct confirmation that mirtrons are independent of this nuclease, as inferred earlier from pasha knockouts. These mutants were further used to demonstrate in vivo cross-regulation of Drosha and Pasha in the intact animal, confirming remarkable conservation of a homeostatic mechanism that aligns their respective levels. Although the loss of core miRNA pathway components is universally lethal in animals, hypomorphic alleles were unexpectedly recovered that gave adult escapers with overtly normal development. However, the mutant photoreceptor neurons exhibited reduced synaptic transmission, without accompanying defects in neuronal development or maintenance. These findings indicate that synaptic function is especially sensitive to optimal miRNA pathway function. These allelic series of miRNA pathway mutants should find broad usage in studies of miRNA biogenesis and biology in the Drosophila system (Smibert, 2011).

This study describes a forward genetic screen for factors involved in miRNA biogenesis or function and validate its utility by characterizing a series of core miRNA pathway mutants. These were used to to investigate Microprocessor cross-regulation in vivo, as well as to study post-developmental roles of miRNAs in neural function. In particular, this study provides the first loss-of-function analysis of Drosophila drosha. As expected, a strong block was observed in canonical miRNA biogenesis in the drosha-null mutant, which accumulated primary miRNA transcripts and was depleted of mature miRNAs, similar to pasha mutants. In addition mirtron biogenesis was found to be unaffected by drosha mutation, providing direct evidence that these splicing-derived miRNAs are completely independent of the Drosha nuclease. Animals null for drosha and pasha are generally similar with respect to all phenotypes examined, supporting the obligate nature of these core protein partners within the Microprocessor complex. The screen generated allelic series for the key Microprocessor components drosha and pasha. These allowed assessment of the sensitivities of development versus function in cells with mildly reduced miRNA levels (Smibert, 2011).

Compared with full knockouts that give only null states, forward genetic screening can yield allelic series of varying phenotypic severity, which can uncover interesting aspects of pathway and gene function. It is noted that the hypomorphic drosha and pasha mutants have very different effects in different assays, as highlighted by the differences in derepression of both an endogenous miRNA target and a transgenic sensor for miRNA activity between weak drosha and pasha alleles. This is the case despite the fact that these mutants have similar effects on total mature miRNA levels as measured by Northern blot. The reason for this phenotypic discrepancy is not yet clear but suggests the possibilities that miRNAs are differentially sensitive to availability of the core biogenesis machinery or that specific biological processes are especially sensitive to optimal miRNA biogenesis. Indeed, electrophysiological evidence of the latter is provided, and both of these hypotheses represent compelling future directions for analysis of these and other mutants that may emerge from genetic screening (Smibert, 2011).

The theme of miRNA pathway autoregulation has emerged at multiple levels in animals and in plants. An in vivo demonstration of the reciprocal regulation of the core Microprocessor components reveals that this mechanism is conserved and occurs within the intact animal. The role of Pasha within the Microprocessor to position Drosha catalytic centers is clearly of crucial importance to miRNA biogenesis. Perhaps the instability of Drosha in the absence of Pasha is a biological safeguard to prevent inappropriate cleavage of transcripts by Drosha in the absence of Pasha. Likewise, the capacity of Drosha to cleave pasha transcripts may also limit Drosha levels by restricting the amount of Pasha for it to associate with (Smibert, 2011).

The studies to date focused on mutants of known miRNA pathway components. This has been a productive effort, as indicated by the first reported Drosophila alleles of drosha, the first allelic series of pasha in any organism, and novel alleles of dicer-1. The screening recovered other suppressor mutations that do not map to known pathway components, as well as enhancer mutations that are dependent on the endogenous 3' UTR of the white gene (w-miR) trigger. It is anticipated that the cloning and characterization of these mutations may provide additional insights into the miRNA biogenesis machinery or the mechanism of miRNA-mediated silencing (Smibert, 2011).

miRNAs in whole organisms have to date mostly been studied for their roles in development. This is at least in part due to the early developmental defects that potentially mask later functional defects. Yet, miRNAs have long been viewed as a potentially key component of neural function and fine-tuning due to their regulatory potential. One tantalizing property of miRNAs is their ability to regulate gene expression locally. In neurons, where a synapse may be a great distance from the nucleus, this could provide a means for rapid post-transcriptional regulation of targets. How this may be regulated in a signal-dependent manner is a topic of ongoing study. This study presents novel hypomorphic mutants that mildly affect miRNA levels and cause synapse function defects without affecting development (Smibert, 2011).

The requirement of neurons for precise miRNA activity is emphasized by the specific synaptic transmission defects in hypomorphic miRNA pathway mutants. In weak alleles of either pasha or drosha, only a mild reduction was observed in miRNA biogenesis. While these animals quickly succumb just before or after adult eclosion, they exhibit grossly normal development of all external structures. Using whole eye clone analysis, normal specification and projection of mutant photoreceptors was document, but substantial synaptic transmission defects wee obtained that were very similar in drosha, pasha, and dicer-1 mutants. The lack of deterioration of this phenotype with age both argues for a specific synaptic transmission defect and also that the reduction in miRNA levels in these photoreceptor neurons does not affect their viability or general health (Smibert, 2011).

The commonality of these phenotypes among the different mutants indicates underlying defective biogenesis of one or more canonical miRNAs, as opposed to mirtrons or other noncanonical species. It is conceivable that the synaptic transmission defect is caused by the cumulative effect of mildly reducing all photoreceptor miRNAs. However, the specificity of this phenotype and its critical dependence on optimal miRNA pathway function may imply that there is some aspect of synaptic transmission machinery that is especially sensitive to a more limited set of miRNAs. As the number of mutant strains for Drosophila miRNA loci steadily increases, it will be productive to screen them using ERGs on mutant eyes. An alternative approach may be to test miRNA sponges expressed presynaptically, perhaps in a candidate screen of head-expressed miRNAs (Smibert, 2011).

Since loss of single alleles is typically well tolerated at the organismal level, dose-sensitive loci are of particular relevance to human disease. DGCR8 is one of about 20 genes within the 22q11.2 locus deleted in patients with diGeorge syndrome, for which heterozygosity results in cognitive and behavioral impairments in humans. In a mouse model of diGeorge syndrome bearing the 22q11.2 microdeletion, heterozygosity of dgcr8 contributes to a reduction in brain miRNAs. More recently, specific heterozygosity of dgcr8 was shown to cause subtle but detectable defects in neural developmental and physiology, providing more direct evidence of dgcr8 haploinsufficiency. In the Drosophila system, cells heterozygous for pasha[KO] have less Pasha protein than homozygous wild-type cells. Accordingly, the levels of the GFP-ban sensor indicated that it was repressed more strongly in homozygous wild-type cells than in neighboring pasha[KO] heterozygous cells. Therefore, despite the operation of feedback mechanisms that regulate Microprocessor levels, pasha exhibits functional haploinsufficiency. Altogether, these studies reaffirm that subtle alterations in miRNA biogenesis can lead to detectable organismal phenotypes, helping to explain the lengths to which animal evolution has gone to maintain Microprocessor homeostasis. Reciprocally, these studies define Drosophila as a suitable system for future exploration of the unique sensitivity of neuronal function to miRNA activity (Smibert, 2011).

Drosophila Argonaute 1 and its miRNA biogenesis partners are required for oocyte formation and germline cell division

Argonaute 1 (Ago1) is a member of the Argonaute/PIWI protein family involved in small RNA-mediated gene regulation. In Drosophila, Ago1 plays a specific role in microRNA (miRNA) biogenesis and function. Previous studies have demonstrated that Ago1 regulates the fate of germline stem cells. However, the function of Ago1 in other aspects of oogenesis is still elusive. This study reports the function of Ago1 in developing egg chambers. Ago1 protein was found to be enriched in the oocytes and is also highly expressed in the cytoplasm of follicle cells. Clonal analysis of multiple ago1 mutant alleles shows that many mutant egg chambers contain only 8 nurse cells without an oocyte; this phenotype is phenocopied in dicer-1, pasha and drosha mutants. These results suggest that Ago1 and its miRNA biogenesis partners play a role in oocyte determination and germline cell division in Drosophila (Azzam, 2012).

Drosophila Ago1 forms a complex with mature miRNAs and acts to repress mRNAs. However, the spatial distribution of Ago1 during development has not been well characterized. The protein trap lines from the Carnegie Protein Trap library provide a powerful way to characterize the spatial and temporal distribution of trapped genes. The distribution of Ago1 in the cytoplasm has been described and shown to be localized in small puncta in the egg chamber. The findings using two independent assays for Ago1 localization have shown that Ago1 is enriched in the oocyte and mutant analysis has revealed a role in oocyte formation and germline cell division (Azzam, 2012).

Nurse cells supply nutrition for oocyte growth. The germline cell division defect described in this study has been previously observed in a cyclin-E mutant where 30% of the egg chambers have 8 cells, but the egg chamber still manages to develop an oocyte. Other studies have also described 8 cell egg chambers when String is over expressed as well as in a tribbles mutant. Both String overexpression and the tribbles mutant have 8 cells per egg chamber, but only a proportion fail to develop an oocyte. This defect occurs in the germarium while the cyst cells are undergoing mitosis. In the wild-type situation, the cystoblast divides four times to produce 16 cyst-cells. In the absence of ago1, some of the cystoblasts undergo only three divisions, producing 8-cell cysts. However, the ago1 mutant ovarioles with this phenotype still express Cyclin E, suggesting that mitosis is still occurring although perhaps at a slower rate. Combined with the oocyte formation defect, the resulting egg chambers only have 8 nurse cells and lack an oocyte. The cyst cell division in the germarium is not well understood. One potential explanation for the observed phenotype is that when Ago1, and presumably miRNA mediated gene regulation, are lost, the signal to stop dividing occurs early. Another possibility is because the egg chamber grows more slowly, the oocyte reaches region 2A before it manages to divide 4 times, thus receiving a premature signal to stop dividing, or being prematurely enclosed by the migrating follicle cells. The smaller germarium of ago1 mutant might also be an effect of cyst-cells dividing slower. The defective egg chamber however still manages to grow. Furthermore, the observation of Orb protein in region 2 of the germarium and in the stage 1 egg chamber could mean that the oocyte is trying to enter meiosis, or has entered meiosis but is unable to maintain the meiotic state because the Orb accumulation is lost in later stage egg chambers and no oocyte is formed. Oocyte differentiation and maintainance in the meiotic cycle are reliant on microtubule based transport of mRNAs and proteins from the nurse cells to the oocyte. Orb, the germline specific RNA-binding protein starts accumulating in the oocyte at region 2a in a microtubule-dependent manner. orb mutant causes the egg chamber to produce 8 nurse cells and no oocyte, similar to the ago1, dcr-1, drosha and pasha mutant phenotype seen in this study. However, since Orb is still expressed, it could be rule out that the phenotype is cause by loss of orb function. The inability to maintain the accumulation of Orb in the oocyte in later stages of oogenesis could relate to defect on maintaining the meiotic cycle (Azzam, 2012).

These results have shown that a greater proportion of older ago1 flies exhibit the 8-nurse cell phenotype than younger mutant flies. This could be due to the level of Ago1 in older flies decreasing to a certain threshold level to show an obvious phenotype. There is also the possibility that the remaining or leaky (due to hypomorphic allele) Ago1 is diluted through GSC division and maintainance such that GSCs from flies at 14 DAE have less Ago1 than GSCs from flies at 7 DAE. Previous studies suggest that GSC loss in ago1 mutants are age-dependent. This could potentially explain the age-dependent 8-nurse cell phenotype that were observed in ago1 mutants. Self-renewed GSC in the absence of Ago1 could be defective, so cystoblasts produced by defective GSC might not be able to divide normally. Although ago1^k08121 and ago1¹⁴ showed a more severe phenotype in older flies, ago1^EMS, as the strongest allele, showed very severe phenotype even in young flies (Azzam, 2012).

Ago1, Dcr-1, Loquacious and PIWI have roles in small RNA biogenesis and all of them have been shown to be important for germline stem cell maintenance. The role of miRNAs regulating GSC division was first reported by Hatfield (2005) who studied null mutants of dcr-1. A similar study looking at ago1 mutants revealed that Ago1 also regulates the fate of the GSC. Both of these studies showed a similar phenotypic defect in the germline. Furthermore, there are some cases where mutations in individual miRNA genes show phenotypes in the germline cells. The miRNA bantam has been previously found to be important for GSC maintenance. Also, miR-184 controls GSC differentiation, dorsoventral patterning of the egg shell and anteroposterior patterning. Although the effect in the GSC is quite reproducible from previous studies, it is not uncommon to see this in that knockouts of miRNA biogenesis factors. This has been shown quite well in the developing wing primordium where clones lacking mir-9a upregulate dLMO and induce wing notching. This phenotype is however not fully reproducible in dcr-1 and pasha mutant clones. The effect of removing all miRNA could cancel the effect of a single miRNA mutation (Azzam, 2012).

This study shows that the dcr-1, pasha and drosha mutants phenocopy the ago1 mutant during oogenesis. However, one Pasha mutant allele, pasha^LL03360, did not phenocopy ago1 and dcr-1. This mutant is a piggyBac insertion into the 5'UTR of pasha and despite showing a convincing loss of pasha protein in adult neurons, it is possible that the allele may only be hypomorphic in the ovary. Pasha has not been studied in the Drosophila germline but it has been shown to play a role in olfactory neuron morphogenesis in the Drosophila adult brain. In that study, Pasha and Dcr-1 were found to be required for arborization of projection neurons but not Ago1. This argues for Ago1-independent roles of Dcr-1 and Pasha. Alternatively, the ago1 mutant used in that study and the current study, ago1^k08121 may not be completely null or the protein from the parental cell could be compensating for the loss of Ago1. Recent studies have suggested that neural processes are exquisitely sensitive to miRNA pathway activity so perhaps a more complete loss of Pasha function is required to produce phenotypic consequences in the ovary compared to neurons. Indeed, the relative phenotypic strength of ago1^k08121 versus ago1^EMS1 and the null mutants of miRNA biogenesis enzymes argues for the hypomorphic nature of ago1^k08121. Mirtrons are another class of small RNAs which bypass Pasha/Drosha processing by utilizing the splicing machinery, but are still processed by Dcr-1 and loaded into Ago1. However, drosha^21K11 and the newly generated pasha^36B2 mutant show the same phenotype, qualitatively and quantitatively, as ago1 and dcr-1 mutants. This argues that the majority of the phenotype we observed is due to loss of canonical miRNAs and that miRtrons have a comparably insignificant role (if any) in the phenotypes analysed. Altogether, this study reaffirms that loss of miRNA function at various stages of biogenesis or effector function has important phenotypic consequences for oogenesis (Azzam, 2012).

Drosha-independent DGCR8/Pasha pathway regulates neuronal morphogenesis

Cleavage of microRNAs and mRNAs by Drosha and its cofactor Pasha/DGCR8 is required for animal development, but whether these proteins also have independent roles in development has been unclear. Known phenotypes associated with loss of either one of these two proteins are very similar and consistent with their joint function, even though both cofactors are involved with additional distinct RNA biogenesis pathways. This study reports clear phenotypic differences between drosha and pasha/dgcr8 null alleles in two postembryonic lineages in the Drosophila brain: elimination of pasha/dgcr8 leads to defects that are not shared by drosha null mutations in the morphology of γ neurons in the mushroom body lineage, as well as many neurons in the anterodorsal projection neuron lineage. These morphological defects are not detected in neurons that are genetically depleted of two additional microRNA pathway components, dicer-1 and argonaute1, indicating that they are not due to loss of microRNA activity. They are, however, phenocopied by a newly identified recessive gain-of-function allele in drosha that probably interferes with the microRNA independent functions of Pasha/DGCR8. These data therefore identify a general Drosha-independent DGCR8/Pasha pathway that promotes proper morphology in multiple neuronal lineages. Given that reduction of human DGCR8/Pasha may contribute to the cognitive and behavioral characteristics of DiGeorge syndrome patients, disruption of this newly described pathway could underlie human neurological disease (Luhur, 2014).

This study delineates the function of two RNA pathways in neuronal lineages. The first, the canonical miRNA pathway, maintains neural progenitors. The relevant miRNAs may include miR-124, a neural-specific miRNA that contributes to neural progenitor proliferation (Weng, 2012). The second, a unique Pasha-dependent and Drosha- and Ago1-independent pathway, promotes dendritic and axonal targeting independent of canonical miRNAs. The RNA effectors of this pathway are not known but may include snoRNAs, because human Pasha is reported to regulate snoRNA biogenesis by an unknown mechanism. The possibility cannot be excluded that this pathway involves Ago1-independent noncanonical miRNAs, because some dcr-1 adPN NB and DL1 clones display mistargeting. However, the low penetrance of mistargeting defects in dcr-1 adPNs and the absence of phenotypes in dcr-1 MB γ neurons indicates that any such contribution to the morphogenesis function of Pasha is minor. Because loss of the human ortholog of Pasha/DGCR8 is reported to contribute to DiGeorge syndrome, this unique Pasha pathway may be directly relevant to the cognitive and behavioral disorders associated with this syndrome. The results strongly suggest that the dendritic defects associated with dgcr8 heterozygosity in mice involve this unique, canonical miRNA-independent function of Pasha/DGCR8 (Luhur, 2014).

The gain-of-function adPN phenotypes of drosha^Δ,E859K illuminate this Pasha pathway, likely because the mutant form of Drosha incapacitates its binding partner and blocks not only miRNA processing but Pasha's other functions as well. Drosha^Δ does not efficiently cleave both strands of pri-miRNA, leading to the accumulation of partially processed intermediates. These intermediates likely sequester Pasha, because current biochemical and in vivo imaging data suggest that pri-miRNA cleavage causes a conformational change in the microprocessor that leads to rapid release of Drosha and a slower release of Pasha. Trapped Pasha would therefore not be available to bind either to other pri-miRNAs or additional RNA targets. The morphological defects in γ MB and adPN neurons are likely due to aberrant metabolism of these other RNAs, because drosha null γ MB and adPN neurons that lack canonical miRNAs are mostly normal. Identifying additional phenotypic differences between drosha^Δ,E859K and drosha null alleles will reveal other contexts where Pasha's miRNA-independent roles are relevant (Luhur, 2014).

The gain-of-function drosha^Δ,E859K phenotypes are also recessive, a relatively rare occurrence that provides genetic support that the microprocessor complex contains two independent Drosha subunits. Biochemical purification and reconstitution experiments indicate that the large microprocessor complex, which is ~650 kDa in humans and ~500 kDa in flies, contains at least one ~70-kDa Pasha/DGCR8 subunit and one ~150-kDa Drosha subunit, although it has been unclear whether the complex contains additional Pasha/Drosha subunits and/or other auxiliary proteins. Recessive gain-of-function mutations have previously been identified in genes whose products contribute several subunits to a protein complex, such as an ion channel. In these cases, the effects of the mutant form are suppressed by redundant WT versions in heterozygotes but lead to phenotypes in homozygotes that are stronger than complete elimination of the protein. Isolation of recessive gain-of-function alleles in drosha suggests that the fly microprocessor complex functions analogously and contains two functionally independent Drosha subunits, either one of which is sufficient for its activity (Luhur, 2014).

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).

SMAD proteins control DROSHA-mediated microRNA maturation

MicroRNAs (miRNAs) are small non-coding RNAs that participate in the spatiotemporal regulation of messenger RNA and protein synthesis. Aberrant miRNA expression leads to developmental abnormalities and diseases, such as cardiovascular disorders and cancer; however, the stimuli and processes regulating miRNA biogenesis are largely unknown. The transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) family of growth factors orchestrates fundamental biological processes in development and in the homeostasis of adult tissues, including the vasculature. Induction of a contractile phenotype in human vascular smooth muscle cells by TGF-β and BMPs is mediated by miR-21. miR-21 downregulates PDCD4 (programmed cell death 4), which in turn acts as a negative regulator of smooth muscle contractile genes. Surprisingly, TGF-β and BMP signalling promotes a rapid increase in expression of mature miR-21 through a post-transcriptional step, promoting the processing of primary transcripts of miR-21 (pri-miR-21) into precursor miR-21 (pre-miR-21) by the DROSHA (also known as RNASEN) complex. TGF-β- and BMP-specific SMAD signal transducers are recruited to pri-miR-21 in a complex with the RNA helicase p68 (also known as DDX5), a component of the DROSHA microprocessor complex. The shared cofactor SMAD4 is not required for this process. Thus, regulation of miRNA biogenesis by ligand-specific SMAD proteins is critical for control of the vascular smooth muscle cell phenotype and potentially for SMAD4-independent responses mediated by the TGF-β and BMP signalling pathways (Davis, 2008).

Drosha regulates gene expression independently of RNA cleavage function

Drosha 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, this study demonstrates 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, 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, this study has uncover a previously unsuspected RNA cleavage-independent function of Drosha in the regulation of human gene expression (Gromak, 2013).

Search PubMed for articles about Drosophila Drosha

Azzam, G., Smibert, P., Lai, E. C. and Liu, J. L. (2012). Drosophila Argonaute 1 and its miRNA biogenesis partners are required for oocyte formation and germline cell division. Dev. Biol.. 365(2): 384-94. PubMed ID: 22445511

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. PubMed ID: 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. PubMed ID: 15531879

Davis, B. N., Hilyard, A. C., Lagna, G. and Hata, A. (2008). SMAD proteins control DROSHA-mediated microRNA maturation. Nature 454(7200): 56-61. PubMed ID: 18548003

Faller, M. et al. (2007). Heme is involved in microRNA processing. Nat. Struct. Mol. Biol. 14: 23-29. PubMed ID: 17159994

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

Gromak, N., Dienstbier, M., Macias, S., Plass, M., Eyras, E., Caceres, J. F. and Proudfoot, N. J. (2013). Drosha regulates gene expression independently of RNA cleavage function. Cell Rep 5: 1499-1510. PubMed ID: 24360955

Hatfield, S. D., et al. (2005). Stem cell division is regulated by the microRNA pathway. Nature 435: 974-978. PubMed ID: 15944714

Han, J., et al. (2009). Posttranscriptional Crossregulation between Drosha and DGCR8. Cell 136: 75-84. PubMed ID: 19135890

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. PubMed ID: 15589161

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

Lee, Y., et al. (2006). The role of PACT in the RNA silencing pathway. EMBO J. 25: 522-532. PubMed ID: 16424907

Luhur, A., Chawla, G., Wu, Y. C., Li, J. and Sokol, N. S. (2014). Drosha-independent DGCR8/Pasha pathway regulates neuronal morphogenesis. Proc Natl Acad Sci U S A 111: 1421-1426. PubMed ID: 24474768

Lund, E., Guttinger, S., Calado, A., Dahlberg, J. E. and Kutay, U. (2004). Nuclear export of microRNA precursors. Science 303: 95-98. PubMed ID: 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. PubMed ID: 16581772

Shiohama, A., et al. (2007). Nucleolar localization of DGCR8 and identification of eleven DGCR8-associated proteins. Exp. Cell Res. 313: 4196-4207. PubMed ID: 17765891

Smibert, P., et al. (2011). A Drosophila genetic screen yields allelic series of core microRNA biogenesis factors and reveals post-developmental roles for microRNAs. RNA 17(11): 1997-2010. PubMed ID: 21947201

Stark, K. L., et al. (2008). Altered brain microRNA biogenesis contributes to phenotypic deficits in a 22q11-deletion mouse model. Nat. Genet. 40: 751-760. PubMed ID: 18469815

Wang, Y., et al. (2007). DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat. Genet. 39: 380-385. PubMed ID: 17259983

Weng, R. and Cohen S. M. (2012) Drosophila miR-124 regulates neuroblast proliferation through its target anachronism. Development 139(8): 1427-1434. PubMed ID: 22378639

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. PubMed ID: 10948199

date revised: 10 June 2014

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