InteractiveFly: GeneBrief

partner of drosha: Biological Overview | References

BIOLOGICAL OVERVIEW

Canonical primary microRNA (miRNA) transcripts and mirtrons are proposed to transit distinct nuclear pathways en route to generating mature ~22 nucleotide regulatory RNAs (see Canonical miRNA and mirtron pathways in Drosophila). A null allele was generated of Drosophila pasha, which encodes a double-stranded RNA-binding protein partner of the RNase III enzyme Drosha. Analysis of this mutant yielded stringent evidence that Pasha is essential for the biogenesis of canonical miRNAs but is dispensable for the processing and function of mirtron-derived regulatory RNAs. The pasha mutant also provided a unique tool to study the developmental requirements for Drosophila miRNAs. While pasha adult somatic clones are similar in many respects to those of dicer-1 clones, pasha mutant larvae revealed an unexpected requirement for the miRNA pathway in imaginal disc growth. These data suggest limitations to somatic clonal analysis of miRNA pathway components (Martin, 2009).

MicroRNAs (miRNAs) are endogenous, ~22 nucleotide (nt), regulatory RNAs that associate with Argonaute proteins to repress target transcripts posttranscriptionally. miRNAs constitute one of the largest gene families in animal genomes, with over 600 members in humans. Although they can regulate perfectly complementary targets, the vast majority of animal miRNA targets are defined by as little as 7 nt of complementarity to positions 2 to 8 of the miRNA, also known as the miRNA seed. Evolutionary conservation of seed matches suggests that 20 to 30% of Drosophila and mammalian transcripts actively maintain functional target sites for one or more miRNAs, and presumably many other transcripts contain functional sites that are either not conserved and/or have seed mismatches (Martin, 2009).

In Drosophila, as in other animals, miRNA biogenesis proceeds in a stepwise, cell-compartmentalized manner. Canonical miRNAs are initially transcribed, mostly by RNA polymerase II, as long primary transcripts (pri-miRNAs) bearing one or more miRNA hairpins. Most of these hairpins are located in the exons or introns of noncoding RNAs, but approximately one-third are located in the introns of protein-coding genes. Pri-miRNA hairpins contain >30 nt of stem, with the basal hairpin duplex serving to recruit the double-strand RNA-binding domain protein Pasha (also known as DGCR8 in mammals) (Denli, 2004; Gregory, 2004; Han, 2006; Landthaler, 2004). Pasha binds the nuclear RNase III enzyme Drosha, which 'crops' the base of the hairpin ~10 nt away from the junction of its single-stranded flanks to yield the pre-miRNA hairpin (Han, 2004, Han, 2006, Lee, 2003). The pre-miRNA is exported to the cytoplasm via Exportin-5, where it is cleaved by the cytoplasmic RNase III enzyme Dicer-1 (Dcr-1) and its double-strand RNA-binding domain partner Loquacious (Loqs). From the resultant ~22-nt duplex, one strand preferentially enters an Argonaute-1 (AGO1) complex and guides it to seed-complementary targets (Martin, 2009).

Recently, the analysis of Drosophila small RNAs revealed that a subclass of miRNAs derives from atypical hairpin precursors termed mirtrons (Okamura, 2007, Ruby, 2007). Their defining feature is that the ends of mirtron hairpins coincide precisely with 5' and 3' splice sites of introns of protein-coding genes. Biogenesis studies carried out primarily using knockdowns of candidate factors in Drosophila S2 cells provided evidence that mirtrons use the splicing machinery to bypass Drosha cleavage. Following their linearization by intron lariat debranching enzyme, mirtrons gain access to Exportin-5 and are subsequently treated in the cytoplasm as conventional pre-miRNA hairpins. The mirtron pathway has been most thoroughly studied in Drosophila, but the analysis of large-scale small RNA sequence catalogs permitted the confident categorization of mirtrons in nematodes (Ruby, 2007), diverse mammals and in chickens (Martin, 2009).

Although the initial studies of mirtron biogenesis were well supported, a potential caveat was their reliance on knockdown strategies. This is potentially significant in light of recent studies of Loqs. This Dcr-1 cofactor was originally classified as a core component of the miRNA biogenesis pathway based on studies of loqs knockdown in S2 cells and a hypomorphic loqs allele. Since these conditions reduced the level of at least some miRNAs and caused pre-miRNA hairpins to accumulate, one might have expected the complete loss of Loqs to confer a stronger effect on miRNA maturation. Perhaps surprisingly then, subsequent analysis of a loqs deletion revealed that the biogenesis of many miRNAs was only subtly affected in the loqs-null condition (Liu, 2007). This is in strong contrast to the loss of dcr-1, for which homozygous mutant cells are unable to generate miRNAs (Liu, 2007) (Martin, 2009).

This study describes the generation of a pasha-null allele and uses it to validate the hypothesis that canonical miRNAs and mirtrons transit distinct nuclear pathways. In particular, mirtrons but not canonical miRNAs are produced and can repress targets in pasha mutants. Because of its maternal contribution, homozygous pasha mutants survive embryogenesis and larval stages. This makes it a particularly useful genetic tool among mutants in core components of the Drosophila miRNA pathway. In particular, the pasha mutant demonstrates that miRNAs are strictly required for the growth of all imaginal discs, a conclusion that cannot be derived from the clonal analysis of either pasha or dcr-1. Since the postembryonic functions of invertebrate dcr-1 and vertebrate DGCR8 and Dicer must be analyzed using mosaics, these findings suggest that caution must be exercised when using this technique to infer whether a given process does or does not require miRNAs (Martin, 2009).

This study describes the first loss-of-function analysis of Drosophila pasha in the animal, using a deletion allele that removes this locus. Evidence is provided for a nearly complete block in the production of canonical miRNAs in this mutant, similar to evidence generated for embryonic stem cells with the pasha ortholog, DGCR8, deleted (Wang, 2007). The effects of pasha deletion on miRNA cropping are more severe than was previously observed using knockdown strategies (Denli, 2004, Landthaler, 2004), validating the status of Pasha as an essential component of the canonical miRNA biogenesis pathway (Martin, 2009).

Importantly, the data provide stringent evidence for the separation of nuclear miRNA sorting pathways in Drosophila. Although Pasha is essential for processing of canonical primary miRNA transcripts, it is dispensable for the processing of mirtrons. Indeed, mirtrons were capable of potent target repression in pasha-mutant cells. The contribution of mirtrons to the miRNA-mediated regulatory network is undoubtedly smaller than that of canonical miRNAs, owing to their generally modest expression levels (Berezikov, 2007; Ruby, 2007). Nevertheless, in light of the supposition that DGCR8 mutant cells are specifically lacking miRNA pathway activity (Wang, 2007), it is important to recognize that Pasha/DGCR8-mutant cells retain this subclass of miRNA regulators (Martin, 2009).

In theory, canonical miRNAs might be functionally reprogrammed into mirtron backbones, realizing that their 3' ends would need to be modified into splice sites. This is plausible given that miRNA 3' ends may be relatively subtly required for major miRNA targeting activities. Despite known roles for miRNA 3' ends in compensatory pairing, all point mutants of endogenous miRNAs isolated in nematodes (lin-4, let-7, and lsy-6) and flies (mir-278) invariably affect the seed region. If successful, such a scheme might enable the genetic rescue of Pasha/DGCR8-mutant phenotypes by single mirtronic-miRNA transgenes, akin to the rescue of maternal-zygotic Dicer mutants in zebrafish by injecting individual miRNA duplexes. It might even prove to be the case that mirtrons are especially active in Pasha/DGCR8 or Drosha mutant cells, given that Dicer would be relieved of its normal role in processing canonical pre-miRNAs in such genetic conditions (Martin, 2009 and references therein).

The general fates of long primary miRNA (pri-miRNA) transcripts that escape Drosha processing are incompletely understood at present. In at least some cases, stable transcripts representing full-length pri-miRNA species have been detected. The strong accumulation of many pri-miRNA fragments is observed using qPCR analysis, and the stable accumulation is also detected of an ~8-kb pri-mir-1 transcript that far exceeded its previously inferred size(s) (which ranged from 0.5 to 1 kb to 3 kb. At the same time, it is relevant to bear in mind that heterogeneous transcripts in the process of degradation are still substrates for qPCR; thus, the accumulation of pri-miRNA species as detected by qPCR need not necessarily be accompanied by single band on a Northern blot. Some invertebrate and vertebrate pri-miRNA transcripts have been suggested to be 50 to 100 kb in length, and it would be perhaps remarkable if such long transcripts were completely immune to degradation by one or more RNases. It will therefore be interesting to examine the fates of pri-miRNA transcripts more systematically in pasha^KO (Martin, 2009).

It is popularly presumed that the clonal loss of a core miRNA biogenesis component can be used to assess the consequences of removing most, if not all, miRNAs from a given developmental setting. Since mutants in core components of the miRNA biogenesis pathway are lethal in all animals, conditional loss is the only way to examine the effects of miRNA pathway loss-of-function mutations on adult tissues. The activity of residual protein and RNA/miRNA products in these conditions has not often been critically assessed. Notably, miRNAs are highly abundant species and directed tests suggested several miRNAs to be very stable and removed only by dilution in dividing cells. The presence of small amounts of mature miRNAs in late third-instar pasha^KO larvae, 5 days after their birth, attests to the stability of maternal Pasha and/or mature miRNAs. Double-stranded RNA-mediated knockdown studies carry similar, if not greater caveats, in light of their inherent capability for partial target suppression (Martin, 2009).

In Drosophila, an allele of dcr-1 was originally isolated in a genetic screen that assayed eye pigment levels in 'whole-eye' mutant animals. The very method of its isolation meant that a substantial amount of eye tissue had to be isolated. It was similarly observed that although 'whole-eye' pasha^KO mutant adults are substantially reduced in size, homozygous mutant adult tissue nonetheless recovers. Such observations seemingly suggest that miRNAs are dispensable for imaginal disc growth. This seems unlikely to be the case, since it has been reported that larvae deleted for the bantam miRNA lack imaginal discs, similar to what was observed in pasha mutant larvae. Thus, the strict genetic requirement for bantam does not appear to be revealed through clonal analysis of dcr-1 or pasha (Martin, 2009).

While it is conceivable that the severe pasha^KO growth defects are due to a greater reduction of global miRNA activity in imaginal discs relative to clonal experiments, a nonautonomous role for miRNAs in promoting imaginal disc growth cannot be excluded. Nevertheless, the observations serve an important reminder of the necessity for caution in interpreting the consequences of conditional Dicer or Pasha/DGCR8 loss. In particular, many studies of Dicer conditional ablation have concluded that many specific developmental events do not require miRNAs. Instead, residual miRNAs may suffice to drive substantial aspects of development in early clones. Perhaps only later in the age of these clones do miRNA levels fall below a threshold that reveals a phenotype, often during differentiation or survival of mutant cells. The extant catalog of conditional Dicer phenotypes is consistent with this interpretation (Martin, 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).

MicroRNA processing pathway regulates olfactory neuron morphogenesis

The microRNA (miRNA) processing pathway produces miRNAs as posttranscriptional regulators of gene expression. The nuclear RNase III Drosha catalyzes the first processing step together with the dsRNA binding protein DGCR8/Pasha generating pre-miRNAs. The next cleavage employs the cytoplasmic RNase III Dicer producing miRNA duplexes. Finally, Argonautes are recruited with miRNAs into an RNA-induced silencing complex for mRNA recognition. This study identified two members of the miRNA pathway, Pasha and Dicer-1, in a forward genetic screen for mutations that disrupt wiring specificity of Drosophila olfactory projection neurons (PNs). The olfactory system is built as discrete map of highly stereotyped neuronal connections. Each PN targets dendrites to a specific glomerulus in the antennal lobe and projects axons stereotypically into higher brain centers. In selected PN classes, pasha and Dicer-1 mutants cause specific PN dendritic mistargeting in the antennal lobe and altered axonal terminations in higher brain centers. Furthermore, Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate dendrite and axon targeting during development. However, Argonaute-1 and Argonaute-2 are dispensable for PN morphogenesis. These findings suggest a role for the miRNA processing pathway in establishing wiring specificity in the nervous system (Berdnik, 2008).

To identify genes that are essential for dendrite targeting in Drosophila olfactory projection neurons (PNs), a MARCM-based mosaic forward genetic screen was performed by using novel piggyBac transposon insertions. The insertions LL03660 and LL06357, integrated in pasha and Dicer-1, respectively, were uncovered. Both alleles are homozygous lethal, likely to be null, and referred to as pasha^−/− and Dicer-1^−/− mutants throughout this study. The pasha^−/− allele is an insertion in the 5'UTR, resulting in undetectable Pasha protein in homozygous mutant neurons. The Dicer-1^−/− allele is an insertion in the coding region resulting in a truncated 740 amino acid protein lacking the RNase III, PAZ, and dsRNA binding domains (Berdnik, 2008).

The MARCM technique allows visualization and manipulation of PNs in neuroblast and single-cell clones in an otherwise heterozygous animal. Gal4-GH146 was used to label PNs from three neuroblast lineages, anterodorsal (ad), lateral (l), and ventral (v) PNs. Wild-type (WT) adPNs, lPNs, and vPNs target stereotyped sets of glomeruli in neuroblast clones. pasha^−/− PNs show two dendrite morphogenesis defects for all neuroblast clones. First, the dendritic density in most glomeruli is drastically reduced. Second, dendritic branches spill into incorrect glomerular classes. Very similar PN dendritic defects were observed in Dicer-1^−/− MARCM clones (Berdnik, 2008).

It was confirmed that the transposon insertions in pasha and Dicer-1 are the cause for the mutant phenotype with two further experiments. First, precise excision of both transposons fully revert PN morphogenesis defects. Second, expression of UAS-pasha-HA or UAS-Dicer-1 transgenes fully rescued pasha or Dicer-1 mutant PN phenotypes, respectively, in MARCM experiments. Because Gal4-GH146 is expressed only in postmitotic neurons, these experiments also demonstrate that Pasha and Dicer-1 act in postmitotic neurons to regulate dendrite morphogenesis (Berdnik, 2008).

As expected, in all rescue experiments, Pasha-HA localizes to the nucleus and Dicer-1 is enriched in the cytoplasm of PNs. Endogenous Pasha protein is found ubiquitously in all cell nuclei in the brain center at 18 hr after puparium formation (APF), when PN dendrites organize the proto-antennal lobe prior to olfactory receptor neuron (ORN) axon entry. Moreover, Pasha is undetectable in pasha^−/− adPNs and DL1 single neurons (Berdnik, 2008).

To study dendrite targeting with a better resolution, single-cell MARCM clones were examined. WT DL1 single-cell clones (hereafter referred to as DL1 single neurons) always target a posterior, dorsolateral glomerulus and fill the glomerulus with dendritic branches. In pasha^−/− PNs, 17/25 DL1 single neurons show stereotyped mistargeting defects: dendrites innervate DL1 more sparsely and also mistarget to several additional glomeruli (VA7m, VC2, VA6, DL2d, and DL5), all of which are partially innervated. 8/25 DL1 single neurons spill their dendrites medially to adjacent glomeruli, mostly D and DL5. Again, Dicer-1 single mutant neurons exhibit similar PN dendrite mistargeting although to a lower frequency. Similar stereotyped mistargeting pattern as in pasha mutants occur in 19/35 DL1 single neurons mutant for Dicer-1, 7/35 single neurons show medially spilled dendrites and 9/35 target normally. The variation of DL1 phenotypes could be caused by perdurance of WT protein in single-cell mutant clones, which might affect Dicer-1 more than Pasha. The stereotyped DL1 targeting defect was not found in more than 1400 other piggyBac insertions screened, supporting the specificity of the mutant phenotype for the miRNA processing pathway (Berdnik, 2008).

MARCM expression of UAS-Pasha-HA in pasha^−/− or UAS-Dicer-1 in Dicer-1^−/− DL1 single neurons fully rescued dendrite targeting, as is the case of neuroblast clones. These experiments demonstrate that Pasha and Dicer-1 act cell autonomously in postmitotic neurons to regulate DL1 dendrite targeting (Berdnik, 2008).

To expand the studies of dendrite targeting to other specific PN classes, Gal4-Mz19 was used to label fewer neurons in neuroblast clones. This Gal4 line labels ~6 adPNs that innervate VA1d and DC3 (posterior to VA1d) in WT. In 21/21 pasha^−/− adPNs, VA1d/DC3 is sparsely innervated and dendrites are incorrectly targeted to variable glomeruli such as DA1, VA2, and VM7. 23/25 Dicer-1^−/− PNs show similar medial mistargeting phenotypes albeit to a milder extent, innervating less distant glomeruli. Similarly, the dendritic density is reduced and incorrect glomeruli are innervated, as in GH146 MARCM experiments. Gal4-Mz19 is also expressed in ~7 lPNs innervating the dorsolateral DA1 glomuerlus in WT. DA1 PN targeting is much less affected in pasha and Dicer-1 mutants. 4/5 pasha mutant and 7/9 Dicer-1 mutant lPNs target normally to DA1 with WT dendrite densities, whereas 1/5 and 2/9 lPNs exhibit additional partial innervation of the adjacent DL3 glomerulus, respectively. Thus, Pasha and Dicer-1 are not required equally in all PN classes, suggesting that potential miRNAs might selectively regulate the targeting of specific classes of PNs (Berdnik, 2008).

In addition to dendrite mistargeting, axon defects were also observed in pasha and Dicer-1 mutants. WT DL1 axons project into the lateral horn (LH) via the mushroom body calyx (MBC) where they form several collateral branches. After entering the LH, DL1 axons always form one characteristic dorsal branch whereas the main branch terminates at the lateral edge of the LH. In pasha and Dicer-1 mutant DL1 single neurons, axons extend along the normal pathway, form collaterals in the MBC, and always reach the LH. However, more than half of the mutant DL1 axons do not reach the lateral edge but stop within the LH. The dorsal branch in the LH is either absent or reduced in length. Adding one copy of a UAS-pasha-HA transgene in pasha or UAS-Dicer-1 in Dicer-1 mutant DL1 single neurons rescued all axon phenotypes: the main branch fully extends to the lateral edge of the LH and the dorsal branch is indistinguishable from WT. Thus, Pasha and Dicer-1 cell autonomously regulate PN axon-terminal elaboration (Berdnik, 2008).

To determine whether the PN dendrite targeting errors are a result of initial mistargeting or failure to maintain stable synaptic connections later, developmental studies were performed. At 18 hr APF, when ORN axons have not yet entered the proto-antennal lobe, WT adPN, lPN, and vPN dendrites have already occupied a large area of the proto-antennal lobe . DL1 single neurons already target their dendrites in the area of the future DL1 glomerulus. In pasha^−/− PNs, dendritic elaboration within the proto-antennal lobe is extremely reduced in all neuroblast or DL1 single-cell clones at 18 hr APF. At 50 hr APF, glomeruli become first visible. In WT adPNs, lPNs, and DL1 single neurons, the same stereotyped innervation patterns as in adults are already evident even though the antennal lobe is smaller in its overall size. Dendrites of pasha^−/− PNs are reduced in density and spill into lineage-inappropriate glomeruli. Moreover, stereotyped mistargeting of DL1 single neurons is already evident in 4/4 pasha^−/− PNs at 50 hr APF (Berdnik, 2008).

These data, in combination with the observation that pasha mutant PN dendrite phenotypes do not vary in brains of 3- and 10-day-old adults indicate that Pasha regulates dendrite elaboration and correct targeting early during development (Berdnik, 2008).

Dicer functions in small RNA maturation across species. Dicer mutants are defective for both transcript destruction and translational repression, suggesting that Dicer is required for the siRNA (small interfering RNA) and miRNA maturation pathway. However, the Drosophila genome contains two Dicer genes, Dicer-1 and Dicer-2, that share similar protein domains but are different in their functions. Dicer-1 and Dicer-2 are both required for siRNA-dependent mRNA cleavage, with Dicer-2 acting in siRNA processing and Dicer-1 acting downstream of siRNA production. However, Dicer-1, but not Dicer-2, is essential for miRNA-induced silencing during translational repression (Berdnik, 2008).

To test whether the siRNA processing pathway is required for PN targeting, use was made of Dicer-2^L811fsX mutants that lack the two RNase III domains essential for dsRNA processing. It was found that Dicer-2^L811fsX mutant PNs exhibit normal dendrite and axon targeting, suggesting that Dicer-2 is dispensable and the siRNA pathway is not required for PN targeting (Berdnik, 2008).

Next it was asked whether Dicer-2 could compensate for Dicer-1's function in PN targeting because their protein domain organization is highly similar. UAS-Dicer-2 was expressed in Dicer-1^−/− PNs to test whether PN mistargeting phenotypes could be rescued as is the case for UAS-Dicer-1 expression. No alteration was seen in the Dicer1^−/− dendrite mistargeting phenotypes in DL1 PNs, adPNs, or lPNs. This observation suggests that Dicer-2 cannot replace Dicer-1's function during PN targeting. It is proposed that Dicer-1-dependent PN targeting defects are caused by the absence of one or several miRNA(s), because Dicer-1, but not Dicer-2, is essential for miRNA-directed translation repression and mRNA turnover (Berdnik, 2008).

Many distinct mechanisms have been described for miRNA-mediated gene silencing. However, for all these, the RNA-induced silencing complex (RISC) containing the Argonaute (AGO) proteins as core components is required. AGO members can be divided into two groups, the ubiquitously expressed AGO and the reproductive cell-specific Piwi subfamily. The AGO subclass containing AGO1 and AGO2 in Drosophila is involved in small RNA loading into the RISC. Both miRNAs and siRNAs act as components of RISCs but use different silencing mechanisms. miRNAs typically contain several mismatches when paired with target mRNAs, causing mostly translational repression, whereas siRNAs are perfectly paired with target mRNAs leading to their degradation. AGO2 is described as a multiple-turnover RNA-directed RNA endonuclease acting in mRNA cleavage, whereas AGO1 functions in translational repression but also plays a role in efficient mRNA degradation. However, mRNAs targeted by almost perfectly paired miRNAs can also be degraded via AGO2. Thus, AGO1 is typically necessary for stable miRNA maturation and is essential for viability, whereas AGO2 is an essential component of the siRNA-directed RNA interference response (Berdnik, 2008).

To determine which AGO member is involved in PN targeting, MARCM clones of the strong loss-of-function allele AGO1^k08121 and the AGO2⁴¹⁴ null allele were examined. Surprisingly, normal PN dendrite and axon targeting were observed in AGO1^k08121 and AGO2⁴¹⁴ adPNs, and DL1 single neurons as dendrites elaborate in the single dorsolateral DL1 glomerulus like in WT. To test whether AGO1 and AGO2 could act in a redundant manner, PN clones were generated homozygous mutant for AGO1 in an AGO2 homozygous mutant background. 7/7 adPNs and 9/9 DL1 PNs exhibit normal targeting. In addition, axon-terminal arborization is normal in AGO1/AGO2 mutant DL1 cells (Berdnik, 2008).

There are several explanations for this surprising result. First, the AGO1^k08121 allele may not be null. Second, perdurance of AGO1 protein from parental cells is capable of compensating for the loss of the AGO1 gene in homozygous mutant clones. AGO1^k08121 mutants have drastically reduced mRNA levels, AGO1 is absent in homozygous AGO1^k08121 embryo lysates, and AGO1^k08121 has been shown to disrupt stable miRNA maturation. AGO1^k08121 mutant wing disc clones miRNA function is disrupted as in pasha^−/− and Dicer-1^−/− clones as shown by using a bantam sensor transgene. Because of these facts and given that WT AGO1 mRNA or protein would be heavily diluted at least in neuroblast clones, the above two explanations imply that a very small amount of AGO1 would be sufficient for PN dendrite targeting. Third, perhaps one or more members of the Piwi subfamily thought to be expressed and function predominantly in the germline could compensate for the loss of AGO1/AGO2 in PNs. However, normal PN morphogenesis was observed in mutants for piwi¹ and aubergine^LL06590, and both are Piwi subfamily members. Lastly, PN dendrite targeting may utilize a novel miRNA-processing mechanism that is Dicer-1 dependent but AGO independent (Berdnik, 2008).

MicroRNA-mediated posttranslational regulation of gene expression has been documented in an increasing number of biological processes. Many miRNAs are developmentally regulated and show tissue-specific expression. In the nervous system, miRNAs have been shown to play roles during neurogenesis, specification of neuronal fate, neuronal morphogenesis, synaptogenesis, and neurodegeneration. This study has demonstrated a new function of the miRNA-processing pathway in regulating wiring specificity of the olfactory circuit (Berdnik, 2008).

The results support the model that one or more miRNA(s) are essential for regulating expression of genes that in turn regulate PN dendrite targeting and axon-terminal elaboration in identified neurons during development. Candidate target genes could be transcription factors that regulate wiring specificity in postmitotic neurons, cell-surface receptors for dendrite targeting, or their regulators. Expression or protein levels of such genes are essential for PN dendrite targeting. However, each miRNA is predicted to target hundreds of mRNAs and several miRNAs can regulate one mRNA, adding much more complexity to their regulatory function. Indeed, 7 miRNAs with available null mutants (out of 152 miRNAs predicted in the Drosophila genome were tested; none of them exhibit PN targeting defects. In flies, techniques that would allow the injection of individual or pools of mature miRNAs to rescue the neural phenotypes in pasha or Dicer-1 mutants, or mimic these phenotypes by injecting 'target protectors' that interfere with miRNA-mRNA interactions as in zebrafish, are currently not available. Therefore, it remains to be a future challenge to identify the miRNA(s), and ultimately their targets, for PN target selection. Looking for mutants with similar phenotypes as pasha and Dicer-1 in forward genetic screens or candidate gene approaches may help to identify specific miRNA and their targets (Berdnik, 2008).

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).

Processing of primary microRNAs by the Microprocessor complex

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, that acts on primary miRNA transcripts (pri-miRNAs) in the nucleus. This study shows that Drosha exists in a multiprotein complex, the Microprocessor, and begins 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; 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, 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 was 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).

MeCP2 suppresses nuclear microRNA processing and dendritic growth by regulating the DGCR8/Drosha complex

Loss- and gain-of-function mutations of the X-linked gene MECP2 (methyl-CpG binding protein 2) lead to severe neurodevelopmental disorders in humans, such as Rett syndrome (RTT) and autism. MeCP2 is previously known as a transcriptional repressor by binding to methylated DNA and recruiting histone deacetylase complex (HDAC). This study reports that MeCP2 regulates gene expression posttranscriptionally by suppressing nuclear microRNA processing. MeCP2 was found to bind directly to DiGeorge syndrome critical region 8 (DGCR8; Drosophila homolog, Partner of Drosha), a critical component of the nuclear microRNA-processing machinery, and interferes with the assembly of Drosha and DGCR8 complex. Protein targets of MeCP2-suppressed microRNAs include CREB, LIMK1, and Pumilio2, which play critical roles in neural development. Gain of function of MeCP2 strongly inhibits dendritic and spine growth, which depends on the interaction of MeCP2 and DGCR8. Thus, control of microRNA processing via direct interaction with DGCR8 represents a mechanism for MeCP2 regulation of gene expression and neural development (Cheng, 2014).

Search PubMed for articles about Drosophila Pasha

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

Berdnik, D., Fan, A. P., Potter, C. J. and Luo, L. (2008). MicroRNA processing pathway regulates olfactory neuron morphogenesis. Curr. Biol. 18(22): 1754-9. PubMed ID: 19013069

Berezikov, E., et al. (2007). Mammalian mirtron genes. Mol. Cell 28: 328-336. PubMed ID: 17964270

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

Cheng, T. L., Wang, Z., Liao, Q., Zhu, Y., Zhou, W. H., Xu, W. and Qiu, Z. (2014). MeCP2 suppresses nuclear microRNA processing and dendritic growth by regulating the DGCR8/Drosha complex. Dev Cell 28: 547-560. PubMed ID: 24636259

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

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: 235-240. PubMed ID: 15531877

Han, J., et al. (2004). The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18: 3016-3027. PubMed ID: 15574589

Han, J., et al. (2006). Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex. Cell 125: 887-901. PubMed ID: 16751099

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date revised: 30 August 2015

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