BIOLOGICAL OVERVIEW

microRNAs (miRNAs) are a large family of 21- to 22-nucleotide non-coding RNAs that interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. miRNAs are excised in a stepwise process from primary miRNA (pri-miRNA) transcripts. The Drosha-Pasha/DGCR8 complex in the nucleus cleaves pri-miRNAs to release hairpin-shaped precursor miRNAs (pre-miRNAs). These pre-miRNAs are then exported to the cytoplasm and further processed by Dicer to mature miRNAs. Drosophila Dicer-1 interacts with Loquacious, a double-stranded RNA-binding domain protein. Depletion of Loquacious results in pre-miRNA accumulation in Drosophila S2 cells, as is the case for depletion of Dicer-1. Immuno-affinity purification experiments have revealed that along with Dicer-1, Loquacious resides in a functional pre-miRNA processing complex, and stimulates and directs the specific pre-miRNA processing activity. Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs^f00791 mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis. These results support a model in which Loquacious mediates miRNA biogenesis and, thereby, the expression of genes regulated by miRNAs (Forstemann, 2005; Saito, 2005).

miRNAs act as RNA guides by binding to complementary sites on target mRNAs to regulate gene expression at the post-transcriptional level in plants and animals, much as small interfering RNAs (siRNAs) do in the RNA interference (RNAi) pathway. The expression of miRNAs is often developmentally regulated in a tissue-specific manner, suggesting an important role for miRNAs in the regulation of endogenous gene expression. The importance of miRNAs for development is also highlighted by a recent computer-based analysis that predicted nearly a thousand miRNA genes in the human genome. Furthermore, recent studies have revealed that miRNAs regulate a large fraction of the protein-coding genes (Saito, 2005 and references therein).

miRNAs are transcribed as long primary miRNA (pri-miRNA) transcripts by RNA polymerase II. miRNA maturation begins with cleavage of the pri-miRNAs by the nuclear RNase III Drosha (Lee, 2002; Lee, 2003; Lee, 2004) to release approximately 70-nucleotide hairpin-shaped structures, called precursor miRNAs (pre-miRNAs). Pre-miRNAs are then exported to the cytoplasm by the protein Exportin 5, which recognizes the two-nucleotide 3' overhang that is a signature of RNase III-mediated cleavage. In the cytoplasm, pre-miRNAs are subsequently cleaved by a second RNase III enzyme, Dicer, into approximately 22-nucleotide miRNA duplexes, with an end structure characteristic of RNase III cleavage. Only one of the two strands is predominantly transferred to the RNA-induced silencing complex (RISC), which mediates either cleavage of the target mRNA or translation silencing, depending on the complementarity of the target by a mechanism that remains unclear (Saito, 2005).

There is a growing list of double-stranded RNA (dsRNA)-binding proteins that play important yet distinct roles in the RNAi pathway. Both Drosha and Dicer contain dsRNA-binding domains (dsRBDs). Drosha requires a dsRNA-binding protein partner known as Pasha in flies and Caenorhabditis elegans, and its ortholog DGCR8 in mammals to convert pri-miRNAs to pre-miRNAs (Denli, 2004; Gregory, 2004; Han, 2005; Landthaler, 2004). In plants, the predominantly nuclear Dicer-like-1, equipped with two dsRBDs, is thought to catalyze both primary-miRNA and pre-miRNA processing. The HYL1 protein, which also contains a tandem dsRBD, is required for miRNA accumulation and may play the same molecular role as Pasha/DGCR8 for Dicer-like-1 in plants (Vazquez, 2004; Han, 2004). In Drosophila, Dicer-2 is required for production of siRNAs, and forms a heterodimeric complex with the dsRNA-binding protein R2D2, which is required for its function in RISC assembly, although Dicer-2 alone suffices to convert long dsRNA into siRNAs. Drosophila Dicer-1 is associated with the processing of pre-miRNAs. However, prior to this study, a dsRNA-binding protein partner for Dicer-1 had not been identified (Saito, 2005).

Drosophila Dicer-1 is shown to interact with the dsRBD protein Loquacious (Loqs). RNAi-based reverse-genetic methods were used to screen a list of Drosophila dsRBD proteins for a protein(s) that has an effect on miRNA biogenesis in Drosophila S2 cells, and a novel protein (CG6866) was found equipped with three dsRBD. A parallel study presents genetic evidence that several types of silencing are lost in CG6866 mutant flies (Förstemann, 2005). Therefore, CG6866 was designated as Loquacious ('very talkative') (Saito, 2005).

The results indicate that Loqs and Dicer-1 form a complex that converts pre-miRNAs into mature miRNAs; so how do they act together in pre-miRNA processing? Sequence comparison reveals that Loqs is a paralog of R2D2. Therefore, Loqs may play the molecular role of R2D2 for Dicer-1. R2D2 forms a stable heterodimeric complex with Dicer-2, while either protein alone seems to be unstable in vivo. In the absence of R2D2, Dicer-2 is still capable of efficiently processing long dsRNA into siRNAs. Therefore, the siRNA generating activity of Dicer-2 is not dependent upon R2D2. However, the resultant siRNAs are not effectively channeled into RISC in the absence of R2D2. The Dicer-2-R2D2 complex, but not Dicer-2 alone, binds to siRNA, which indicates that siRNA binding by the heterodimer is important for RISC entry. In the case of Loqs, this protein alone is not capable of converting pre-miRNAs into mature miRNAs, but it clearly stimulates and directs the specific pre-miRNA processing activity of Dicer-1. Furthermore, knocking down Loqs markedly reduces the pre-miRNA processing activity in cytoplasmic lysates in vitro, but does not cause a significant reduction of the level of Dicer-1 protein, implying that Dicer-1 may largely depend on Loqs for its pre-miRNA processing activity. Thus, the molecular role of Loqs for Dicer-1 is not simply similar to that of R2D2 for Dicer-2 (Saito, 2005).

It can be envisioned that Loqs may have one of several roles in pre-miRNA processing. Dicer-1 contains only one dsRBD, which may not be sufficient for strong interaction with and/or specific recognition of the pre-miRNA substrate. Loqs, containing three dsRBDs with no other identifiable domains being apparent, could provide the additional RNA-binding modules required for specific recognition of the pre-miRNA, and thereby stabilize pre-miRNA binding for Dicer-1. Loqs could also organize binding of Dicer-1 on the pre-miRNA, contributing to the specific positioning of the Dicer-1 cleavage site. Alternatively, since dsRBDs are known to not only bind dsRNAs but also to mediate protein-protein interactions, Loqs may directly bind Dicer-1 through its dsRBDs. This protein-protein interaction may trigger a conformational change of Dicer-1 that facilitates either the formation of an intramolecular dimer of its two RNase III domains: this creates either a pair of catalytic sites, or the handover of the Dicer-1 cleaved mature miRNAs to the RISC (Saito, 2005).

Sequence analysis has revealed that protein activator of protein kinase dsRNA dependent (PKR) (PACT) (Patel, 1998) and HIV TAR RNA binding protein (TRBP) (Gatignol,1991) in mammals bear 34% identity to Loqs, and share a highly similar domain structure with it. Both PACT and TRBP are thought to play a role in the regulation of translation through modulating PKR that also contains two dsRBDs. PACT interacts with PKR and enhances the autophosphorylation of PKR, which in turn, phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2α) and leads to an inhibition of mRNA translation in response to viral infection and other stimuli. TRBP prevents PKR-mediated inhibition of protein synthesis through binding to PKR. Considered together, it will be important to find out Loqs' partners other than Dicer-1 for possible involvement of Loqs in miRNA-mediated translational regulation in Drosophila (Saito, 2005).

Dicer partner proteins tune the length of mature miRNAs in flies and mammals

Drosophila Dicer-1 produces microRNAs (miRNAs) from pre-miRNA, whereas Dicer-2 generates small interfering RNAs (siRNAs) from long dsRNA. Alternative splicing of the loquacious (loqs) mRNA generates three distinct Dicer partner proteins. To understand the function of each, flies were constructed expressing Loqs-PA, Loqs-PB, or Loqs-PD. Loqs-PD promotes both endo- and exo-siRNA production by Dicer-2. Loqs-PA or Loqs-PB is required for viability, but the proteins are not fully redundant: a specific subset of miRNAs requires Loqs-PB. Surprisingly, Loqs-PB tunes where Dicer-1 cleaves pre-miR-307a, generating a longer miRNA isoform with a distinct seed sequence and target specificity. The longer form of miR-307a represses glycerol kinase and taranis mRNA expression. The mammalian Dicer-partner TRBP, a Loqs-PB homolog, similarly tunes where Dicer cleaves pre-miR-132. Thus, Dicer-binding partner proteins change the choice of cleavage site by Dicer, producing miRNAs with target specificities different from those made by Dicer alone or Dicer bound to alternative protein partners (Fukunaga, 2012).

The simplest interpretation of these data that Loqs-PA and Loqs-PB both decrease the KM of Dcr-1 for pre-miRNA substrates is that Loqs partner proteins increase the affinity of Dcr-1 for some pre-miRNA substrates. For other pre-miRNAs, Loqs-PB can also increase Dcr-1 enzyme turnover. Thus preventing Dcr-2 from processing pre-miRNAs (R2D2), promoting the processing of 'foreign' dsRNA and of esiRNAs and other endo-siRNA precursors (Loqs-PD), enhancing the efficiency of pre-miRNA processing by Dcr-1 (Loqs-PA and PB), and tuning cleavage site choice by Dcr-1 for a small number of pre-miRNAs (Loqs-PB) (Fukunaga, 2012).

The last two functions are surprising because the number of pre-miRNAs that require Loqs-PB and not Loqs-PA for the efficient production of their miRNAs is small and even fewer require Loqs-PB to produce the correct isomir. Nonetheless, the results suggest that the loss of GSCs in flies lacking Loqs- PB reflects the reduced abundance of specific miRNAs such as miR-79, miR-283, miR-305, miR-311, and miR-318 or perhaps the production of the wrong isomirs for miR-307a, miR-87, or miR-316. Among these, miR-318 is the most abundant miRNA in ovaries (140,000 ppm in w1118 ovaries but only 8 ppm in heads). Future experiments using Loqs-independent versions of pre-miR-318 and other miRNAs should help test this idea (Fukunaga, 2012).

The amino acid sequence of Loqs-PB suggests it is slightly more related to PACT (28% identity; 34% similarity) than to TRBP (24% identity; 33% similarity). Nonetheless, the data suggest that the functional homolog of Loqs-PB in mammals is TRBP. Like Loqs-PA and Loqs-PB, TRBP may act generally to enhance the binding of Dicer to pre-miRNA. Such a role for TRBP is consistent with earlier observations that the two cleavage reactions catalyzed by mammalian Dicer are less well coordinated in the absence of TRBP. But like Loqs-PB, TRBP also helps Dicer produce specific isoforms of a one or more miRNAs. This novel function of Dicer partner proteins may be widely conserved, enabling plants and animals to effectively and accurately dice difficult but important pre-miRNA substrates. Perhaps the Drosha-binding partner Pasha in flies or DGCR8 in mammals similarly tunes pri-miRNA cleavage site choice by Drosha (Fukunaga, 2012).

The stem, but not the loop, of pre-miR-307a enables Loqs-PB to influence where Dcr-1 cleaves. One possible explanation for how Loqs-PB and TRBP change where Dcr-1 and Dicer cleave is that the mismatches and internal loop causes the stems of pre-miR-307a, pre-miR-87, and mammalian pre-miR-132 to be longer than the corresponding A-form helix; binding of Loqs-PB or TRBP might then 'shrink' the stems. In contrast, Loqs-PB binding may extend of the stem of premiR-316. Because Loqs-PB does not change where Dcr-2 cleaves pre-miR-307a, Loqs-PB likely acts only when bound to Dcr-1 (Fukunaga, 2012).

The data suggest that the effect of Loqs-PB is biologically relevant. The long miR-307a isomir predominates in wild-type flies, and miR-307^a23-mer but not miR-307^a21-mer can repress the Gk and tara mRNAs in vivo. Why has evolution failed to select for easier-to-dice variants of pre-miR-307a and premiR- 87 in flies and pre-miR-132 in mice, so that a specific partner is no longer needed to ensure production of the 'right' isomir? The persistence of the miR-307^a21-mer in wild-type flies, for example, suggests that most, if not all, abundant isomirs have distinct biological functions and that the optimal function of miRNAs like miR-307a, miR-87, and miR-132 requires a defined ratio of isomirs (Fukunaga, 2012).

Although this study failed to detect altered regulation of predicted miR-307a^21-mer targets in ovaries from flies lacking Loqs-PB, miR-307a^21-mer may have functions in other tissues or times in development. In flies, mice, and humans, the relative abundance of miRNA isomirs generated by different Dicer cleavage sites, including 50 isomirs with distinct seed sequences, varies among tissues and developmental stages. Perhaps the abundance of Loqs-PB versus Loqs-PA or TRBP versus PACT is regulated across development and differentiation to ensure the correct relative abundance of isomirs from various pre-miRNAs, much as the ratios of alternatively spliced mRNAs are regulated in different tissues and cell types. The loqsKO flies rescued with transgenes producing individual Loqs isoforms should facilitate the testing of this idea in vivo (Fukunaga, 2012).

Functionally diverse microRNA effector complexes are regulated by extracellular signaling

Because microRNAs (miRNAs) influence the expression of many genes in cells, discovering how the miRNA pathway is regulated is an important area of investigation. This study found that the Drosophila miRNA-induced silencing complex (miRISC) exists in multiple forms. A constitutive form, called G-miRISC, is comprised of Ago1, miRNA, and GW182. Two distinct miRISC complexes that lack GW182 are regulated by mitogenic signaling. Exposure of cells to serum, lipids, or the tumor promoter PMA suppressed formation of these complexes. P-miRISC is comprised of Ago1, miRNA, and Loqs-PB, and it associates with mRNAs assembled into polysomes. The other regulated Ago1 complex associates with membranous organelles and is likely an intermediate in miRISC recycling. The formation of these complexes is correlated with a 5- to 10-fold stronger repression of target gene expression inside cells. Taken together, these results indicate that mitogenic signaling regulates the miRNA effector machinery to attenuate its repressive activities (Wu, 2013).

This study found that different miRISC complexes are present in S2 cells, depending upon extracellular signals received by the cells. A constitutive G-miRISC complex composed of Ago1, miRNA, and GW182 is present under all signaling conditions tested. Other groups have shown that G-miRISC in S2 cells suppresses target mRNAs via inhibition of translation initiation and enhanced mRNA decay. This study found that lipid signaling does not affect G-miRISC but blocks other miRISC complexes from forming. This signaling is likely mediated by PKC because a phorbol ester mimics the effect of lipids on miRISC formation. Signaling blocks the formation of P-miRISC, which contains Ago1, miRNA, and Loqs-PB, but not GW182. P-miRISC represses translation of target mRNAs, which is manifested in polysome association of the complex. Thus, this work reveals a mechanistic shift in miRISC-executed translation repression under the influence of extracellular lipid signals. In the presence of lipid signaling, initiation is inhibited, and this occurs by G-miRISC. In the absence of lipid signaling, it is proposed that cells generate two levels of translational repression: one mediated by G-miRISC that inhibits initiation, and one mediated by P-miRISC that inhibits elongation. It is proposed that each miRISC complex independently represses the same target, and because they act in series (initiation - elongation), the net result on protein synthesis is the product (not sum) of each inhibitory step. This would provide the strongly synergized repression of reporter protein synthesis that was observed after serum withdrawal (Wu, 2013).

P-miRISC resembles the miRNA loading complex (miRLC) complex in terms of subunit composition (Ago1, Loqs-PB), but the two differ in one important way. Whereas miRLC contains premiRNA, P-miRISC contains mature miRNA. Thus, P-miRISC has an inherent potential to engage target mRNAs via base pairing interactions. It is suggested that P-miRISC is formed by the processing and loading of mature miRNA into Ago1 within the miRLC. Rather than releasing Loqs-PB/Dcr-1 and recruiting GW182, the loaded Ago1 retains Loqs-PB and never recruits GW182. P-miRISC can then engage target mRNAs, but its subunit composition dictates a different mode of repression upon the target (Wu, 2013).

Although GW182 and Loqs-PB binding to Ago1 are mutually exclusive, P-miRISC is not simply a default state when GW182 recruitment fails to occur. Knockdown of GW182 was insufficient to induce formation of P-miRISC. Moreover, formation of P-miRISC did not appear to occur at the expense of G-miRISC levels, as measured in sedimentation and immunoprecipitation experiments. This suggests a mechanism in which stable loading of miRNA is limited by the availability of cofactors for Ago1. Under serum-fed conditions, only GW182 is available, whereas both GW182 and Loqs-PB are available under serum-free conditions. This possibly offers a rapid way to modulate miRISC levels without the need for synthesis of more cofactors (Wu, 2013).

The switch in miRISC formation is regulated by PKC, but how this switch occurs is not clear. A recent study demonstrated that the mammalian homolog of Drosophila Ago1 can be phosphorylated by Akt3, which contributes to increased miRISC-mediated translation repression (Horman, 2013). However, no evidence was found for differential phosphorylation of Ago1 in S2 cells. A study of the mammalian ortholog of Loqs-PB, called TRBP, found it to be phosphorylated by ERK kinase in response to PKC. Phosphorylation stabilized miRLC and increased processing of growth-promoting miRNAs. The same mechanism was not shown for Loqs-PB, and examination of the Loqs-PB sequence failed to find strict conservation of those sites (Wu, 2013).

A second Ago1 complex also appears when lipid signaling is absent. Membrane-associated Ago1 likely contains miRNA, but not Loqs-PB or GW182. Association of mammalian Ago proteins with late endosomes has been previously observed. Drosophila Ago1 has also been observed to associate with endosomes in vivo. Endosomes have been proposed to serve as sites for miRISC turnover whereby miRISC continuously associates and releases from endosomes, constituting a mechanism that promotes miRISC recycling onto new targets. Thus, membrane-associated Ago1 may represent an intermediate in miRISC turnover. If so, where does the membrane- associated Ago1 originate? Several lines of evidence suggest that it originates from P-miRISC. First, its appearance precisely correlates with P-miRISC. Second, it is sensitive to puromycin treatment, which also disrupts association of P-miRISC with polysomes. However, membrane-associated Ago1 does not sediment in ribosome-containing fractions. Third, insulin specifically inhibits membrane-associated Ago1, arguing that membrane-associated Ago1 is not an obligate precursor of P-miRISC. The simplest interpretation of these data is that membrane- associated Ago1 is formed from a P-miRISC precursor. If so, then Loqs-PB dissociation must be involved in the conversion because Loqs-PB is not found in the membrane-associated complex. A similar manner of cofactor stripping was observed for GW182, which dissociated from Ago-miRNA complexes when they associated with endosomes. Perhaps, cofactor dissociation is a fundamental part of the recycling mechanism (Wu, 2013).

This model might provide some insights into a long-standing controversy in the miRNA field. Some studies have found evidence for translation initiation as the regulated step, whereas others have found evidence for translation elongation. This work provides a potential explanation for these differences. That is, experimental model systems experiencing diverse extracellular signals might respond accordingly to form distinct types of miRISC complexes, which regulate different steps of translation. Thus, all studies have depicted an accurate picture of miRISC activity because signals that dictate miRISC subunit composition affect its mode of action (Wu, 2013).

GENE STRUCTURE

cDNA clone length - 1886 (isoform A)

Bases in 5' UTR - 184

Exons - 4

Bases in 3' UTR - 444

PROTEIN STRUCTURE

Amino Acids - 419 (isoform A), 465 (isoform B)

Structural Domains

RNAi-based reverse-genetic methods were used to screen a list of Drosophila dsRBD proteins for a protein(s) that has an effect on miRNA biogenesis in Drosophila S2 cells, and a novel protein was found equipped with three dsRBDs (two canonical dsRBDs at the N-terminal half, and one non-canonical dsRBD at the C-terminal), originally dubbed CG6866, which has a role in pre-miRNA processing. This protein bears high similarity to R2D2 and to the C. elegans RNAi protein RDE-4, both of which contain dsRBDs and interact with Dicer. Thus the sequence data show that CG6866 is a paralog of R2D2. A parallel study presents genetic evidence that several types of silencing are lost in CG6866 mutant flies (Förstemann, 2005). Therefore, CG6866 was designated as Loquacious ('very talkative') (Saito, 2005).

Depletion of Loqs results in accumulation of pre-miRNAs in Drosophila S2 cells. Loqs is predominantly cytoplasmic and is conserved in mammals. Immuno-affinity purification experiments, together with the use of recombinant Loqs, reveal that along with Dicer-1, Loqs resides in a functional pre-miRNA processing complex, and stimulates and directs specific pre-miRNA processing activity. These results support a model in which Loqs mediates miRNA biogenesis and, thereby, the expression of genes regulated by miRNAs (Saito, 2005).

To identify a dsRBD protein partner for Dcr-1, a search was performed of a conserved domain database for all Drosophila proteins that contain dsRBDs. The protein encoded by the gene CG6866 has two dsRBDs that are most closely related to dsRBD 1 and 2 of R2D2, suggesting that the two genes are paralogs. CG6866 and R2D2 are 37% similar and 25% identical in the region of the two dsRBDs. A third dsRBD at the C-terminus of CG6866 was detected using the PFam collection of protein sequence motifs. This truncated domain deviates from the canonical dsRBD sequence. Because loss of CG6866 function de-silences both endogenous silencing and reporter expression in vivo, the gene was named loquacious.loqs is located on the left arm of Chromosome 2 at polytene band 34B9. loqs produces at least three different mRNA isoforms through alternative splicing. The shortest transcript, loqs RNA splice variant A (RA), encodes a 419-amino-acid protein, Loqs protein isoform A (PA), with a predicted molecular mass of 45 kDa. The transcript loqs RNA splice variant B (RB) contains one additional exon and encodes a protein of 465 amino acids, Loqs protein isoform B (PB), with a predicted molecular mass of 50 kDa. These two mRNA species were identified as cDNAs in the Drosophila genome sequencing project and annotated in FlyBase among the Drosophila proteins that contain dsRBDs. Using non-quantitative RT-PCR, a third splice variant, loqs RNA splice variant C (RC), was detected in which an alternative splice acceptor site for exon 4 was used. Use of the alternative splice site created a 5'-extended fourth exon and changed the reading frame, resulting in a truncated protein, Loqs protein isoform C (PC), 383 amino acids long. Loqs PC has a predicted molecular mass of 41 kDa and lacks the entire third dsRBD of Loqs PA and PB. loqs RA is the predominant mRNA species in dissected testes, whereas loqs RB is the most abundant species in ovaries. Both isoforms are expressed in the carcasses of males and females after removal of the gonads. Using two independent antibodies raised against an N-terminal Loqs peptide, but not using pre-immune sera, a candidate protein for Loqs PC was detected in S2 cells, suggesting that the three loqs transcripts give rise to distinct Loqs protein isoforms (Forstemann, 2005).

date revised: 25 October 2023

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