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).

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: 3 July 2005

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