Dicer-1 and R3D1-L catalyze microRNA maturation in Drosophila

Abstract

In Drosophila melanogaster, Dicer-2/R2D2 and Dicer-1 generate small interfering RNA (siRNA) and microRNA (miRNA), respectively. Here we identify a novel dsRNA-binding protein, which we named R3D1-L, that forms a stable complex with Dicer-1 in vitro and in vivo. While depletion of R3D1-L by RNAi causes accumulation of precursor miRNA (pre-miRNA) in S2 cells, recombinant R3D1-L enhances miRNA production by Dicer-1 in vitro. Furthermore, R3D1 deficiency causes miRNA-generating defect and severe sterility in male and female flies. Therefore, R3D1-L functions in concert with Dicer-1 in miRNA biogenesis and is required for reproductive development in Drosophila

Principles and effects of microRNA-mediated post-transcriptional gene regulation

“…In addition, orthologues of R2D2 are important for RNAi and miRNA processing in worms flies and mammals. The miRNA specific fly Dicer-1 requires Loquacious, for proper miRNA processing, Dicer in worms coimmunoprecipitates with RDE-4, a dsRNA-binding protein essential for RNAi initiated with long dsRNA, and human Dicer requires two R2D2 orthologues, TRBP and PACT, for miRNA-mediated gene silencing (Tabara et al, 2002;Forstemann et al, 2005;Haase et al, 2005;Jiang et al, 2005;Saito et al, 2005;Lee et al, 2006). Furthermore, Argonautes seem to interact physically with the miRNA maturation and loading machinery as immunoprecipitated hAgo2 complexes contain functional Dicer activity that can asymmetrically process exogenous pre-miRNAs, generating mature RISC that can cleave a perfect complementary target RNA Maniataki and Mourelatos, 2005;Meister et al, 2005).…”

Biochemical mechanisms of the RNA-induced silencing complex

“…Dicer-1 was required for processing precursor miRNA (pre-miRNA) to miRNA, and Dicer-2 was needed for the conversion of long dsRNA to siRNA [18,19]. Recombinant Drosophila Dicer-1 was found to preferentially process pre-miRNA, and Dicer-2 preferentially processed long dsRNA [20].…”

“…Sequence alignment and biochemical purification of miRNA-generating activity revealed a novel protein R3D1 [20,33,34]. This nomenclature derives from its three dsRNA-binding domains (R3) and its association with Dicer-1 (D1).…”

“…R3D1 is also known as loquacious as mutant flies were found to de-repress the expression of a miRNA-regulated reporter gene [33,34]. Genetic mutation and RNAi knockdown of R3D1 resulted in defective pre-miRNA processing in vitro and accumulation of endogenous pre-miRNAs [20,33,34]. Recombinant R3D1 facilitated Dicer-1-mediated miRNA production, possibly by enhancing the affinity of Dicer-1 for its substrate premiRNAs [20].…”

“…However, dsRBP-mediated enhancement of Dicer activity [20] and their requirement in partially reconstituted RISC systems [15,32] indicate that these components are important regulators of the catalytic RNAi machinery. …”