Functional analysis of dicer-2 missense mutations in the siRNA pathway of Drosophila

Abstract

The Drosophila RNase III enzyme Dicer-2 processes double-stranded RNA (dsRNA) precursors into small interfering RNAs (siRNAs). It also interacts with the siRNA product and R2D2 protein to facilitate the assembly of an RNA-induced silencing complex (RISC) that mediates RNA interference. Here, we characterized six independent missense mutations in the dicer-2 gene. Four mutations (P8S, L188F, R269W, and P365L) in the DExH helicase domain reduced dsRNA processing activity. Two mutations were located within an RNase III domain. P1496L caused a loss of dsRNA processing activity comparable to a null dicer-2 mutation. A1453T strongly reduced both dsRNA processing and RISC activity, and decreased the levels of Dicer-2 and R2D2 proteins, suggesting that this mutation destabilizes Dicer-2. We also found that the carboxylterminal region of R2D2 is essential for Dicer-2 binding. These results provide further insight into the structure-function relationship of Dicer, which plays a critical role in the siRNA pathway. KeywordsRNA interference; siRNA; Drosophila; Mutation; Dicer-2; R2D2 RNA interference (RNAi) is a biological process in which double-stranded RNA (dsRNA) triggers gene silencing in a sequence-specific manner [1]. RNAi can be divided into two phases. During the initiation phase, dsRNA is processed by a multidomain RNase III enzyme called Dicer, into 21-23 nucleotide (nt) siRNA duplexes. In Drosophila, Dicer-2 (Dcr-2) is responsible for generating siRNAs [2,3] and is present as a complex with the dsRNA-binding protein R2D2, which does not regulate the dsRNA processing activity of Dcr-2 [4].During the effector phase, the siRNA product assembles into an RNA-induced silencing complex (RISC) via an ordered pathway by which one of the two RNA strands is selectively retained to direct RISC-mediated mRNA cleavage and degradation [5]. In Drosophila, RISC

Loqs-PD and R2D2 define independent pathways for RISC generation in Drosophila

“…Binding of Loqs-PD and R2D2 could be mutually exclusive if the same binding site is used; alternatively, Loqs-PD and R2D2 could simultaneously bind Dcr-2 but induce different conformations. Reduced co-precipitation of both Loqs-PD and R2D2 with the Δhel-Flag-Dcr-2 construct (Figure 2B) substantiates the competition hypothesis as both proteins seem to interact with the helicase region of Dcr-2 (24,42). On the other hand, a recent finding by the Siomi lab (21) reported association of R2D2 with the Loqs-PD/Dcr-2 complex upon overexpression of Loqs-PD.…”

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

“…A similar strategy was previously used successfully to identify genes involved in RNAi in Drosophila Kim et al 2007;Lim et al 2008;Lee et al 2009;Marques et al 2010). To begin, we reprogrammed the mir-6-1 backbone (Chen et al 2007) to generate an miRNA that targets a single location in the endogenous 39 UTR of the white gene (w-miR) (Fig.…”

“…The paucity of mutants limits the flexibility of genetic analysis for miRNA pathway function, especially in ways that would otherwise be facilitated by the availability of allelic series. For example, an extensive collection of dicer-2 alleles includes mutants that permit genetic separation of the role of this enzyme for siRNA genesis from its role in siRNA loading into AGO2 complex Tomari et al 2004;Lim et al 2008).…”

‘Black sheep’ that don’t leave the double-stranded RNA-binding domain fold

“…We discovered DHX30 dsRBD2 by searching the poorest of dsRBD profile hits in Prosite followed by secondary structure predictions (JPred3), suggesting that other dsRBD-like folds are also hidden within known protein sequences. For example, the C-terminal region of D. melanogaster R2D2, which interacts with the helicase domain of Dicer-2 [90] analogously to how D. melanogaster Loqs interacts with the helicase domain of Dicer-1 [100], contains a predicted (MeDor) ~60-amino acid α 1 -β 1 -β 2 -β 3 -α 2 topology. No one to our knowledge has interpreted this predicted secondary structure as a dsRBD, and it is not identified as such using low-fidelity Prosite parameters that were able to define STAU1 dsRBD5 (Table 1).…”