Abstract
The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs (siRNAs) and catalyzes target mRNA cleavage during RNA interference (RNAi). Dicer-2, R2D2, Loquacious, and Argonaute-2 are examples of RISC-associated factors that are involved in RNAi. Holo-RISC is an approximately 80 S small interfering ribonucleoprotein, which suggests that there are many additional proteins that participate in the RNAi pathway. In this study, we used siRNA affinity capture combined with mass spectrometry to identify novel components of the Drosophila RNAi machinery. Our study identified both established RISC components and novel siRNA-associated factors, many of which contain domains that are consistent with potential roles in RNAi. Functional analysis of these novel siRNA-associated proteins suggests that these factors may play an important role in RNAi.
Highlights
The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs and catalyzes target mRNA cleavage during RNA interference (RNAi)
SiRNA Affinity Capture of Drosophila RISC—Previous studies have used a model small interfering RNAs (siRNAs) directed against firefly luciferase (Pp-luc) to direct efficient cleavage of a cognate mRNA target in vitro [21, 36]
Some proteins could conceivably co-purify based on their association with RISC-bound mRNAs that are present in the extract, but this potential problem is minimized by the use of distinct siRNAs (including one (Pp-luc) that lacks any natural mRNA targets in Drosophila) that would likely associate with different populations of mRNAs
Summary
The Drosophila melanogaster RNA-induced silencing complex (RISC) forms a large ribonucleoprotein particle on small interfering RNAs (siRNAs) and catalyzes target mRNA cleavage during RNA interference (RNAi). These siRNAs form an initiator complex with Dicer-2 and the dsRNA-binding domain (dsRBD)-containing protein R2D2 (19 –23). These observations indicate that additional protein factors associate with siRNAs. In this study, we identified siRNA-binding proteins from Drosophila embryo extracts.
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