Molecular Requirements for RNA-induced Silencing Complex Assembly in the Drosophila RNA Interference Pathway

John W Pham,Erik J Sontheimer

doi:10.1074/jbc.m509202200

John W Pham, Erik J Sontheimer

Open Access

https://doi.org/10.1074/jbc.m509202200

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Abstract

Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). Here we show that R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer alone can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute 2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading complex, which also requires Argonaute 2 for assembly into RISC. We have compared R2 to the RISC-loading complex and show that the two complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. We have examined the requirements for RISC formation and show that the siRNA 5'-termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2'-position of the 5'-terminal nucleotide also affects RISC assembly, because an siRNA strand bearing a 2'-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2'-deoxyribose-modified strand has enhanced activity in the RNA interference pathway.

Highlights

The short interfering RNA (siRNA) produced by Dicer may determine the outcome of this transition
Dcr-2 and R2D2 Comprise R1 and Can Initiate RNA-induced silencing complex (RISC) Assembly— RISC assembly in Drosophila proceeds in a stepwise manner and is first detected when proteins bind siRNA within the R1 complex [14]
These results indicate that R1 is not likely to contain any additional factors, so we refer to it as the R2D2/Dcr-2 initiator (RDI) complex to distinguish it from the unbound Dcr-2/R2D2 heterodimer

Summary

Introduction

The siRNAs produced by Dicer may determine the outcome of this transition. Newly generated siRNAs have characteristic 5Ј-phosphoryl-. When synthetic siRNAs are used to initiate RNAi, the siRNA strand whose 5Ј-end is less stably base-paired is more frequently incorporated into the RISC [17, 18], whereas the cognate strand is more frequently degraded. R1 is an ATP-independent complex that cannot form on siRNAs that lack 5Ј-phosphorylated ends. It contains at least two protein factors: Dicer-2 (Dcr-2), the Drosophila enzyme responsible for generating siRNAs, and presumably R2D2, a Dcr-2-associated factor that is a key player in bridging the initiation and effector phases of RNAi [19]. The siRNA in R2 is unwound, leading to the formation of a large, ATP-dependent, ϳ80 S complex known as holoRISC (formerly R3) This complex can cleave targeted mRNAs, likely acting as the RNAi effector in Drosophila [14]. We have defined in greater detail the molecular events of the assembly pathway, providing a model that includes multiple 5Ј-recognition events and reconciles the reported differences in the R2 and RLC complexes

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