RDE-2 interacts with MUT-7 to mediate RNA interference in Caenorhabditis elegans

Abstract

In Caenorhabditis elegans, the activity of transposable elements is repressed in the germline. One of the mechanisms involved in this repression is RNA interference (RNAi), a process in which dsRNA targets cleavage of mRNAs in a sequence-specific manner. The first gene found to be involved in RNAi and transposon silencing in C.elegans is mut-7, a gene encoding a putative exoribonuclease. Here, we show that the MUT-7 protein resides in complexes of ∼250 kDa in the nucleus and in the cytosol. In addition, we find that upon triggering of RNAi the cytosolic MUT-7 complex increases in size. This increase is independent of the presence of target RNA, but does depend on the presence of RDE-1 and RDE-4, two proteins involved in small interfering RNA (siRNA) production. Finally, using a yeast two-hybrid screen, we identified RDE-2/MUT-8 as one of the other components of this complex. This protein is encoded by the rde-2/mut-8 locus, previously implicated in RNAi and transposon silencing. Using genetic complementation analysis, we show that the interaction between these two proteins is required for efficient RNAi in vivo. Together these data support a role for the MUT-7/RDE-2 complex downstream of siRNA formation, but upstream of siRNA mediated target RNA recognition, possibly indicating a role in the siRNA amplification step.

ACaenorhabditis elegansRNA-Directed RNA Polymerase in Sperm Development and Endogenous RNA Interference

“…An important result of this analysis was that RRF-3 had little or no effect on gene-specific siRNA counts for the majority of the 4723 genes with spermatogenesis siRNAs (Figure 8): ,4% of the genes had reproducible, greater-than-twofold decreases in siRNA levels in rrf-3(pk1426) relative to control him-8(e1489) males (69 of 1784 genes; to exclude background noise from genes with low siRNA alignment counts, only genes with counts of at least 25 in both sets of control males were included). This result is consistent with the existence of many other RNAi-related factors with substantial germline phenotypes (Cox et al 1998;Smardon et al 2000;Knight and Bass 2001;Tops et al 2005;Chen et al 2007).…”

Interacting endogenous and exogenous RNAi pathways in Caenorhabditis elegans

“…We show that both protein-coding and noncoding sequences are involved in ongoing RNAi in C. elegans and that the mRNA levels of many diverse protein-coding genes of C. elegans are affected, in some cases profoundly, by endog- a Previously published roles in silencing phenomena in C. elegans (Ketting et al 1999;Tabara et al 1999;Dernburg et al 2000;Grishok et al 2000;Ketting and Plasterk 2000;Bernstein et al 2001;Grishok et al 2001;Ketting et al 2001;Timmons et al 2001;Simmer et al 2002;Tabara et al 2002;Tijsterman et al 2002a;Kennedy et al 2004;Chen et al 2005;Tops et al 2005 Measurement shown is for tncR35; similar results were obtained for tncR36 and for tncR4 (except that tncR36 and tncR4 were not assayed in eri-1). e Measurement shown is for mir-238; similar results were obtained for mir-38, mir-42, mir-52, mir-58, mir-71, mir-79, and mir-229 (except that only mir-238 was assayed in eri-1 total RNA).…”

“…In other animals, the same Argonaute can function with both siRNAs and microRNAs (Williams and Rubin 2002;Meister et al 2004;Okamura et al 2004). Other proteins associated with the generation of siRNAs in C. elegans (Table 1) include RdRp, a nucleotidyltranferase b family protein (RDE-3), and various helicases and RNA-binding proteins (Ketting et al 1999;Tabara et al 1999;Fagard et al 2000;Smardon et al 2000;Sijen et al 2001;Morel et al 2002;Simmer et al 2002;Tabara et al 2002;Tijsterman et al 2002a;Tops et al 2005).…”

TheCaenorhabditis elegans rsd-2andrsd-6Genes Are Required for Chromosome Functions During Exposure to Unfavorable Environments

“…Another common feature of these five genes is that they are required for RNAi in the intestine and germline, as revealed when dsRNA is delivered by feeding. While it is known that RSD-2 and RSD-6 proteins can physically interact (Tijsterman et al 2004), that RDE-2 and MUT-7 proteins interact (Tops et al 2005), and that rde-2, mut-7, and haf-6 mutants genetically interact (Sundaram et al 2008), the precise function of these genes in RNAi remains to be elucidated. One common feature for all the mutants is a temperaturesensitive response to ingestion of unc-22 food; this phe- notype may serve as a hallmark for genes with similar functions in RNAi.…”

“…Proteins with Tudor domains 1 localize to diverse intracellular regions; for example, the proteins have been variously associated with mitochondria, nuclei, and kinetochores (Bardsley et al 1993;Amikura et al 2001;Hiyoshi et al 2005). RSD-2 is an unfamiliar protein that physically associates with RSD-6 (Tops et al 2005).…”