Yates III, John R., Chen, Chun-Chieh G., Gu, Weifeng, Tsai, Hsin-Yue, Moresco, James J., Mello, Craig
[
International Worm Meeting,
2009]
Several classes of small RNAs have been identified in mammals, zebrafish, Drosophila, and the nematode C. elegans. These include Dicer-dependent products (primary siRNAs and miRNAs) as well as secondary siRNAs produced by RNA-dependent RNA polymerase (RdRP). We have recently identified a new RNAi pathway component, RDE-8, which is essential for the accumulation of RdRP-derived small RNAs in both the exo- and endo- RNAi pathways. RDE-8 contains a novel protein motif that is highly conserved throughout all three kingdoms of life. Interestingly, in other organisms the majority of proteins containing the RDE-8 motif also contain RNA binding domains, most often of the CCCH zinc finger or KH domain families. Although RDE-8 itself is not highly conserved, one C. elegans RDE-8 family member (C30F12.1) has a human homolog in which both the amino acid composition and organization of its CCCH and RDE-8 motifs are well conserved. A third C. elegans RDE-8-motif protein also contains a Cytidine deaminase motif and is required for male fertility (See abstract by Colin Conine). These findings suggest that proteins containing the RDE-8 motif function in RNA binding and modification. To explore the role of RDE-8 in small-RNA pathways, we have utilized mass-spectrometry-based proteomics to identify protein interactors, and small-RNA deep sequencing to identify endogenous small RNAs whose biogenesis/stability depend on RDE-8. Our data suggests that RDE-8 is required for several distinct small-RNA pathways and resides in at least two protein complexes. RDE-8 interacts with the sap-domain exonuclease ERI-1b, and is required for the accumulation of ERI-pathway small RNAs. Interestingly, RDE-8 not only co-purifies with ERI-1b, but is required for a ssRNA trimming activity associated with the native ERI-1b/RDE-8 complex. We speculate that this trimming activity is necessary for the processing of substrates (or products) of the RdRP RRF-3. RDE-8 appears to form a second complex with components of the RNAi and transposon silencing small RNA pathway, including RDE-3, MUT-15, and MUT-16. We have not yet identified specific biochemical activities associated with this complex. However, it may also be involved in recruiting RdRPs: in this case, RRF-1 and EGO-1, which function to amplify silencing signals in the exo- and endo-RNAi pathways. We are currently investigating the biochemical properties of the RDE-8 complexes, and are exploring the activity of the RDE-8 motif.