A Poly(UG) RNA Modifying Complex Mediates Viral Immunity in C. elegans

Abstract

RNA interference (RNAi) is a major component of innate immune systems in many eukaryotes, including C. elegans. During RNAi in C. elegans, dsRNA triggers the production of small RNAs via the nucleotidyltransferase RDE-3 which modifies the 3’ termini of mRNAs with polyUG (pUG) tails to generate RNA-dependent RNA Polymerase (RdRP) templates to drive gene silencing. During normal growth and development, RDE-3 pUGylates transposon RNAs to silence transposons and protect genomic integrity. How C. elegans identifies specific RNAs for pUGylation and whether the pUGylation system has tissue-specific functions is not known. In Chapter 2, I show that RNA pUGylation contributes to antiviral immunity in the soma of C. elegans. I present evidence that RDE-8, NYN-1/2, MUT-15, and the pUGylase RDE-3 form an RNA modifying complex that we term the pUGasome. During infection of C. elegans with Orsay virus, the pUGasome adds pUG tails to viral RNAs which convert these RNAs into RdRP templates for antiviral siRNA production, limiting viral replication. The data show that pUGylation contributes to antiviral immunity in C. elegans and suggest that RDE-3 is part of a four-protein complex, which provides molecular insights into how C. elegans identifies and neutralizes its internal and external parasitic threats in the soma. In Chapter 3, I show that piRNAs are necessary and sufficient for the production of the majority of endogenous pUG RNAs in C. elegans germ cells. Interestingly, piRNA-initiated pUG RNAs often possess short non-templated oligoA tails preceding their pUG tails, which I termed oligoA::pUG tails. I present evidence that the oligoA::pUG tailed RNAs are likely generated via piRNA-directed CCR4/NOT-based deadenylation of mRNAs followed by RDE-3-based pUGylation, suggesting that piRNA-directed mRNA deadenylation, which has been described previously in mice and flies, is conserved in C. elegans. Finally, in the appendix of the dissertation, I describe the discovery of another class of pUG RNA, termed the U(pUG) RNAs, which are produced during Orsay virus infection as the result of sequential terminal uridylation and then terminal pUGylation of full-length viral RNAs. In summary, the dissertation provides novel insights into mechanisms of tissue-specific RNA pUGylation, which protect C. elegans from internal and external nucleic acid parasites.