poly(UG)-tailed RNAs in Genome Protection and Epigenetic Inheritance [other]

Mobile genetic elements threaten genome integrity in all organisms. MUT-2/RDE-3 is a ribonucleotidyltransferase required for transposon silencing and RNA interference (RNAi) in C. elegans. When tethered to RNAs in heterologous expression systems, RDE-3 can add long stretches of alternating non-templated uridine (U) and guanosine (G) ribonucleotides to the 3' termini of these RNAs (polyUG or pUG tails). Here, we show that, in its natural context in C. elegans, RDE-3 adds pUG tails to transposon RNAs, as well as to targets of RNAi. pUG tails with more than 16 perfectly alternating 3' U and G nucleotides convert RNA fragments into agents of gene silencing. pUG tails promote gene silencing by recruiting RNA-dependent RNA Polymerases (RdRPs), which use pUG-tailed RNAs (pUG RNAs) as templates to synthesize small interfering RNAs (siRNAs). Our results show that cycles of pUG RNA-templated siRNA synthesis and siRNA-directed mRNA pUGylation underlie dsRNA-directed transgenerational epigenetic inheritance in the C. elegans germline. We speculate that this pUG RNA/siRNA silencing loop allows parents to inoculate progeny against the expression of unwanted or parasitic genetic elements. Overall design: oma-1 pUG RNAs were sequenced in WT (N2) vs. rde-3(ne3370) mutant (YY1449) adult animals using an Illumina MiSeq. Animals were fed either an empty vector control (L4440) or dsRNA targeting the gene oma-1 (Ahringer library clone or custom clone pAS74, which targets exon 6 of oma-1). oma-1 pUG RNAs from F1-F4 descendants of pAS74-fed animals were also sequenced.