Nuclear RNAi Contributes to the Silencing of Off-Target Genes and Repetitive Sequences in Caenorhabditis elegans

Abstract

Small RNAs recognize, bind, and regulate other complementary cellular RNAs. The introduction of small RNAs to eukaryotic cells frequently results in unintended silencing of related, but not identical, RNAs: a process termed off-target gene silencing. Off-target gene silencing is one of the major concerns during the application of small RNA-based technologies for gene discovery and the treatment of human disease. Off-target gene silencing is commonly thought to be due to inherent biochemical limitations of the RNAi machinery. Here we show that following the introduction of exogenous sources of double-stranded RNA, the nuclear RNAi pathway, but not its cytoplasmic counterparts, is the primary source of off-target silencing in Caenorhabditis elegans. In addition, we show that during the normal course of growth and development the nuclear RNAi pathway regulates repetitive gene families. Therefore, we speculate that RNAi off-target effects might not be "mistakes" but rather an intentional and genetically programmed aspect of small RNA-mediated gene silencing, which might allow small RNAs to silence rapidly evolving parasitic nucleic acids. Finally, reducing off-target effects by manipulating the nuclear RNAi pathway in vivo might improve the efficacy of small RNAbased technologies.

GTSF ‐1 is required for formation of a functional RNA ‐dependent RNA Polymerase complex in Caenorhabditis elegans

“…Accordingly, we did not find strongly enriched gene ontology terms for the targets defined in the oxidized and TAP‐treated libraries (Table EV1). Furthermore, consistent with a role for GTSF‐1 upstream of NRDE‐3, we observed a significant overlap between GTSF‐1‐dependent sRNA targets and NRDE‐3 targets (Zhou et al , ; Fig EV4B).…”

“… AVenn diagram showing overlap between the 1% FDR GTSF‐1 targets defined in the three different NGS library preparation conditions. BVenn diagram depicting the overlap between the GTSF‐1 targets defined in the oxidized and TAP‐treated libraries in this study, and NRDE‐3 targets as defined elsewhere (Zhou et al , ). C–EExemplary UCSC genome browser tracks with RPM‐normalized sRNA coverage profiles. Three biological replicates of wild‐type N2 worms (Rep. 1–Rep.…”

GTSF-1 is required for the formation of a functional RNA-dependent RNA Polymerase complex in C. elegans

“…Accordingly, we did not find strongly enriched gene ontology terms for the targets defined in the oxidized and TAP-treated libraries (Table S1). Furthermore, consistent with a role for GTSF-1 upstream of NRDE-3, we observed a significant overlap between GTSF-1-dependent sRNA targets and NRDE-3 targets (Zhou et al, 2014) (Figure S4B).…”

The IDR-containing protein PID-2 affects Z granules and is required for piRNA-induced silencing in the embryo

“…The effect on 275 overall bulk 22G RNA levels was only minor ( Figure S2A). Also when we split the 22G RNAs into previously defined sub-categories (Almeida et al, 2019a;Bagijn et al, 2012;Conine et al, 2013;Gu et al, 2009;Phillips et al, 2014;Zhou et al, 2014), we only identified relatively small differences in all pathways analysed ( Figure S2C).…”