Diversification of small RNA pathways underlies germline RNAi incompetence in wild C. elegans strains

Abstract

The discovery that experimental delivery of dsRNA can induce gene silencing at target genes revolutionized genetics research, by both uncovering essential biological processes and creating new tools for developmental geneticists. However, wild-type C. elegans strains vary dramatically in their response to exogenous RNAi, challenging our characterization of RNAi in the lab relative to its activity and significance in nature. Here, we investigate why some strains fail to mount a robust RNAi response to germline targets. We observe diversity in mechanism: in some strains, the response is stochastic, either on or off among individuals, while in others the response is consistent but delayed. Increased activity of the Argonaute PPW-1, which is required for germline RNAi in the laboratory strain N2, rescues the response in some strains, but dampens it further in others. Across strains, we observe variability in expression of known RNAi genes and strain-specific instances of pseudogenization and allelic divergence. Our results support the conclusions that Argonautes share overlapping functions, that germline RNAi incompetence is strain-specific but likely caused by genetic variants at common genes, and that RNAi pathways are evolving rapidly and dynamically. This work expands our understanding of RNAi by identifying conserved and variable pathway components, and it offers new access into characterizing gene function, identifying pathway interactions, and elucidating the biological significance of RNAi.

Beyond the reference: gene expression variation and transcriptional response to RNAi inC. elegans

“…However, overlap among strains was sparse (Figure S6): no genes with differential expression to both par-1 and pos-1 RNAi were shared across all five strains, and the only gene responsive to both treatments in the competent strains (JU1088, N2, and EG4348) was asp-14, a predicted aspartyl protease involved in innate immunity (Harris et al, 2020). Such strain-specific patterns fit with our observations of RNAi variability: not only does C. elegans exhibit substantial natural variation in germline RNAi competence (Elvin et al, 2011;Felix, 2008;Felix et al, 2011;Paaby et al, 2015;Tijsterman et al, 2002), but the genetic basis for RNAi failure appears strain-specific as well (Chou et al, 2022). We posit that even among competent strains, C. elegans varies in details of the RNAi biological response mechanism, including which genes are affected, the magnitude or functionality of their activity, and their timing.…”

“…This majority represents a slight enrichment relative to the proportion of missing or low coverage genes within the complete set of 'off' genes (286/411 or 70%) (onesided proportion test with continuity correction: c 2 = 3.05, df = 1, p = 0.04). Enrichment of genome divergence among RNAi-responsive 'off' genes supports the hypothesis that genes associated with RNAi are evolving rapidly in C. elegans (Chou et al, 2022). By adding the missing and low DNA coverage filters, we infer that, of genes with an RNAi effect in another strain, zero (in N2) to 12 (in QX1211) were missing from the strain's genome and 1-6 genes per strain were present but truly unexpressed at the RNA level.…”

“…As previously described (Chou et al, 2022), we used SNPs and INDELs from CeNDR (release 20210121) (Cook et al, 2017) to update the N2 reference genome (release ws276) (Harris et al, 2020) to generate strain-specific transcriptomes using the software g2gtools (v0.1.31 via conda v4.7.12,Python v2.7.16) (https://github.com/churchill-lab/g2gtools). Specifically, INDELS were added to the reference genome with g2gtools vcf2chain and SNPs with g2gtools patch.…”

“…RNAi was discovered in C. elegans (Fire et al, 1998), but competency in response to environmental triggers is highly variable across wild C. elegans strains (Elvin et al, 2011; Felix, 2008; Felix et al, 2011; Paaby et al, 2015; Tijsterman et al, 2002). Previous work showed that a loss-of-function mutation in Argonaute RNAi effector gene ppw-1 is largely responsible for the near-complete failure of Hawaiian strain CB4856 to mount an RNAi response against germline targets (Tijsterman et al, 2002), and later work characterized the failure in CB4856 as a much delayed, rather than absent, response (Chou et al, 2022). Other strains incompetent for germline RNAi exhibit distinct modes of RNAi failure with distinct genetic bases (Chou et al, 2022; Elvin et al, 2011; Pollard & Rockman, 2013).…”

“…RNAi(Paaby et al 2015, Chou et al 2022: JU1088 (highly competent), EG4348 (moderately competent), and CB4856 and QX1211 (largely incompetent). These wild strains also vary in divergence from N2, representing some of the least (JU1088) and most (QX1211) divergent strains (variants per kilobase vs. N2 genome: 0.82, 1.40, 1.99, and 4.20, respectively, from Caenorhabditis elegans Natural Diversity Resource [CeNDR] data(Cook et al, 2017)).…”