C. elegans ADARs antagonize silencing of cellular dsRNAs by the antiviral RNAi pathway

Abstract

Cellular dsRNAs are edited by adenosine deaminases that act on RNA (ADARs). While editing can alter mRNA-coding potential, most editing occurs in noncoding sequences, the function of which is poorly understood. Using dsRNA immunoprecipitation (dsRIP) and RNA sequencing (RNA-seq), we identified 1523 regions of clustered A-to-I editing, termed editing-enriched regions (EERs), in four stages of development, often with highest expression in embryos. Analyses of small RNA-seq data revealed 22- to 23-nucleotide (nt) siRNAs, reminiscent of viral siRNAs, that mapped to EERs and were abundant in mutant animals. Consistent with roles for these siRNAs in silencing, EER-associated genes (EAGs) were down-regulated in embryos, and this was dependent on associated EERs and the RNAi factor RDE-4. We observed that ADARs genetically interact with the 26G endogenous siRNA (endo-siRNA) pathway, which likely competes for RNAi components; deletion of factors required for this pathway ( or ) in mutant strains caused a synthetic phenotype that was rescued by deleting antiviral RNAi factors. Poly(A) RNA-seq revealed EAG down-regulation and antiviral gene induction in embryos, and these expression changes were dependent on and Our data suggest that ADARs restrict antiviral silencing of cellular dsRNAs.

DAF-16/Foxo suppresses the transgenerational sterility ofprg-1piRNA mutants via a systemic small RNA pathway

“…In contrast, loss of the dsRNA degrading adenosine deaminases adr-1 and adr-2 results to high levels of endogenous dsRNAs that, in rrf-3 or eri-1 mutant backgrounds that eliminate the endogenous 26G RNA pathway and nuclear NRDE-3 (46), are converted into siRNAs that promote strong nuclear localization of NRDE-3 ( Fig. 7B) (59). Nuclear NRDE-3 can be similarly induced in response to exogenous dsRNA triggers (46).…”

“…The requirement for DCR-1 and RDE-4 in the suppression of sterility in prg-1; daf-2 double mutants suggests the involvement of a dsRNA intermediate, because RDE-4 interacts with its targets via a dsRNA interaction domain (57). Adenosine deaminases modify double stranded RNAs by converting adenine to inosine, which results in dsRNA destruction (58) and therefore precludes dsRNA processing into 1 o siRNAs by RDE-4/DCR-1 and subsequent creation of 2 o 22G RNAs (59). To investigate potential daf-2-dependent changes in dsRNA processing, we re-analysed previously published daf-2 RNA-seq data (60) and lists of previously defined daf-16dependent loci based on RNA microarrays (61).…”

“…To investigate potential daf-2-dependent changes in dsRNA processing, we re-analysed previously published daf-2 RNA-seq data (60) and lists of previously defined daf-16dependent loci based on RNA microarrays (61). Loss of adenosine deaminase activity results in targeting of endogenous dsRNA by the RNAi machinery (59), which is one possible mechanism by which loss of daf-2 could drive changes in dsRNA processing. However, we found that the C. elegans adenosine deaminases adr-1 and adr-2 were unchanged in daf-2 mutants when compared to wildtype (S3 Table), indicating that transcriptional repression of these genes is not responsible for altered dsRNA metabolism in daf-2 mutants.…”

“…6A). Most dsRNAs were found to occur in introns of protein-coding genes (59). The observed differences in siRNA abundance upon loss of daf-2 could lead to alterations in the expression of the respective genes.…”

Disruption in A-to-I editing levels affects C. elegans development more than a complete lack of editing

“…We previously suggested that downregulation of genes in ADAR mutants could be a consequence of the antagonistic relationship between RNA editing and RNAi (Goldstein et al, 2017). The possibility that ADAR genes and RNA editing itself are antagonistic to the RNAi process was raised previously as editing and RNAi both involve dsRNA substrates, transgene silencing was observed in ADAR double mutants, and changes in the amount of siRNAs generated in wild-type worms and those lacking both ADARs (Goldstein et al, 2017, Knight and Bass, 2002, Reich et al, 2018, Tonkin and Bass, 2003, Warf et al, 2012, Whipple et al, 2015, Wu et al, 2011. Hypersensitivity to exogenous RNAi is a phenotype related to the ERI/RRF-3 endogenous RNAi pathway (Simmer et al, 2002), which is also reflected by transgene silencing.…”