Genetic dissection of endogenous siRNA involvement in the nonstop decay pathway in C. elegans

Abstract

In Caenorhabditis elegans, endogenous small interfering RNAs known as 22G RNAs regulate expression of thousands of mRNAs and other transcripts in developing sperm and egg cells, and loss of 22G RNAs leads to reduced fertility. We are working to understand the molecular triggers that control which genes are targeted for regulation by 22G RNAs. Previous research in the Youngman lab identified a gene of unknown function, F43E2.6 (F43) that is targeted by 22G RNAs only in polymorphic wild strains of worms that lack a stop codon at F43. Conversely, when the stop codon is present, 22G RNA production is low. While it is well documented that mRNAs without a stop codon enter the nonstop decay pathway, resulting in destruction of the readthrough transcript, nonstop decay has not previously been linked to small silencing RNA pathways.In addition to 22G RNAs, the F43 transcript is targeted by a Dicer product, and RDE‐1‐bound Dicer products trigger 22G RNA synthesis in the classical exogenous RNAi pathway. This leads to a critical question about how 22G RNA production is triggered at this locus: is it caused by the nonstop polymorphism, targeting by the Dicer product, or by both pathways acting in cooperation? Further, how does this novel biogenesis pathway integrate into known pathways for 22G RNA production in the C. elegans germline? To determine whether production of 22G RNAs at the F43 locus is independent of the Dicer product and dependent solely on the nonstop polymorphism, we first generated rde‐1; F43 nonstop(ns) homozygotes, which carry the nonstop allele in absence of the Dicer‐dependent RNAi pathway, and have completed deep sequencing of 22G RNAs from this strain. To further define the genetic requirements for production of 22G RNAs targeting this locus, we have sequenced 22G RNAs from strains that carry the nonstop polymorphism but lack either portions of the small RNA biogenesis machinery or of the known nonstop decay machinery (mut‐7; F43ns, prg‐1; F43ns, skih‐2; F43ns, and dom‐34; F43ns). Bioinformatic analyses to assess small RNA levels targeting F43 are underway and will be presented. Although it is well established that 22G RNAs serve to defend the genome against foreign or invasive nucleic acids, this work represents the first evidence that they may also serve to defend the transcriptome from the deleterious effects of low‐quality mRNAs, and suggests the intriguing possibility that quality control of mRNAs may play a role in animal fertility.Support or Funding InformationNSF/MRI #1531855This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.