Small noncoding RNAs play diverse roles in the regulation of gene expression. In Caenorhabditis elegans, endogenous small-interfering RNAs known as 22G RNAs are synthesized from mRNA templates and form mRNA-targeting complexes that execute sequence-specific regulation of gene expression. 22G RNAs target thousands of mRNAs expressed in the germline of C. elegans, and their loss leads to impaired development of sperm and eggs. However, the full complement of upstream events that can trigger the biosynthesis of these 22G RNAs remains unclear. Our lab identified a locus (F43E2.6) at which the loss of the stop codon correlates with an increased production of 22G RNAs. The F43 mRNA 3'UTR has no further in frame stop codons, which introduces the possibility of a novel link between 22G RNAs and a quality control decay pathway characteristically triggered by a lack of a stop codon. mRNA quality control involves the recognition of aberrant mRNA transcripts followed by degradation via three decay pathways including nonsense-mediated decay. Previously, these decay pathways were defined as distinct from each other. However, a recent study demonstrated that nonsense-mediated decay products are directed into the nonstop decay pathway with subsequent mRNA degradation (Arribere & Fire, 2018). To determine whether nonsense alleles are associated with increased production and targeting by 22G RNAs, we have used next-generation sequencing to quantitate 22G RNA production from nonsense alleles of the germline-expressed gene
sel-1. We find that 22G RNA levels targeting
sel-1 in these strains are unchanged relative to the wild-type, in spite of a 3- to 14-fold reduction in
sel-1 mRNA levels in these strains. In order to compare 22G RNA levels under conditions where the concentration of the
sel-1 mRNA - the template for 22G RNA production - is expected to be comparable, we have used feeding RNAi to knock down expression of either
xrn-1, the exonuclease responsible for mRNA degradation in the 5' - 3' direction or
smg-2, an upstream protein critical to the recognition of premature termination codons. Sequencing of 22G RNAs from these strains is currently underway, and results of these experiments will be presented. Though small noncoding RNA molecules are implicated in mechanisms to defend the genome from invasive genetic elements, small RNAs may also play roles in protection against genetic errors in the form of low-quality mRNAs.