Regulation of microRNA degradation in Caenorhabditis elegans via the E3 ubiquitin ligase EBAX-1

Abstract

microRNAs (miRNAs) are short, ~22-nucleotide noncoding RNAs that base-pair to messenger RNAs (mRNAs) to direct their post-transcriptional repression through their associated Argonaute (AGO) proteins. Animal genomes encode hundreds of miRNAs that, together, regulate a majority of mRNAs and tune spatiotemporal gene expression programs. The production and degradation of many miRNAs occurs in a regulated manner, but molecular pathways of miRNA degradation are relatively poorly understood. Some rapidly degraded miRNAs owe their instability to a mechanism termed target-directed miRNA degradation (TDMD), whereby unusual miRNA binding sites with extensive complementarity to the miRNA promote a conformational shift in AGO, leading to the recruitment of an E3 ubiquitin ligase complex containing the substrate receptor ZSWIM8. The subsequent polyubiquitination and proteolysis of AGO liberates the miRNA, rendering it vulnerable to nucleases. TDMD underlies the instability of many miRNAs in diverse cell lines and animals. In this work, I probe the biological scope of TDMD as a regulatory mechanism in the nematode Caenorhabiditis elegans, which tolerates homozygous loss of the ZSWIM8 ortholog, EBAX-1, and expresses some miRNAs that are subject to rapid, developmentally regulated decay. I have confidently identified at least 22 miRNAs destabilized by EBAX-1 across the worm life cycle. These included the embryonic miR-35–42 family as well as certain stress-responsive miRNAs that together constitute some of the shortest-lived miRNAs in this organism. In mutants of ebax-1, the accumulated miR-35–42 excessively repressed predicted target mRNAs and underwent 3′ trimming as they aged, though no consistent signature of 3′ trimming or tailing emerged for EBAX-1-sensitive miRNAs. A recent study reports that the destabilization of miR-35 at the embryo-to-L1 transition does not depend on that miRNA’s 3′ region, unlike canonical mammalian TDMD. To test the generality of this result for other EBAX-1 sensitive miRNAs, I assayed the behavior of seed- or 3′-based miR-43 variants in the presence and absence of EBAX-1. Intriguingly, the miR-43 3′ variants showed substantially reduced propensity to be regulated by EBAX-1. The requirement for 3′ pairing therefore varies between EBAX-1 sensitive miRNAs, raising questions about the molecular features of TDMD trigger RNAs that recruit EBAX-1 when extensive pairing is not crucial.