Widespread destabilization of C. elegans microRNAs by the E3 ubiquitin ligase EBAX-1 [miRNA-Seq]

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to form a complex that directs mRNA repression. One way that miRNAs are regulated is at the level of their stability. Some animal miRNAs are destabilized through a pathway in which pairing to specialized RNA transcripts recruits the ZSWIM8 E3 ubiquitin ligase, which polyubiquitinates AGO, leading to its degradation and exposure of the miRNA to cellular nucleases. We found that 22 miRNAs in C. elegans are sensitive to loss of EBAX-1, the ZSWIM8 ortholog in nematodes, implying that these 22 miRNAs might be subject to this pathway of target-directed miRNA degradation (TDMD). These miRNAs included the embryonic miR-35–42 family, as well as other miRNAs among the least stable in the worm, suggesting that TDMD is a major pathway of miRNA destabilization in the worm. The excess miR-35–42 molecules that accumulated in ebax-1 mutants caused increased repression of their predicted target mRNAs and underwent 3' trimming over time. In general, however, miRNAs sensitive to loss of EBAX-1 did not have a consistent pattern of either trimming or tailing. A previous study reports that in contrast to TDMD substrates in mammals and flies, the 3' region of miR-35 is not required for its destabilization, raising the question of whether the 3' regions of other miRNAs are dispensable for EBAX-1 sensitivity in nematodes. To address this question, we replaced the 3' region of another EBAX-sensitive miRNA, miR-43, and found that this replacement greatly reduced EBAX-sensitivity in the worm. Together, these findings broaden the implied biological scope of TDMD-like regulation of miRNA stability in animals, and indicate that the requirement for miRNA 3' sequences in this process is variable in the worm. Overall design: To profile microRNAs destabilized by EBAX-1, we sequenced small RNAs from synchronized wild-type and ebax-1 mutant worms at eight different developmental time points. For mutagenesis studies of miR-43, we sequenced small RNAs from L4 larvae with or without miR-43 mutations and/or ebax-1 mutations in triplicate.