The miR-43 3′ region contributes to EBAX sensitivity. (A) Sequence alignment of the four members of the C. elegans miR-2 family. (B) EBAX-1 sensitivity of the miR-2 family members. (C) Schematic of the miR-42–44 cluster, indicating Cas9 cut sites (red lines), and repair template boundaries (blue lines) used for in vivo genome editing of this locus. (D) miR-43 variants generated through genome editing. Seed nucleotides are in bold, substitutions are in magenta. (E) The effect of EBAX-1 loss on the abundance of the miR-43 variants shown in (D) at the L4 stage. Points show fold change upon EBAX-1 loss in each miR-43 guide-strand variant (green), each miR-43 passenger-strand variant (blue), or each miR-44 passenger strand (purple) in wild-type worms or worms carrying the indicated mir-43 mutations. Plotted are values for each replicate, with horizontal lines indicating mean values of replicates. To assess statistical significance, we performed a two-way ANOVA followed by Dunnett's multiple comparisons test. n.s., not significant. (*) P < 0.05. (***) P < 0.0001. For one of three replicates in worms without miR-43 mutations, the fold change in miR-43, miR-43*, and miR-44* represents the mean of L4-stage data in the sRNA-seq experiment shown in Figure 1 and Supplemental Table S1. (F) Molecular models for EBAX-1–mediated miRNA destabilization. In the canonical model of TDMD (top), which is consistent with observations in mammalian and insect cells, extensive pairing between the miRNA 3′ region and a trigger RNA (either an mRNA or ncRNA) causes a conformational change in AGO, leading to the recruitment of EBAX-1 and subsequent polyubiquitination of AGO. In an alternative model (bottom), which is consistent with observations for miR-35–42 in C. elegans, EBAX-1 is recruited to the trigger RNA by an RNA cis-acting element, either directly or with the help of an adapter protein. In this alternative model, either strong seed pairing or weak seed pairing supplemented by pairing to the miRNA 3′ region recruits the trigger, thereby recruiting EBAX-1 to the vicinity of AGO-like proteins.
