microRNAs are frequently modified by addition of untemplated nucleotides to the 3′ end, but the role of this tailing is often unclear. Here we characterize the prevalence and functional consequences of microRNA tailing in vivo, using Caenorhabditis elegans. MicroRNA tailing in C. elegans consists mostly of mono-uridylation of mature microRNA species, with rarer mono-adenylation which is likely added to microRNA precursors. Through a targeted RNAi screen, we discover that the TUT4/TUT7 gene family member CID-1/CDE-1/PUP-1 is required for uridylation, whereas the GLD2 gene family member F31C3.2—here named GLD-2-related 2 (GLDR-2)—is required for adenylation. Thus, the TUT4/TUT7 and GLD2 gene families have broadly conserved roles in miRNA modification. We specifically examine the role of tailing in microRNA turnover. We determine half-lives of microRNAs after acute inactivation of microRNA biogenesis, revealing that half-lives are generally long (median = 20.7 h), as observed in other systems. Although we observe that the proportion of tailed species increases over time after biogenesis, disrupting tailing does not alter microRNA decay. Thus, tailing is not a global regulator of decay in C. elegans. Nonetheless, by identifying the responsible enzymes, this study lays the groundwork to explore whether tailing plays more specialized context- or miRNA-specific regulatory roles.

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AbstractmicroRNAs are frequently modified by addition of untemplated nucleotides to the 3’ end, but the role of this tailing is often unclear. Here we characterize the prevalence and functional consequences of microRNA tailing in vivo, using the C. elegans model. MicroRNA tailing in C. elegans consists mostly of mono-uridylation of mature microRNA species, with rarer mono-adenylation which is likely added to microRNA precursors. Through a targeted RNAi screen, we discover that the TUT4/TUT7 gene family member CID-1 is required for uridylation, whereas the GLD2 gene family member F31C3.2 and, to a lesser extent, PAP-1 are required for adenylation. Thus, the TUT4/TUT7 and GLD2 gene families have broadly conserved roles in miRNA modification. We specifically examine the role of tailing in microRNA turnover. We determine half-lives of microRNAs after acute inactivation of microRNA biogenesis, revealing that half-lives are generally long (median=20.7h), as observed in other systems. Although we observe that tails are more prevalent on older microRNAs, disrupting tailing does not alter microRNA abundance or decay. Thus, tailing is not a global regulator of decay in C. elegans. Nonetheless, by identifying the responsible enzymes, this study lays the groundwork to explore whether tailing plays more specialized context- or miRNA-specific regulatory roles.

The germline sex determination pathway in C. elegans determines whether germ cells develop as oocytes or sperm, with no previously known effect on viability. The mir-35 family of microRNAs are expressed in the C. elegans germline and embryo and are essential for both viability and normal hermaphroditic sex determination, preventing aberrant male gene expression in XX hermaphrodite embryos. Here we show that combining feminizing mutations with partial loss of function of the mir-35 family results in enhanced penetrance embryonic lethality that preferentially kills XO animals. This lethal phenotype is due to altered signaling through the germline sex determination pathway, and maternal germline feminization is sufficient to induce enhanced lethality. These findings reveal a surprising pleiotropy of sperm-fate promoting pathways on organismal viability. Overall, our results demonstrate an unexpectedly strong link between sex determination and embryonic viability, and suggest that in wild type animals, mir-35 family members buffer against misregulation of pathways outside the sex determination program, allowing for clean sex reversal rather than deleterious effects of perturbing sex determination genes.