Most micro-RNAs (miRNA) result from the processing of longer primary transcripts transcribed from miRNA genes by the RNA polymerase Pol II. These transcripts harbor hairpin structures which are recognized and processed twice to yield mature miRNAs: (1) once in the nucleus by Drosha, with the support of Pasha (PASH -1 in C. elegans) and (2) again in the cytoplasm by Dicer. The resulting ~22nt mature miRNAs are effective regulators of gene expression. When loaded onto the RNA-induced silencing complex (RISC), mature miRNAs direct RISC activity to transcripts with complementary sequence elements to repress their expression. In turn, their levels must also be regulated. Despite this, the processes responsible for miRNA turnover remain poorly understood. In instances where they are turned over, miRNAs are frequently modified by the addition of untemplated nucleotides to their 3' end, but the role of this tailing is often unclear. Here we characterized the prevalence and functional consequences of microRNA tailing in vivo, using the C. elegans model. Our data showed that miRNA tailing in C. elegans consists mostly of mono-uridylation of mature miRNA species, with rarer mono-adenylation - likely added to microRNA precursors. Through a targeted RNAi screen, we discovered that the TUT4/TUT7 gene family member CID-1 is required for uridylation, whereas the GLD2 gene family member F31C3.2 is required for adenylation. Thus, the TUT4/TUT7 and GLD2 gene families have broadly conserved roles in miRNA modification. We also examined the role of tailing in turnover in the absence of ongoing miRNA production, using a temperature sensitive
pash-1 strain. At permissive temperatures, PASH-1 is functional, and biogenesis is uninterrupted. When upshifted to 25°C, PASH-1 activity is abrogated, and mature miRNA production ceases. We determined the half-lives of miRNAs after acute inactivation of microRNA biogenesis, revealing that half-lives are generally long (median=20.7h), as observed in other systems. Despite an increased proportion of tailed species in older microRNAs, we detected no changes to microRNA abundance or decay dynamics upon disrupting tailing. Thus, tailing is not a global regulator of miRNA decay in C. elegans. Nonetheless, by identifying the responsible enzymes, this work lays the groundwork to explore whether tailing plays more specialized context- or miRNA-specific regulatory roles.