Screening by deep sequencing reveals mediators of microRNA tailing in C. elegans

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. To determine the role of microRNA tailing (i.e. untemplated addition of nucleotides to the 3' end), an RNAi screen was performed to identify enzymes required for microRNA tailing in C. elegans (screen group samples). To determine the role of tailing in rates of microRNA decay, decay rates were profiled in the presence of tailing (sample group 1), or in the context of disrupted tailing by knockdown of tailing enzymes CID-1 or F31C3.2/GLDR-2 (sample group 2). To further determine redundancy of CID-1 or F31C3.2/GLDR-2 with their paralogs, tailing was profiled in double mutant conditions for CID-1/PUP-1 and PUP-2 (sample group 3) or in F31C3.2/GLDR-2 mutant with GLD-2 RNAi (sample group 4). Additional experiments were performed, which were ultimately inconclusive due to technical limitations, to determine requirements for TDMD in C. elegans (sample group 5).