Abstract
Terminal nucleotidyltransferases add nucleotides to the 3' end of RNA to modify their stability and function. In Caenorhabditis elegans, the terminal uridyltransferases/poly(U) polymerases PUP-1 (aka CID-1, CDE-1), PUP-2, and PUP-3 affect germline identity, survival, and development. Here, we identify small RNA (sRNA) and mRNA targets of these PUPs and of a fourth predicted poly(U) polymerase, F43E2.1/PUP-4. Using genetic and RNA sequencing approaches, we identify RNA targets of each PUP and the U-tail frequency and length of those targets. At the whole organism level, PUP-1 is responsible for most sRNA U-tailing, and other PUPs contribute to modifying discrete subsets of sRNAs. Moreover, the expression of PUP-2, PUP-3, and especially PUP-4 limits uridylation on some sRNAs. The relationship between uridylation status and sRNA abundance suggests that U-tailing can have a negative or positive effect on abundance depending on context. sRNAs modified by PUP activity primarily target mRNAs that are ubiquitously expressed or most highly expressed in the germline. mRNA data obtained with a Nanopore-based method reveal that the addition of U-tails to nonadenylated mRNA is substantially reduced in the absence of PUP-3. Overall, this work identifies PUP RNA targets, defines the effect of uridylation loss on RNA abundance, and reveals the complexity of PUP regulation in C. elegans development.