Poly(U) polymerases (PUPs) add uridine to the 3’end of RNAs, a modification that correlates with reduced RNA stability in many organisms. Targets of uridylation include mRNAs and small RNAs, including small-interfering (si) RNAs and unmethylated micro (mi) RNAs. Despite the prevalence of uridylation, our knowledge of its developmental importance is limited. Three members of the PUP family have been identified in Caenorhabditis elegans [1]. Two have been studied in some detail. PUP-1/CDE-1 (co-suppression defective) targets a subset of siRNAs and is expressed in the germ line [2]; PUP-2 activity regulates a subset of miRNAs that act in the soma [3, 4]. Despite these distinct functions, we find that PUP-1/CDE-1 and PUP-2 are required redundantly in meiotic germ cells. We used the CRISPR-Cas9 system to generate a double knockout of pup-1/cde-1 and pup-2 [pup-1(0) pup-2(0)] and then compared its phenotype to those of pup-1(0) and pup-2(0) single mutants. The single mutants have numerous defects that are present additively in the double mutant. In addition, we observe unique phenotypes in the double mutant. Under conditions of temperature stress, pup-1(0) pup-2(0) hermaphrodites produce progressively smaller numbers of progeny and become sterile within three generations. The majority (~93%) of those sterile adults contain no germ cells. In addition, meiotic chromatin regulation is abnormal: in pup-1(0) pup-2(0) males, the histone H3 lysine 9 dimethylation (H3K9me2) modification is elevated from pachytene stage of first meiotic prophase until late spermiogenesis compared with controls. In keeping with delayed H3K9me2 removal, developing pup-1(0) pup-2(0) sperm progress through spermiogenesis relatively slowly compared with controls. Small RNAs are known to impact both germline survival and H3K9me2 regulation. We observe PUP-2 protein expression in germ line cytoplasm, particularly in developing oocytes. Taken together, our findings suggest that PUP-1 and PUP-2 function redundantly to ensure germline survival and chromatin regulation, perhaps by modulating small RNA levels. We are working to distinguish between alternative hypotheses for how PUP-1 and PUP-2 activities promote germline survival and chromatin regulation.
[1] Kwak & Wickens (2007) RNA 13: 860-867. [2] van Wolfswinkel et al. (2009) Cell 139: 135-148. [3] Lehrbach et al. (2009) Nat Struct Mol Biol 16: 1016-1020. [4] Ha & Kim (2014) Nat Rev Mol Cell Biol 15: 509-524.