GSE123478
Unbiased screen of RNA tailing enzymes reveals a poly(UG) polymerase required for genome integrity and RNA silencing
Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed a screening strategy in S. cerevisiae to identify sequences added by candidate enzymes from different organisms at single-nucleotide resolution. The rNTase activities of 19 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a C-adding enzyme that is likely part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, C. elegans MUT-2, which adds alternating U and G nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutations in the enzyme that are defective in silencing also fail to add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing. Overall design: We developed a screening approach to identify enzymes that add non-templated nucleotides to RNAs. Candidate rNTases were tethered in vivo to a reporter RNA in S. cerevisiae, and the number and identity of nucleotides they added were determined at single-nucleotide resolution using high-throughput sequencing.