Unbiased screen of RNA tailing enzymes at single-nucleotide resolution reveals a poly(UG) polymerase required for genome integrity and RNA silencing

Abstract

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.

Unbiased screen of RNA tailing activities reveals a poly(UG) polymerase

“…Both enzymes are essential in S. pombe 35 and members of the CCA-adding enzyme subfamily. We propose this is the first identified dual-enzyme CCA-addition system in a eukaryote; our data are supported by a recent report 36,37 .…”

“…We propose that Sp SPAC1093.04 and Sp SPCC645.10 constitute a two-enzyme system that catalyzes CCA addition to tRNAs in S. pombe . This is strongly suggested by their nucleotide specificities and ability to jointly complement a yeast strain lacking a functional CCA-adding enzyme, consistent with a recent report 36,37 . These studies are the first observations of a two-enzyme system in a eukaryote, and await verification in S. pombe .…”