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

Abstract

Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed TRAID-Seq, a screening strategy in S. cerevisiae to identify sequences added to a reporter RNA at single-nucleotide resolution by overexpressing candidate enzymes from different organisms. The rNTase activities of 22 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 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.

Records of RNA localization through covalent tagging

“…APEX-seq, as currently configured, is not suitable in intact organisms. Conversely, in cells with endogenous uridylation activities, application of tagging would be simplified through enzymes with different nucleotide specificities 98…”

Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes

“…Here, we present an in-depth phylogenetic analysis indicating that the occurrence of multiple nucleotidyltransferases is not limited to Bacteria, but is equally found in Eukaryotes. Similar to the situation in S. pombe [37,38], the enzymes of S. rosetta (group of Choanoflagellata) exhibit separate activities for CC-and A-addition and have to collaborate for complete CCA synthesis. A detailed bioinformatical analysis allowed us to pin down the unusual evolution of eukaryotic CC-and A-adding enzymes in detail.…”

“…The examination of the genomic sequence of Schizosaccharomyces pombe revealed two sequences with high similarity (>32% at protein level) to the known CCA-adding enzyme of Saccharomyces cerevisiae [8,37,38]. To address whether this observation is restricted to S. pombe or whether it is more widespread among Fungi, we performed database searches and phylogenetic analyses on all available genomes of the genus Schizosaccharomyces as well as closely related species.…”

“…Both enzyme copies in S. rosetta as well as in S. pombe show all five elements of the catalytic core [10] and do not carry a deletion of the flexible loop element, the hallmark for bacterial CC-adding enzymes ( Figures 4A and 5A) [11,22]. However, Reid et al and Preston et al demonstrated that the S. pombe enzymes exhibit partial activities and represent the first example of eukaryotic CCand A-adding enzymes [37,38]. The CC-adding enzyme lacks the B/A motif that positions the 3 -end of the reaction intermediate tRNA-CC for the terminal A-addition [11,22,27,37].…”

“…In these organisms, the original eukaryotic gene was not replaced and still exists in parallel with the alphaproteobacterial-like version [8]. In addition, representatives of the group of Fungi (e.g., Schizosaccharomyces pombe) also possess two versions of nucleotidyltransferases [35][36][37][38]. However, they are both of ancestral eukaryotic (e-type) origin [8].…”