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

ABSTRACT: Ribonucleotidyl transferases (rNTases) add untemplated ribonucleotides to diverse RNAs. We have developed TRAID-seq, a screening strategy in Saccharomyces cerevisiae to identify sequences added to a reporter RNA at single-nucleotide resolution by overexpressed 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 cytidine-adding enzyme that is likely to be 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, Caenorhabditis elegans MUT-2, that adds alternating uridine and guanosine nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutants of the enzyme that result in defective silencing did not 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.

SUBMITTER: Preston MA

PROVIDER: S-EPMC6613791 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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

Preston Melanie A MA Porter Douglas F DF Chen Fan F Buter Natascha N Lapointe Christopher P CP Keles Sunduz S Kimble Judith J Wickens Marvin M

Nature methods 20190415 5

Ribonucleotidyl transferases (rNTases) add untemplated ribonucleotides to diverse RNAs. We have developed TRAID-seq, a screening strategy in Saccharomyces cerevisiae to identify sequences added to a reporter RNA at single-nucleotide resolution by overexpressed 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 cytidine-adding enzyme that is likely to be part of ...[more]

PMID: 30988468

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Project description:Influenza A viruses counteract the cellular innate immune response at several steps, including blocking RIG I-dependent activation of interferon (IFN) transcription, interferon (IFN)-dependent upregulation of IFN-stimulated genes (ISGs), and the activity of various ISG products; the multifunctional NS1 protein is responsible for most of these activities. To determine the importance of other viral genes in the interplay between the virus and the host IFN response, we characterized populations and selected mutants of wild-type viruses selected by passage through non-IFN-responsive cells. We reasoned that, by allowing replication to occur in the absence of the selection pressure exerted by IFN, the virus could mutate at positions that would normally be restricted and could thus find new optimal sequence solutions. Deep sequencing of selected virus populations and individual virus mutants indicated that nonsynonymous mutations occurred at many phylogenetically conserved positions in nearly all virus genes. Most individual mutants selected for further characterization induced IFN and ISGs and were unable to counteract the effects of exogenous IFN, yet only one contained a mutation in NS1. The relevance of these mutations for the virus phenotype was verified by reverse genetics. Of note, several virus mutants expressing intact NS1 proteins exhibited alterations in the M1/M2 proteins and accumulated large amounts of deleted genomic RNAs but nonetheless replicated to high titers. This suggests that the overproduction of IFN inducers by these viruses can override NS1-mediated IFN modulation. Altogether, the results suggest that influenza viruses replicating in IFN-competent cells have tuned their complete genomes to evade the cellular innate immune system and that serial replication in non-IFN-responsive cells allows the virus to relax from these constraints and find a new genome consensus within its sequence space.In natural virus infections, the production of interferons leads to an antiviral state in cells that effectively limits virus replication. The interferon response places considerable selection pressure on viruses, and they have evolved a variety of ways to evade it. Although the influenza virus NS1 protein is a powerful interferon antagonist, the contributions of other viral genes to interferon evasion have not been well characterized. Here, we examined the effects of alleviating the selection pressure exerted by interferon by serially passaging influenza viruses in cells unable to respond to interferon. Viruses that grew to high titers had mutations at many normally conserved positions in nearly all genes and were not restricted to the NS1 gene. Our results demonstrate that influenza viruses have fine-tuned their entire genomes to evade the interferon response, and by removing interferon-mediated constraints, viruses can mutate at genome positions normally restricted by the interferon response.

Dataset Information

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

Publications

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

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