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Viral Nucleases Induce an mRNA Degradation-Transcription Feedback Loop in Mammalian Cells.

ABSTRACT: Gamma-herpesviruses encode a cytoplasmic mRNA-targeting endonuclease, SOX, that cleaves most cellular mRNAs. Cleaved fragments are subsequently degraded by the cellular 5'-3' mRNA exonuclease Xrn1, thereby suppressing cellular gene expression and facilitating viral evasion of host defenses. We reveal that mammalian cells respond to this widespread cytoplasmic mRNA decay by altering RNA Polymerase II (RNAPII) transcription in the nucleus. Measuring RNAPII recruitment to promoters and nascent mRNA synthesis revealed that the majority of affected genes are transcriptionally repressed in SOX-expressing cells. The transcriptional feedback does not occur in response to the initial viral endonuclease-induced cleavage, but instead to degradation of the cleaved fragments by cellular exonucleases. In particular, Xrn1 catalytic activity is required for transcriptional repression. Notably, viral mRNA transcription escapes decay-induced repression, and this escape requires Xrn1. Collectively, these results indicate that mRNA decay rates impact transcription and that gamma-herpesviruses use this feedback mechanism to facilitate viral gene expression.

SUBMITTER: Abernathy E

PROVIDER: S-EPMC4538998 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Viral Nucleases Induce an mRNA Degradation-Transcription Feedback Loop in Mammalian Cells.

Abernathy Emma E Gilbertson Sarah S Alla Ravi R Glaunsinger Britt B

Cell host & microbe 20150723 2

Gamma-herpesviruses encode a cytoplasmic mRNA-targeting endonuclease, SOX, that cleaves most cellular mRNAs. Cleaved fragments are subsequently degraded by the cellular 5'-3' mRNA exonuclease Xrn1, thereby suppressing cellular gene expression and facilitating viral evasion of host defenses. We reveal that mammalian cells respond to this widespread cytoplasmic mRNA decay by altering RNA Polymerase II (RNAPII) transcription in the nucleus. Measuring RNAPII recruitment to promoters and nascent mRNA ...[more]

PMID: 26211836

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Project description:BackgroundCpf1 nucleases have recently been repurposed for site-specific genome modification. Two members of the Cpf1 family, the AsCpf1 from Acidaminococcus sp. and the LbCpf1 from Lachnospiraceae bacterium were shown to induce higher indel frequencies than SpCas9 when examining four randomly-selected target sequences for each type of nuclease. Whether they are a real match for Cas9 nucleases, however, remains to be verified.ResultsHere, we used AsCpf1 and LbCpf1 to induce homology directed repair, either single strand annealing (SSA) or homologous recombination (HR), in N2a mouse neuroblastoma cells. Exploiting a plasmid that contains two GFP halves with overlapping sequences and exploring 20 targets, on all but one both nucleases consistently performed with above 10 % efficiency. Several Cas9 nucleases have been previously characterised in order to find an orthogonal counterpart for the most widely used promiscuous SpCas9. Here, we found that AsCpf1 and LbCpf1 might be better candidates than three of the best such counterparts: Cas9 from Staphylococcus aureus, from Streptococcus thermophilus and from Neisseria meningitidis, when assessed for inducing efficient SSA mediated repair in N2a cells. When tested on genomic targets exploiting HR, both nucleases were able to induce the integration of a donor cassette with 1000 bp-long homologous arms. We also generated plasmids that express these Cpf1 nucleases together with their cognate crRNAs and that are equipped with type IIS restriction enzyme sites to facilitate spacer cloning.ConclusionsOur results suggest that employing As- or LbCpf1 nuclease to induce homology directed repair in N2a cells, although is less effective at present than employing SpCas9, it is an equally or more effective tool than the most frequently used orthogonal Cas9 counterparts of SpCas9. These findings support the position of Cpf1 nucleases on the side of SpCas9 on the palette of effective genome engineering tools.ReviewersThis article was reviewed by Eugene Koonin, Haruhiko Siomi and Jean-Yves Masson.

Project description:Gene silencing mediated by either microRNAs (miRNAs) or Adenylate/uridylate-rich elements Mediated mRNA Degradation (AMD) is a powerful way to post-transcriptionally modulate gene expression. We and others have reported that the RNA-binding protein KSRP favors the biogenesis of select miRNAs (including let-7 family) and activates AMD promoting the decay of inherently labile mRNAs. Different layers of interplay between miRNA- and AMD-mediated gene silencing have been proposed in cultured cells, but the relationship between the two pathways in living organisms is still elusive. We conditionally deleted Dicer in mouse pituitary from embryonic day (E) 9.5 through Cre-mediated recombination. In situ hybridization, immunohistochemistry, and quantitative reverse transcriptase-PCR revealed that Dicer is essential for pituitary morphogenesis and correct expression of hormones. Strikingly, αGSU (alpha glycoprotein subunit, common to three pituitary hormones) was absent in Dicer-deleted pituitaries. αGSU mRNA is unstable and its half-life increases during pituitary development. A transcriptome-wide analysis of microdissected E12.5 pituitaries revealed a significant increment of KSRP expression in conditional Dicer-deleted mice. We found that KSRP directly binds to αGSU mRNA, promoting its rapid decay; and, during pituitary development, αGSU expression displays an inverse temporal relationship to KSRP. Further, let-7b/c downregulated KSRP expression, promoting the degradation of its mRNA by directly binding to the 3'UTR. Therefore, we propose a model in which let-7b/c and KSRP operate within a negative feedback loop. Starting from E12.5, KSRP induces the maturation of let-7b/c that, in turn, post-transcriptionally downregulates the expression of KSRP itself. This event leads to stabilization of αGSU mRNA, which ultimately enhances the steady-state expression levels. We have identified a post-transcriptional regulatory network active during mouse pituitary development in which the expression of the hormone αGSU is increased by let7b/c through downregulation of KSRP. Our study unveils a functional crosstalk between miRNA- and AMD-dependent gene regulation during mammalian organogenesis events.

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Viral Nucleases Induce an mRNA Degradation-Transcription Feedback Loop in Mammalian Cells.

Publications

Viral Nucleases Induce an mRNA Degradation-Transcription Feedback Loop in Mammalian Cells.

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