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Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

ABSTRACT: Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

SUBMITTER: Jha S

PROVIDER: S-EPMC5526112 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

Jha Sujata S Rollins Madeline G MG Fuchs Gabriele G Procter Dean J DJ Hall Elizabeth A EA Cozzolino Kira K Sarnow Peter P Savas Jeffrey N JN Walsh Derek D

Nature 20170621 7660

Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphoryla ...[more]

PMID: 28636603

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Project description:UnlabelledThe more than 200 closely spaced annotated open reading frames, extensive transcriptional read-through, and numerous unpredicted RNA start sites have made the analysis of vaccinia virus gene expression challenging. Genome-wide ribosome profiling provided an unprecedented assessment of poxvirus gene expression. By 4 h after infection, approximately 80% of the ribosome-associated mRNA was viral. Ribosome-associated mRNAs were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h. Cluster analysis identified a subset of early mRNAs that continued to be translated at the later times. At 2 h, there was excellent correlation between the abundance of individual mRNAs and the numbers of associated ribosomes, indicating that expression was primarily transcriptionally regulated. However, extensive transcriptional read-through invalidated similar correlations at later times. The mRNAs with the highest density of ribosomes had host response, DNA replication, and transcription roles at early times and were virion components at late times. Translation inhibitors were used to map initiation sites at single-nucleotide resolution at the start of most annotated open reading frames although in some cases a downstream methionine was used instead. Additional putative translational initiation sites with AUG or alternative codons occurred mostly within open reading frames, and fewer occurred in untranslated leader sequences, antisense strands, and intergenic regions. However, most open reading frames associated with these additional translation initiation sites were short, raising questions regarding their biological roles. The data were used to construct a high-resolution genome-wide map of the vaccinia virus translatome.ImportanceThis report contains the first genome-wide, high-resolution analysis of poxvirus gene expression at both transcriptional and translational levels. The study was made possible by recent methodological advances allowing examination of the translated regions of mRNAs including start sites at single-nucleotide resolution. Vaccinia virus ribosome-associated mRNA sequences were detected for most annotated early genes at 2 h and for most intermediate and late genes at 4 and 8 h after infection. The ribosome profiling approach was particularly valuable for poxviruses because of the close spacing of approximately 200 open reading frames and extensive transcriptional read-through resulting in overlapping mRNAs. The expression of intermediate and late genes, in particular, was visualized with unprecedented clarity and quantitation. We also identified novel putative translation initiation sites that were mostly associated with short protein coding sequences. The results provide a framework for further studies of poxvirus gene expression.

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Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

Publications

Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

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