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. 2013 Jan 2;110(1):324-9.
doi: 10.1073/pnas.1216454109. Epub 2012 Nov 19.

A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs

Amy Si-Ying Lee  1 , Rebeca Burdeinick-Kerr, Sean P J Whelan
Affiliations

VSports最新版本 - A ribosome-specialized translation initiation pathway is required for cap-dependent translation of vesicular stomatitis virus mRNAs

Amy Si-Ying Lee et al. Proc Natl Acad Sci U S A. .

Abstract

Initiation is the primary target of translational control for all organisms. Regulation of eukaryotic translation is traditionally thought to occur through initiation factors and RNA structures VSports手机版. Here, we characterize a transcript-specific translation initiation mechanism that is mediated by the ribosome. By studying vesicular stomatitis virus (VSV), we identify the large ribosomal subunit protein rpL40 as requisite for VSV cap-dependent translation but not bulk cellular or internal ribosome entry site-driven translation. This requirement is conserved among members of the order Mononegavirales, including measles virus and rabies virus. Polysome analyses and in vitro reconstitution of initiation demonstrate that rpL40 is required for 80S formation on VSV mRNAs through a cis-regulatory element. Using deep sequencing, we further uncover a subset of cellular transcripts that are selectively sensitive to rpL40 depletion, suggesting VSV may have usurped an endogenous translation pathway. Together, these findings demonstrate that the ribosome acts as a translational regulator outside of its catalytic role during protein synthesis. .

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Conflict of interest statement

The authors declare no conflict of interest.

Figures (VSports手机版)

Fig. 1.
Fig. 1.
RpL40 is required for VSV gene expression. (A) Fluorescence microscopy of cells transfected with a nontargeting (NT) siRNA or indicated ribosomal protein-targeting siRNA and infected with VSV-eGFP. Nuclei are Hochst stained (blue). (B) Rescue of VSV replication by exogenous expression of rpL40. HeLa cells were cotransfected with pcDNA3.1-rpL40, encoding a wild-type or siRNA-resistant form of the gene, and either NT or rpL40 targeting siRNA. Cells were infected and examined by epifluorescence microscopy. (C) Expression of luciferase from purified rVSV-Luc ribonucleoprotein cores transfected into cells treated with an siRNA targeting rpL40. Luciferase activity was normalized to activity from cells treated with a NT siRNA. The results are given as the mean ± SD of three independent experiments performed in triplicate. (D) VSV primary transcription in rpL40 siRNA-transfected cells. Abundance of VSV N mRNA was measured by quantitative RT-PCR. The results are given as the mean ± SD from a single representative quantitative RT-PCR experiment performed in duplicate. (E) Viral RNA synthesis in infected cells treated with rpL40 siRNA. Total [3H]-uridine–labeled cellular RNA was analyzed by electrophoresis on an acid-agarose gel. (F) Quantitative RT-PCR of total viral RNA synthesis. Results were analyzed as in D.
Fig. 2.
Fig. 2.
RpL40-dependent translation is transcript-specific. (A) VSV protein synthesis. Total [35S]-methionine-cysteine–labeled cytoplasmic proteins were analyzed by SDS/PAGE and detected by phosphorimager. Transfection of cells with nontargeting (NT) or rpL40-targeting siRNA is indicated. (B) Schematic diagram of bicistronic CrPV IRES construct. Translation of firefly luciferase is cap-dependent, whereas translation of renilla luciferase is driven by the CrPV IRES. (C) Firefly luciferase protein synthesis driven from pFR-CrPV. Firefly luciferase was measured from pFR-CrPV transfected lysates at 12 h after transfection and normalized to luciferase units from NT-treated cells. The results are given as the mean ± SD of three independent experiments each performed in triplicate. (D) Renilla luciferase protein synthesis driven from pFR-CrPV. Luciferase levels were measured and normalized as in C. (E) Poliovirus protein synthesis. Cells were infected and total cytoplasmic proteins were analyzed as in A. (F) Microscopy of cells infected with rabies virus-mCherry. (G) Microscopy of cells infected with measles virus-GFP. (H) Microscopy of cells infected with Newcastle disease virus-GFP.
Fig. 3.
Fig. 3.
RpL40 is required for translation initiation on VSV mRNAs as a constituent of the large subunit. (A) Sedimentation profile of mock-infected rpL40-targeting or nontargeting (NT) siRNA-transfected cells. (B) Distribution of β-actin mRNA with ribosomal complexes in cells depleted of rpL40. Transcript number, as determined by quantitative RT-PCR, is expressed as the percentage of total β-actin transcripts recovered and plotted against fraction number. The results are given as the mean ± SD from a representative quantitative RT-PCR experiment performed in duplicate. (C) Sedimentation profile of VSV-infected siRNA-transfected cells. (D) Distribution of VSV N mRNA with ribosomal complexes in cells depleted of rpL40. Lysates were resolved and fractionated, and mRNA distribution was determined as in B. (E) Translation of cellular or VSV-derived luciferase mRNA in yeast extracts expressing or lacking rpL40. Cytoplasmic mRNA was isolated from cells transfected with a luciferase expression plasmid (pRL-CMV) or cells infected with rVSV-Luc and used to program yeast extracts. The results are given as the mean ± SD of three independent experiments performed in triplicate. (F) Distribution of rpL40 with ribosomal complexes in VSV-infected cells.
Fig. 4.
Fig. 4.
RpL40-dependent translation is used by select cellular mRNAs. (A) Functional classification of mRNAs whose polysome association is altered by rpL40 depletion. The gene in each category whose association with polysomes was most decreased by knockdown of rpL40 is listed. (B) Levels of polysome-associated CLG1 mRNA upon rpL40 depletion identified by sequencing analysis. (C) Levels of polysome-associated DDR2 mRNA identified by sequencing analysis. Average reads per kilobase of exon per million mapped reads (RPKM) from biological replicates is graphed in B and C. (D) In vitro translation of CLG1 mRNA. (E) In vitro translation of DDR2 mRNA. The results of D and E are given as the mean ± SD of three independent experiments, performed in triplicate. Conditions that are statistically significant from the +rpL40 conditions are indicated with an asterisk (P < 0.0001).
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Comment in

  • Ribosomes take control.
    Barna M. Barna M. Proc Natl Acad Sci U S A. 2013 Jan 2;110(1):9-10. doi: 10.1073/pnas.1218764110. Epub 2012 Dec 14. Proc Natl Acad Sci U S A. 2013. PMID: 23243144 Free PMC article. No abstract available.

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