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. 2010 Feb;17(2):244-7.
doi: 10.1038/nsmb.1734. Epub 2010 Jan 31.

Nonsense-mediated mRNA decapping occurs on polyribosomes in Saccharomyces cerevisiae

Wenqian Hu  1 , Christine Petzold, Jeff Coller, Kristian E Baker
Affiliations

Nonsense-mediated mRNA decapping occurs on polyribosomes in Saccharomyces cerevisiae

Wenqian Hu et al. Nat Struct Mol Biol. 2010 Feb.

Abstract

Nonsense-mediated decay (NMD) degrades mRNA containing premature translation termination codons. In yeast, NMD substrates are decapped and digested exonucleolytically from the 5' end. Despite the requirement for translation in recognition, degradation of nonsense-containing mRNA is considered to occur in ribosome-free cytoplasmic P bodies. We show decapped nonsense-containing mRNA associate with polyribosomes, indicating that recognition and degradation are tightly coupled and that polyribosomes are major sites for degradation of aberrant mRNAs.

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Figures

Figure 1
Figure 1
NMD substrates are bound by polyribosomes when mRNA decapping is inhibited. (a) Polyribosome analysis of WT, upf1Δ and dcp2Δ cells. RNPs, 80S ribosomes and polyribosomes are indicated. (b) RT-PCR analysis for GFPPTC67 mRNA is quantitative for the analysis shown in c. (c) Distribution of GFP mRNA (lacking a premature termination codon, – PTC) and GFPPTC67 mRNA (harboring a premature termination codon, + PTC) in sucrose-gradient fractions. Arbitrary levels of GFPPTC67 mRNA are compared for WT, upf1Δ and dcp2Δ cells. (d) Northern blot analysis to monitor the sedimentation in sucrose gradients of CYH2 pre-mRNA and PGK1PTC225 reporter mRNA in WT, upf1Δ and dcp2Δ cells. The abundance of mRNA in each fraction in terms of the percentage of the total is shown.
Figure 2
Figure 2
The association of decapped NMD substrates with polyribosomes. (a) The splinted ligation RT-PCR assay for detecting decapped mRNA. (b) Detection of decapped CYH2 pre-mRNA, PGK1PTC225 mRNA and RPL41A mRNA in WT, dhh1Δ/pat1Δ and dcp2Δ cells. Addition of tobacco alkaline phosphatase before splinted ligation RT-PCR (dcp2Δ + TAP), without splinted ligation step (– Ligation), without reverse transcription step (– RT) and without cDNA template added to the PCR reaction (– cDNA). (c) Detection of decapped PGK1PTC225 mRNA in WT and upf1Δ cells. (d,e) Splinted ligation RT-PCR analysis on RNA recovered from sucrose-gradient fractionation of wild-type (d) or dhh1Δ/pat1Δ (e) cells. RNPs, 40S and 60S ribosomal subunits, 80S ribosomes and polyribosomes are indicated. The abundance of mRNA in each fraction in terms of the percentage of the total is shown.
Figure 3
Figure 3
Decapped products of nonsense-containing mRNA co-purify with ribosomes. Immunoprecipitation of ribosomes (mediated by affinity-tagged large ribosomal protein, RPL25) co-purifies decapped CYH2 pre-mRNA as detected by splinted ligation and RT-PCR analysis. Co-purification of CYH2 mRNA and ribosomal RNA (rRNA) were detected by 1.4% agarose gel followed by northern blot and ethidium bromide staining, respectively. U1 snRNA did not co-purify with ribosomes as monitored by northern blot.
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