mRNA made during heat shock enters the first round of translation.
To determine whether mRNA synthesized during a heat shock is translated at least once in spite of the strong inhibition of translation by heat shock, we used nonsense-mediated decay (NMD) as an assay since NMD requires a round of translation. As NMD substrate we used the human psigammaE-crystallin gene, which contains a premature termination codon, and as control, its close relative, the human gammaD-crystallin gene, both placed under control of the Hsp70 promoter. We show that no spliced psigammaE-crystallin mRNA can be detected in heat shocked cells, suggesting that NMD resumes as soon as splicing is restored. We further show that newly synthesized mRNAs co-sediment with the 40S ribosomal subunits, indicating that the transcripts are recruited to the translation machinery but are stalled at the translation initiation stage. Using fluorescence loss in photobleaching (FLIP) we show that cytoplasmic EGFP-CBP20 is immobile in heat shocked cells. CBP20 is part of the cap binding complex which is thought to direct the first round of translation. Together our data suggest that all mRNAs made during heat shock enter the pioneer round of translation.
Marín-Vinader
Laura
L
Department of Biochemistry 271, Radboud University of Nijmegen. P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
van Genesen
Siebe T
ST
Lubsen
Nicolette H
NH
D015153:Blotting, Western; D003459:Crystallins / Q000235:genetics / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D039502:Nuclear Cap-Binding Protein Complex / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection
D003459:Crystallins; D039502:Nuclear Cap-Binding Protein Complex; D021122:Protein Subunits; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011691:Puromycin; D003513:Cycloheximide