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Analysis of Translation Initiation During Stress Conditions by Polysome Profiling
Published on: May 19, 2014
mRNA made during heat shock enters the first round of translation
Laura Marín-Vinader1, Siebe T van Genesen, Nicolette H Lubsen
1Department of Biochemistry 271, Radboud University of Nijmegen. P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Abstract:
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.
Insights
Newly synthesized heat shock mRNAs enter translation but stall at initiation. Nonsense-mediated decay (NMD) assays reveal that despite translation inhibition, these transcripts engage the ribosome, suggesting a "pioneer round" of translation occurs.
Area of Science:
- Molecular Biology
- Cellular Stress Response
- Gene Expression Regulation
Background:
- Heat shock severely inhibits cellular translation.
- Newly synthesized mRNAs during heat shock face translation challenges.
- The role of translation initiation in heat shock response is not fully understood.
Purpose of the Study:
- To investigate if heat shock-induced mRNA undergoes translation.
- To elucidate the stage at which translation is inhibited during heat shock.
- To determine the fate of newly synthesized transcripts under heat stress.
Main Methods:
- Utilized nonsense-mediated decay (NMD) assay with engineered psigammaE-crystallin and gammaD-crystallin genes.
- Analyzed mRNA co-sedimentation with 40S ribosomal subunits.
- Employed fluorescence loss in photobleaching (FLIP) to assess cytoplasmic EGFP-CBP20 mobility.
Main Results:
- No spliced psigammaE-crystallin mRNA was detected in heat-shocked cells, indicating NMD resumption post-splicing.
- Newly synthesized mRNAs were found to co-sediment with 40S ribosomal subunits, indicating recruitment to the translation machinery.
- Immobility of cytoplasmic EGFP-CBP20, a key translation initiation factor, was observed in heat-shocked cells, suggesting stalled initiation.
Conclusions:
- All mRNAs synthesized during heat shock appear to enter the initial "pioneer round" of translation.
- Translation is stalled at the initiation stage for heat shock-induced mRNAs.
- The cap binding complex component CBP20 plays a critical role in directing this initial translation round under stress.
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