Double-stranded RNA-dependent protein kinase (pkr) is essential for thermotolerance, accumulation of HSP70, and stabilization of ARE-containing HSP70 mRNA during stress.
We have investigated the role of the double-stranded RNA-dependent protein kinase gene (pkr) in the regulation of the heat shock response. We show that the pkr gene is essential for efficient activation of the heat shock response and that pkr disruption profoundly inhibits heat shock protein 70 (HSP70) synthesis and blocks the development of thermotolerance. Despite these profound effects, pkr disruption did not markedly affect the activation of heat shock factor 1 by heat and did not reduce the rate of transcription of the HSP70 gene after heat shock. However, despite the lack of effect of pkr disruption on HSP70 gene transcription, we found a significant decrease in the expression of HSP70 mRNA in pkr-/- cells after heat shock. Kinetic studies of mRNA turnover suggested a block in the thermal stabilization of HSP70 mRNA in pkr-/- cells. As the thermal stabilization of HSP70 mRNA is thought to involve cis-acting A+U rich (ARE) elements in the 3'-untranslated region (UTR), we examined a potential role for pkr in this process. We found that a reporter beta-galactosidase mRNA destabilized by introduction of a functional ARE into the 3'-UTR became stabilized by heat but only in cells containing an intact pkr gene. Our studies suggest therefore that pkr plays a significant role in the stabilization of mRNA species containing ARE destruction sequences in the 3'-UTR and through this mechanism, contributes to the regulation of the heat shock response and other processes.
Department of Radiation Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Meijuan
M
Zhao
Dan
D
Tang
Stanislav
S
Lechpammer
Alexander
A
Hoffman
Alexzander
A
Asea
Mary Ann
MA
Stevenson
Stuart K
SK
Calderwood
D020413:3' Untranslated Regions; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D000076249:Heat Shock Transcription Factors; D006358:Hot Temperature; D051379:Mice; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection; D019892:eIF-2 Kinase / Q000502:physiology*
D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D000076249:Heat Shock Transcription Factors; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D014157:Transcription Factors; D012313:RNA; D019892:eIF-2 Kinase