RESUMO
A heat stress causes a rapid inhibition of splicing. Exogenous expression of Hsp27 did not prevent that inhibition but enhanced the recovery of splicing afterward. Another small heat shock protein, alphaB-crystallin, had no effect. Hsp27, but not alphaB-crystallin, also hastened rephosphorylation of SRp38-dephosphorylated a potent inhibitor of splicing-after a heat shock, although it did not prevent dephosphorylation by a heat shock. The effect of Hsp27 on rephosphorylation of SRp38 required phosphorylatable Hsp27. A Hsp90 client protein was required for the effect of Hsp27 on recovery of spicing and on rephosphorylation of SRp38. Raising the Hsp70 level by either a pre-heat shock or by exogenous expression had no effect on either dephosphorylation of SRp38 during heat shock or rephosphorylation after heat shock. The phosphatase inhibitor calyculin A prevented dephosphorylation of SRp38 during a heat shock and caused complete rephosphorylation of SRp38 after a heat shock, indicating that cells recovering from a heat shock are not deficient in kinase activity. Together our data show that the activity of Hsp27 in restoring splicing is not due to a general thermoprotective effect of Hsp27, but that Hsp27 is an active participant in the (de)phosphorylation cascade controlling the activity of the splicing regulator SRp38.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico/fisiologia , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Splicing de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico HSP70/fisiologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/fisiologia , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Toxinas Marinhas , Chaperonas Moleculares , Oxazóis/farmacologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Fatores de Processamento de Serina-Arginina , Cadeia B de alfa-Cristalina/fisiologiaRESUMO
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.
Assuntos
Temperatura Alta , Biossíntese de Proteínas , RNA Mensageiro/genética , Western Blotting , Cristalinas/genética , Cristalinas/metabolismo , Cicloeximida/farmacologia , Citoplasma/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico , Puromicina/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribossomos/metabolismo , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
The beta-crystallins are a family of long-lived, abundant structural proteins that are coexpressed in the vertebrate lens. As beta-crystallins form heteromers, a process that involves transient exposure of hydrophobic interfaces, we have examined whether in vivobeta-crystallin assembly is enhanced by protein chaperones, either small heat shock proteins, Hsp27 or alphaB-crystallin, or Hsp70. We show here that betaA4-crystallin is abundantly expressed in HeLa cells, but rapidly degraded, irrespective of the presence of Hsp27, alphaB-crystallin or Hsp70. Degradation is even enhanced by Hsp70. Coexpression of betaA4-crystallin with betaB2-crystallin yielded abundant soluble betaA4-betaB2-crystallin heteromers; betaB1-crystallin was much less effective in solubilizing betaA4-crystallin. As betaB2-crystallin competed for betaA4-crystallin with Hsp70 and the proteasomal degradation pathway, betaB2-crystallin probably captures an unstable betaA4-crystallin intermediate. We suggest that the proper folding of betaA4-crystallin is not mediated by general chaperones but requires a heteromeric partner, which then also acts as a dedicated chaperone towards betaA4-crystallin.