RESUMO
The 3' poly(A) structure improves translation of a eukaryotic mRNA by 50-fold in vivo. This enhancement has been suggested to be due to an interaction of the poly(A) binding protein, Pab1p, with eukaryotic translation initiation factor 4G (eIF4G). However, we find that mutation of eIF4G eliminating its interaction with Pab1p does not diminish the preference for poly(A)(+) mRNA in vivo, indicating another role for poly(A). We show that either the absence of Fun12p (eIF5B), or a defect in eIF5, proteins involved in 60S ribosomal subunit joining, specifically reduces the translation of poly(A)(+) mRNA, suggesting that poly(A) may have a role in promoting the joining step. Deletion of two nonessential putative RNA helicases (genes SKI2 and SLH1) makes poly(A) dispensable for translation. However, in the absence of Fun12p, eliminating Ski2p and Slh1p shows little enhancement of expression of non-poly(A) mRNA. This suggests that Ski2p and Slh1p block translation of non-poly(A) mRNA by an effect on Fun12p, possibly by affecting 60S subunit joining.
Assuntos
Proteínas Fúngicas/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Poli A/genética , Biossíntese de Proteínas , RNA Helicases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae , RNA Helicases DEAD-box , Eletroporação , Fator de Iniciação 5 em Eucariotos , Deleção de Genes , Genes Reporter , Cinética , Modelos Biológicos , Mutação , Proteínas de Ligação a Poli(A) , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Supressão Genética , Fatores de TempoRESUMO
In wild-type cells, the 3' poly(A) structure is necessary for translation of mRNA and for mRNA stability. The superkiller 2 (ski2), ski3, ski6, ski7, and ski8 mutations enhance the expression of the poly(A)(-) mRNAs of yeast RNA viruses. Ski2p is a DEVH-box RNA helicase and Slh1p resembles Ski2p. Both repress L-A double-stranded RNA (dsRNA) virus copy number, further suggesting that their functions may overlap. We find that slh1Delta ski2Delta double mutants are healthy (in the absence of viruses) and show normal rates of turnover of several cellular mRNAs. The slh1Delta ski2Delta strains translate electroporated nonpoly(A) mRNA with the same kinetics as polyA(+) mRNA. Thus, the translation apparatus is inherently capable of efficiently using nonpoly(A) mRNA even in the presence of normal amounts of competing poly(A)(+) mRNA, but is normally prevented from doing so by the combined action of the nonessential proteins Ski2p and Slh1p.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte , Poli A/metabolismo , Biossíntese de Proteínas , Proteínas de Saccharomyces cerevisiae , Exorribonucleases/genética , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fosfoglicerato Quinase/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Transativadores/genética , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose TumoralRESUMO
We establish that masked mRNAs synthesized from exogenous plasmid templates microinjected into the nuclei of Xenopus oocytes are translationally activated (unmasked) on oocyte maturation concomitant with polyadenylation. Synthetic mRNA injected into the cytoplasm of the oocyte is translated over an order of magnitude more efficiently than is the cognate mRNA synthesized in vivo. Both mRNA synthesized in vivo and mRNA microinjected into the oocyte cytoplasm require a cytoplasmic polyadenylation element in the 3'-untranslated region to activate translation on maturation. Although polyadenylation upon oocyte maturation can relieve the translational repression of mRNA synthesized in vivo, the excision of an intron within the nucleus does not relieve repression. We suggest that the translational repression coupled to the transcription process will more effectively repress inappropriate gene expression in the oocyte and offer the potential to achieve a wider range of gene regulation.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , RNA Mensageiro/metabolismo , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Oogênese , Poli A/metabolismo , Biossíntese de Proteínas , Splicing de RNA , Transcrição Gênica , Xenopus laevisRESUMO
The translational regulation of maternal mRNAs is the primary mechanism by which stage-specific programs of protein synthesis are executed during early development. Translation of a variety of maternal mRNAs requires either the maintenance or cytoplasmic elongation of a 3' poly(A) tail. Conversely, deadenylation results in translational inactivation. Although its precise function remains to be elucidated, the highly conserved poly(A) binding protein I (PABP) mediates poly(A)-dependent events in translation initiation and mRNA stability. Xenopus oocytes contain less than one PABP per poly(A) binding site suggesting that the translation of maternal mRNAs could be either limited by or independent of PABP. In this report, we have analyzed the effects of overexpressing PABP on the regulation of mRNAs during Xenopus oocyte maturation. Increased levels of PABP prevent the maturation-specific deadenylation and translational inactivation of maternal mRNAS that lack cytoplasmic polyadenylation elements. Overexpression of PABP does not interfere with maturation-specific polyadenylation, but reduces the recruitment of some mRNAs onto polysomes. Deletion of the C-terminal basic region and a single RNP motif from PABP significantly reduces both its binding to polyadenylated RNA in vivo and its ability to prevent deadenylation. In contrast to a yeast PABP-dependent poly(A) nuclease, PABP inhibits Xenopus oocyte deadenylase in vitro. These results indicate that maturation-specific deadenylation in Xenopus oocytes is facilitated by a low level of PABP consistent with a primary function for PABP to confer poly(A) stability.