Bablanian R, Goswami S K, Esteban M, Banerjee A K, Merrick W C
Department of Microbiology and Immunology, SUNY Health Science Center, Brooklyn 11203.
J Virol. 1991 Aug;65(8):4449-60. doi: 10.1128/JVI.65.8.4449-4460.1991.
We have recently demonstrated that the poly(A) moieties of short RNAs obtained from both in vitro transcription and from vaccinia virus (VV)-infected cells exhibit dissimilar effects on the in vitro translation of cellular and VV mRNAs (R. Bablanian, G. Coppola, P. Masters, and A. K. Banerjee, Virology 148:375-380, 1986; M. J. Su and R. Bablanian, Virology 179:679-693, 1990). In the present study, we have investigated the roles of poly(A), m7GTP, and initiation factors in the mechanism of selective translation of VV mRNAs. The effects of unfractionated poly(A) [termed poly(A)un, with various chain lengths up to 3,000 nucleotides] and a 150- to 300-nucleotide fraction of synthetic poly(A) [termed poly(A)150-300] on the translation of HeLa cell mRNAs and early and late VV mRNAs were studied. Both the poly(A)un and the poly(A)150-300 completely inhibited the translation of HeLa cell mRNAs obtained from total cytoplasmic RNA in the nuclease-treated reticulocyte lysates. Viral mRNAs from total cytoplasmic RNA also were slightly inhibited (15 to 38%) by the poly(A)un, whereas the poly(A)150-300 had no significant effect on their translation. The translation of oligo(dT)-cellulose-selected HeLa mRNAs was as sensitive to inhibition by poly(A)150-300 as the mRNAs found in total cytoplasmic RNA. However, the translations of oligo(dT)-cellulose-selected viral mRNAs become more sensitive to the inhibitory effect of poly(A)150-300 than the translations of viral mRNAs found in the total cytoplasmic RNA. Both HeLa and VV mRNAs became more resistant to the poly(A)-mediated inhibition when these mRNAs were deadenylated, but the relative resistance to inhibition by poly(A)150-300 of deadenylated VV mRNAs was much greater than that of HeLa cell mRNAs. The translation of VV mRNAs was significantly less inhibited than the translation of HeLa mRNAs when the cap analog, m7GTP, was added to the cell-free system. The inhibition of HeLa cell mRNA translation by both poly(A)un and poly(A)150-300 was completely restored when poly(A)-binding protein (PAB) was added to the cell-free translational system. The addition of eukaryotic initiation factor 4A (eIF-4A) did not restore translation when poly(A)un was used to inhibit translation; however, inhibition by poly(A)150-300 was significantly reversed by this initiation factor. The reversal of poly (A)-mediated inhibition of HeLa cell mRNA translation was additive when PAB was used together with eIF-4A. Early VV mRNA translation was only slightly inhibited by poly(A)un (15%), and this inhibition was completely reversed by either PAB or eIF-4A.(ABSTRACT TRUNCATED AT 400 WORDS)
我们最近证实，从体外转录以及痘苗病毒（VV）感染细胞中获得的短RNA的聚腺苷酸（poly(A)）部分，对细胞和VV mRNA的体外翻译表现出不同的影响（R. Bablanian、G. Coppola、P. Masters和A. K. Banerjee，《病毒学》148:375 - 380，1986；M. J. Su和R. Bablanian，《病毒学》179:679 - 693，1990）。在本研究中，我们研究了聚腺苷酸、7-甲基鸟苷三磷酸（m7GTP）和起始因子在VV mRNA选择性翻译机制中的作用。研究了未分级的聚腺苷酸[称为poly(A)un，链长可达3000个核苷酸]和150至300个核苷酸的合成聚腺苷酸片段[称为poly(A)150 - 300]对HeLa细胞mRNA以及VV早期和晚期mRNA翻译的影响。在经核酸酶处理的网织红细胞裂解物中，poly(A)un和poly(A)150 - 300都完全抑制了从总细胞质RNA中获得的HeLa细胞mRNA的翻译。总细胞质RNA中的病毒mRNA也受到poly(A)un的轻微抑制（15%至38%），而poly(A)150 - 300对其翻译没有显著影响。寡聚(dT)-纤维素选择的HeLa mRNA的翻译对poly(A)150 - 300抑制的敏感性与总细胞质RNA中的mRNA相同。然而，寡聚(dT)-纤维素选择的病毒mRNA的翻译比总细胞质RNA中的病毒mRNA翻译对poly(A)150 - 300的抑制作用更敏感。当HeLa和VV mRNA去腺苷酸化后，它们对聚腺苷酸介导的抑制作用更具抗性，但去腺苷酸化的VV mRNA对poly(A)150 - 300抑制的相对抗性远大于HeLa细胞mRNA。当向无细胞系统中添加帽类似物m7GTP时，VV mRNA的翻译受到的抑制明显小于HeLa mRNA的翻译。当向无细胞翻译系统中添加聚腺苷酸结合蛋白（PAB）时，poly(A)un和poly(A)150 - 300对HeLa细胞mRNA翻译的抑制作用完全恢复。当使用poly(A)un抑制翻译时，添加真核起始因子4A（eIF - 4A）并不能恢复翻译；然而，这种起始因子能显著逆转poly(A)150 - 300的抑制作用。当PAB与eIF - 4A一起使用时，聚腺苷酸介导的HeLa细胞mRNA翻译抑制的逆转是相加的。VV早期mRNA的翻译仅受到poly(A)un的轻微抑制（15%），这种抑制可被PAB或eIF - 4A完全逆转。（摘要截断于400字）
