Gunnery S, Mathews M B
Cold Spring Harbor Laboratory, New York 11724, USA.
Mol Cell Biol. 1995 Jul;15(7):3597-607. doi: 10.1128/MCB.15.7.3597.
Eukaryotic cellular mRNA is believed to be synthesized exclusively by RNA polymerase II (pol II), whereas pol I produces long rRNAs and pol III produces 5S rRNA, tRNA, and other small RNAs. To determine whether this functional differentiation is obligatory, we examined the translational potential of an artificial pol III transcript. The coding region of the human immunodeficiency virus type 1 tat gene was placed under the control of a strong pol III promoter from the adenovirus type 2 VA RNAI gene. The resultant chimera, pVA-Tat, was transcribed accurately in vivo and in vitro and gave rise to Tat protein, which transactivated a human immunodeficiency virus-driven chloramphenicol acetyltransferase reporter construct in transfected HeLa cells. pol III-specific mutations down-regulated VA-Tat RNA production in vivo and in vitro and dramatically reduced chloramphenicol acetyltransferase transactivation. As expected for a pol III transcript, VA-Tat RNA was not detectably capped at its 5' end or polyadenylated at its 3' end, but, like mRNA, it was associated with polysomes in a salt-stable manner. Mutational analysis of a short open reading frame upstream of the Tat-coding sequence implicates scanning in the initiation of VA-Tat RNA translation despite the absence of a cap. In comparison with tat mRNA generated by pol II, VA-Tat RNA was present on smaller polysomes and was apparently translated less efficiently, which is consistent with a relatively low initiation rate. Evidently, human cells are capable of utilizing pol III transcripts as functional mRNAs, and neither a cap nor a poly(A) tail is essential for translation, although they may be stimulatory. These findings raise the possibility that some cellular mRNAs are made by pol I or pol III.
真核细胞的信使核糖核酸（mRNA）被认为仅由RNA聚合酶II（pol II）合成，而pol I产生长链核糖体RNA（rRNA），pol III产生5S rRNA、转运RNA（tRNA）和其他小RNA。为了确定这种功能分化是否是必然的，我们检测了人工合成的pol III转录本的翻译潜力。人类免疫缺陷病毒1型（HIV-1）tat基因的编码区置于来自腺病毒2型VA RNAI基因的强pol III启动子的控制之下。产生的嵌合体pVA-Tat在体内和体外均能准确转录，并产生Tat蛋白，该蛋白在转染的HeLa细胞中可反式激活由HIV驱动的氯霉素乙酰转移酶报告基因构建体。pol III特异性突变在体内和体外均下调了VA-Tat RNA的产生，并显著降低了氯霉素乙酰转移酶的反式激活作用。正如对pol III转录本的预期，VA-Tat RNA在其5'端未检测到帽状结构，在其3'端也未进行多聚腺苷酸化，但与mRNA一样，它以盐稳定的方式与多核糖体相关联。对Tat编码序列上游一个短开放阅读框的突变分析表明，尽管没有帽状结构，但在VA-Tat RNA翻译起始过程中存在扫描现象。与pol II产生的tat mRNA相比，VA-Tat RNA存在于较小的多核糖体上，且明显翻译效率较低，这与相对较低的起始速率一致。显然，人类细胞能够将pol III转录本用作功能性mRNA，帽状结构和多聚腺苷酸尾巴对于翻译都不是必需的，尽管它们可能具有促进作用。这些发现增加了一些细胞mRNA由pol I或pol III产生的可能性。
