3' RNA processing efficiency plays a primary role in generating termination-competent RNA polymerase II elongation complexes.
3' RNA processing efficiency plays a primary role in generating termination-competent RNA polymerase II elongation complexes.
复制标题
3 RNA 加工效率在生成具有终止能力的 RNA 聚合酶 II 延伸复合物中起主要作用。
DOI：
10.1128/mcb.13.6.3472-3480.1993
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发表时间：
1993
影响因子：
5.3
通讯作者：
Falck-Pedersen,E
中科院分区：
文献类型：
--
作者：
Edwalds-Gilbert,G;Prescott,J;Falck-Pedersen,E
在一些哺乳动物转录单位中，已经鉴定出一种转录终止机制，其中有效终止取决于完整的3'RNA加工位点的存在。小鼠βmaj-珠蛋白转录单位就是这样的一个例子，其中完整的多聚腺苷酸位点是有效转录终止所必需的。现在很明显，3' mRNA 加工位点并不总是以相同的效率进行加工。在这项研究中，我们鉴定了几种作为 3' mRNA 切割和聚腺苷酸化加工反应底物的前体 mRNA。然后我们确定加工效率不同的聚腺苷酸位点是否支持聚腺苷酸位点依赖性终止事件。体外加工效率水平通过测量前mRNA切割事件的效率的测定来确定，体内加工效率水平通过瞬时转染测定中产生的poly(A)位点依赖性mRNA和基因产物表达的水平来确定。 βmajglobin pre-mRNA 的加工非常有效。这种有效的处理与其在核连续测定中使用重组腺病毒终止载体的终止测定中的功能相关。当β大珠蛋白poly(A)位点被腺病毒主要晚期转录单位(Ad-ml)的L1 poly(A)位点取代时，这是一种较差的加工底物，终止效率急剧下降。当β大珠蛋白poly(A)位点被Ad-ml L3 poly(A)位点取代时，其加工效率比Ad-ml L1 poly(A)位点高10至20倍，终止效率仍然很高。因此，终止取决于处理事件的产量。然后，我们测试了包含 L3 核心 AAUAAA 但不同下游富含 GU 元件的嵌合 Poly(A) 位点。下游富含 GU 的元素的变化以与终止效率相关的方式影响处理效率。这些实验提供的证据表明，3'加工复合物形成的效率与哺乳动物转录单位3'端RNA聚合酶II终止的效率直接相关。
In several mammalian transcription units, a transcription termination mechanism in which efficient termination is dependent on the presence of an intact 3' RNA processing site has been identified. The mouse βmaj-globin transcription unit is one such example, in which an intact poly(A) site is required for efficient transcription termination. It is now evident that 3' mRNA processing sites are not always processed with the same efficiency. In this study, we characterized several pre-mRNAs as substrates for the 3' mRNA processing reaction of cleavage and polyadenylation. We then determined whether poly(A) sites which vary in processing efficiency support a poly(A) site-dependent termination event. The level of processing efficiency was determined in vitro by assays measuring the efficiency of the pre-mRNA cleavage event and in vivo by the level of poly(A) site-dependent mRNA and gene product expression generated in transient transfection assays. The βmajglobin pre-mRNA is very efficiently processed. This efficient processing correlates with its function in termination assays using recombinant adenovirus termination vectors in nuclear run-on assays. When the βmajglobin poly(A) site was replaced by the L1 poly(A) site of the adenovirus major late transcription unit (Ad-ml), which is a poor processing substrate, termination efficiency decreased dramatically. When the βmajglobin poly(A) site was replaced by the Ad-ml L3 poly(A) site, which is 10- to 20-fold more efficiently processed than the Ad-ml L1 poly(A) site, termination efficiency remained high. Termination is therefore dependent on the yield of the processing event. We then tested chimeric poly(A) sites containing the L3 core AAUAAA but varied downstream GU-rich elements. The change in downstream GU-rich elements affected processing efficiency in a manner which correlated with termination efficiency. These experiments provide evidence that the efficiency of 3' processing complex formation is directly correlated to the efficiency of RNA polymerase II termination at the 3' end of a mammalian transcription unit.