Bennett M, Piñol-Roma S, Staknis D, Dreyfuss G, Reed R
Program in Cell and Developmental Biology, Harvard Medical School, Boston, Massachusetts 02115.
Mol Cell Biol. 1992 Jul;12(7):3165-75. doi: 10.1128/mcb.12.7.3165-3175.1992.
We have investigated the composition of the earliest detectable complex (H) assembled on pre-mRNA during the in vitro splicing reaction. We show that most of the proteins in this complex correspond to heterogeneous nuclear ribonucleoproteins (hnRNP), a set of abundant RNA-binding proteins that bind nascent RNA polymerase II transcripts in vivo. Thus, these studies establish a direct parallel between the initial events of RNA processing in vitro and in vivo. In contrast to previous studies, in which total hnRNP particles were isolated from mammalian nuclei, we determined the hnRNP composition of complexes assembled on individual RNAs of defined sequence. We found that a unique combination of hnRNP proteins is associated with each RNA. Thus, our data provide direct evidence for transcript-dependent assembly of pre-mRNA in hnRNP complexes. The observation that pre-mRNA is differentially bound by hnRNP proteins prior to spliceosome assembly suggests the possibility that RNA packaging could play a central role in the mechanism of splice site selection, as well as other posttranscriptional events.
我们研究了体外剪接反应过程中在pre-mRNA上组装的最早可检测复合物（H）的组成。我们发现，该复合物中的大多数蛋白质对应于不均一核核糖核蛋白（hnRNP），这是一组丰富的RNA结合蛋白，在体内可结合新生的RNA聚合酶II转录本。因此，这些研究在体外和体内RNA加工的初始事件之间建立了直接的平行关系。与之前从哺乳动物细胞核中分离总hnRNP颗粒的研究不同，我们确定了在特定序列的单个RNA上组装的复合物的hnRNP组成。我们发现，每种RNA都与hnRNP蛋白的独特组合相关联。因此，我们的数据为hnRNP复合物中pre-mRNA的转录本依赖性组装提供了直接证据。在剪接体组装之前，pre-mRNA被hnRNP蛋白差异性结合的观察结果表明，RNA包装可能在剪接位点选择机制以及其他转录后事件中发挥核心作用。
