A cellular protein, hnRNP H, binds to the negative regulator of splicing element from Rous sarcoma virus
A cellular protein, hnRNP H, binds to the negative regulator of splicing element from Rous sarcoma virus
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DOI：
10.1074/jbc.m005000200
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发表时间：
2000-10-13
影响因子：
4.8
通讯作者：
McNally, MT
中科院分区：
文献类型：
--
作者：
Fogel, BL;McNally, MT
不完全RNA剪接是逆转录病毒生命周期的一个关键特征。这与大多数细胞前mRNA的加工形成对比，其通常被剪接完成。在劳斯肉瘤病毒中，剪接控制部分通过称为剪接负调节因子（NRS）的顺式作用RNA元件实现。NRS在功能上分为两部分，称为NRS 5 '和NRS 3'，其结合许多剪接因子。U1和U11小核核糖核蛋白与NRS 3 '中的序列相互作用，而NRS 5'结合几种蛋白质，包括蛋白质家族的成员。在特异性结合NRS 5 '的蛋白质中，有一种以前未鉴定的55-kDa蛋白（p55）。通过UV交联将p55结合位点定位于31个核苷酸的片段，通过RNA亲和选择分离特异性结合p55的蛋白质，并通过质谱鉴定为hnRNP H。针对hnRNP H的抗体免疫沉淀交联p55，并诱导在HeLa细胞核提取物中形成的含有p55的复合物的超位移。紫外交联和电泳迁移率变动分析表明，重组hnRNP H与p55结合位点特异性结合，证实hnRNP H为p55。
Incomplete RNA splicing is a key feature of the retroviral life cycle. This is in contrast to the processing of most cellular pre-mRNAs, which are usually spliced to completion. In Rous sarcoma virus, splicing control is achieved in part through a cis-acting RNA element termed the negative regulator of splicing (NRS). The NRS is functionally divided into two parts termed NRS5' and NRS3', which bind a number of splicing factors. The U1 and U11 small nuclear ribonucleoproteins interact with sequences in NRS3', whereas NRS5' binds several proteins including members of the family of proteins. Among the proteins that specifically bind NRS5' is a previously unidentified 55-kDa protein (p55), In this report we describe the isolation and identification of p55. The p55 binding site was localized by UV cross-linking to a 31-nucleotide segment, and a protein that binds specifically to it was isolated by RNA affinity selection and identified by mass spectrometry as hnRNP H. Antibodies against hnRNP H immunoprecipitated crosslinked p55 and induced a supershift of a p55-containing complex formed in HeLa nuclear extract. Furthermore, UV cross-linking and electrophoretic mobility shift assays indicated that recombinant hnRNP H specifically interacts with the p55 binding site, confirming that hnRNP H is p55, The possible roles of hnRNP H in NRS function are discussed.