Markovtsov V, Nikolic J M, Goldman J A, Turck C W, Chou M Y, Black D L
Department of Microbiology and Molecular Genetics, University of California, Los Angeles, California 90095, USA.
Mol Cell Biol. 2000 Oct;20(20):7463-79. doi: 10.1128/MCB.20.20.7463-7479.2000.
Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins.
神经元细胞中c-src N1外显子的剪接部分取决于一个名为下游控制序列（DCS）的RNA调控元件内含子簇。通过位点特异性交联、RNA凝胶迁移和DCS RNA亲和色谱分析，我们对几种蛋白质与DCS RNA上特定位点的结合进行了表征。异质性核核糖核蛋白（hnRNP）H、多嘧啶序列结合蛋白（PTB）和KH型剪接调节蛋白（KSRP）各自结合到该序列内的不同元件上。我们还鉴定出一种新的60 kDa组织特异性蛋白，它与DCS RNA的CUCUCU剪接抑制元件结合。该蛋白被纯化、部分测序并克隆。这种新蛋白（PTB的神经富集同源物[nPTB]）与PTB高度同源。与PTB不同，nPTB在大脑和一些神经细胞系中富集。尽管nPTB和PTB在序列上相似，但它们在性质上存在显著差异。nPTB比PTB更稳定地结合DCS RNA，但在体外是一种较弱的剪接抑制因子。nPTB还极大地增强了另外两种蛋白hnRNP H和KSRP与DCS RNA的结合。这些实验确定了组装到复杂剪接调控序列上的蛋白质之间的特定协同相互作用，并展示了通过纳入不同但高度相关的蛋白质，这种hnRNP组装在不同细胞类型中是如何改变的。
