Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein
Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein
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DOI：
10.1128/mcb.20.20.7463-7479.2000
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发表时间：
2000-10-01
影响因子：
5.3
通讯作者：
Black, DL
中科院分区：
文献类型：
--
作者：
Markovtsov, V;Nikolic, JM;Black, DL
神经元细胞中c-src N1外显子的剪接部分取决于称为下游控制序列（DCS）的RNA调控元件的内含子簇。使用位点特异性交联，RNA凝胶移位，和DCS RNA亲和层析测定，我们的特点是结合的几种蛋白质的特定位点沿着DCS RNA。异质核核糖核蛋白（hnRNP）H、多聚嘧啶束结合蛋白（PTB）和KH型剪接调节蛋白（KSRP）各自与该序列内的不同元件结合。我们还确定了一个新的60 kDa的组织特异性蛋白，结合到DCS RNA的CUCUCU剪接阻遏元件。该蛋白质被纯化，部分测序，并克隆。新蛋白质（PTB的神经富集同系物[nPTB]）与PTB高度同源。与PTB不同，nPTB在脑和一些神经细胞系中富集。尽管序列相似，但nPTB和PTB在性质上显示出显著差异。nPTB比PTB更稳定地结合DCS RNA，但在体外是较弱的剪接阻遏物。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一个复杂的剪接调控序列上的蛋白质之间的特定合作相互作用，并显示了这种hnRNP组装是如何通过掺入不同但高度相关的蛋白质在不同细胞类型中改变的。
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.