Distinct sets of adjacent heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 binding sites control 5′ splice site selection in the hnRNP A1 mRNA precursor
Distinct sets of adjacent heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 binding sites control 5′ splice site selection in the hnRNP A1 mRNA precursor
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DOI：
10.1074/jbc.m203633200
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发表时间：
2002-08-16
影响因子：
4.8
通讯作者：
Chabot, B
中科院分区：
文献类型：
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作者：
Hutchison, S;LeBel, C;Chabot, B
在异质核核糖核蛋白（hnRNP）A1前mRNA中，内含子侧翼选择性外显子7 B的不同区域与A1和A1 B mRNA剪接异构体的产生有关。其中，分别位于共同外显子7和选择性外显子7 B下游的CE 1a和CE 4元件被hnRNP A1结合以促进外显子7 B在体内的跳跃和远端5'剪接位点选择。在这里，我们报告说，CE 1a的两侧是一个额外的高亲和力A1结合位点（CE 1d）。以类似于CE 1a的方式，CE 1d在体外影响5'剪接位点选择。与hnRNP A1在CE 1d活性中的作用一致，废除A1结合的突变废除了远端5'剪接位点激活。此外，CE 1d刺激远端5'剪接位点利用的能力在耗尽hnRNP A/B蛋白的HeLa提取物中丧失，并且重组A1的添加恢复了CE 1d的活性。值得注意的是，由A1结合位点介导的远端5'剪接位点选择在从hnRNP A1蛋白严重缺陷的小鼠细胞制备的提取物中不受损害。在这种情况下，我们表明，hnRNP A2补偿A1缺陷。对CE 4元件的进一步研究表明，它也由两个不同的部分（CE 4 m和CE 4p）组成，每个部分都能够以hnRNPA 1依赖性方式促进远端5'剪接位点的使用。常见外显子7和选择性外显子7 B下游存在多个A1/A2结合位点可能在最大化hnRNP A1/A2蛋白的活性中起重要作用。
In the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 pre-mRNA, different regions in the introns flanking alternative exon 7B have been implicated in the production of the A1 and A1B mRNA splice isoforms. Among these, the CE1a and CE4 elements, located downstream of common exon 7 and alternative exon 7B, respectively, are bound by hnRNP A1 to promote skipping of exon 7B in vivo and distal 5' splice site selection in vitro. Here, we report that CE1a is flanked by an additional high affinity A1 binding site (CE1d). In a manner similar to CE1a, CE1d affects 5' splice site selection in vitro. Consistent with a role for hnRNP A1 in the activity of CE1d, a mutation that abrogates A1 binding abolishes distal 5' splice site activation. Moreover, the ability of CE1d to stimulate distal 5' splice site usage is lost in an HeLa extract depleted of hnRNP A/B proteins, and the addition of recombinant A1 restores the activity of CE1d. Notably, distal 5' splice site selection mediated by A1 binding sites is not compromised in an extract prepared from mouse cells that are severely deficient in hnRNP A1 proteins. In this case, we show that hnRNP A2 compensates for the A1 deficiency. Further studies with the CE4 element reveal that it also consists of two distinct portions (CE4m and CE4p), each one capable of promoting distal 5' splice site use in an hnRNP A1-dependent manner. The presence of multiple A1/A2 binding sites downstream of common exon 7 and alternative exon 7B probably plays an important role in maximizing the activity of hnRNP A1/A2 proteins.