Reprogramming alternative pre-messenger RNA splicing through the use of protein-binding antisense oligonucleotides
Reprogramming alternative pre-messenger RNA splicing through the use of protein-binding antisense oligonucleotides
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DOI：
10.1074/jbc.m308897200
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发表时间：
2003-12-12
影响因子：
4.8
通讯作者：
Chabot, B
中科院分区：
文献类型：
--
作者：
Villemaire, J;Dion, I;Chabot, B
选择性前信使RNA剪接是高等真核生物中蛋白质组多样性的主要贡献者，并且代表了在多种生物系统中控制蛋白质功能的关键步骤。作为人工改变剪接位点选择的一种手段，我们研究了在5'剪接位点附近定位蛋白质的影响。我们发现重组GST-MS 2蛋白干扰5'剪接位点的使用，当它结合该位点的上游时最有效。为了拓宽蛋白质作为剪接的空间抑制剂的用途，我们测试了携带异质核核糖核蛋白A1/A2蛋白结合位点的反义寡核苷酸的活性。在HeLa细胞提取物中，与外显子序列互补的加尾寡核苷酸引起5'剪接位点选择的强烈移位。在四种不同的人类细胞系中，携带A1/A2结合位点的干扰寡核苷酸也比仅通过双链体形成起作用的寡核苷酸更有效地改变Bcl-x前mRNA的选择性剪接。因此，使用干扰U1小核核糖核蛋白结合的蛋白结合寡核苷酸代表了一种新的和强大的方法来控制细胞中的剪接位点选择。
Alternative pre-messenger RNA splicing is a major contributor to proteomic diversity in higher eukaryotes and represents a key step in the control of protein function in a large variety of biological systems. As a means of artificially altering splice site choice, we have investigated the impact of positioning proteins in the vicinity of 5' splice sites. We find that a recombinant GST-MS2 protein interferes with 5' splice site use, most efficiently when it binds upstream of that site. To broaden the use of proteins as steric inhibitors of splicing, we have tested the activity of antisense oligonucleotides carrying binding sites for the heterogeneous nuclear ribonucleoprotein A1/A2 proteins. In a HeLa cell extract, tailed oligonucleotides complementary to exonic sequences elicit strong shifts in 5' splice site selection. In four different human cell lines, an interfering oligonucleotide carrying A1/A2 binding sites also shifted the alternative splicing of the Bcl-x pre-mRNA more efficiently than oligonucleotides acting through duplex formation only. The use of protein-binding oligonucleotides that interfere with U1 small nuclear ribonucleoprotein binding therefore represents a novel and powerful approach to control splice site selection in cells.