The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers
The splicing factors 9G8 and SRp20 transactivate splicing through different and specific enhancers
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DOI：
10.1017/s1355838299981967
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发表时间：
1999-03-01
期刊：
影响因子：
4.5
通讯作者：
Stévenin, J
中科院分区：
文献类型：
--
作者：
Cavaloc, Y;Bourgeois, CF;Stévenin, J
在结构性剪接或选择性剪接中，SR蛋白家族剪接因子的活性需要与前mRNA底物直接相互作用。因此，重要的是要确定各种SR物种的高亲和力靶标，并评估它们区分所定义的RNA靶标的能力。我们分析了30 kDa SR蛋白9G8的结合特异性，除了RNA结合域(RBD)外，它还含有一个锌指关节。使用SELEX方法，我们证明了9G8选择由GAC三联体形成的RNA序列，而突变的锌指关节变体选择不同的RNA序列，以(A/U)C(A/U)(A/U)C基序为中心，表明锌指关节参与了908的RNA识别特异性。相反，SC35选择由嘧啶或富嘌呤基序组成的序列。通过UV交联和免疫沉淀对纯化的重组30 kDa SR蛋白或核提取液中的RNA-蛋白质相互作用的分析表明，908、SC35和ASF/SF2具有高度特异性地识别其特异性RNA靶标。有趣的是，由突变的锌指关节908变异体选择的RNA序列被SRp20有效地识别，这与908的红色与SRp20相似的事实一致。最后，我们证明了908及其锌指关节变异体SRp20通过其特定的RNA靶标作为有效的剪接反式激活因子的能力。我们的结果为红色指节和锌指关节在定义RNA结合域的特异性方面的合作提供了第一个证据。
The activity of the SR protein family of splicing factors in constitutive or alternative splicing requires direct interactions with the pre-mRNA substrate. Thus it is important to define the high affinity targets of the various SR species and to evaluate their ability to discriminate between defined RNA targets. We have analyzed the binding specificity of the 30-kDa SR protein 9G8, which contains a zinc knuckle in addition to the RNA binding domain (RBD). Using a SELEX approach, we demonstrate that 9G8 selects RNA sequences formed by GAC triplets, whereas a mutated zinc knuckle variant selects different RNA sequences, centered around a (A/U)C(A/U)(A/U)C motif, indicating that the zinc knuckle is involved in the RNA recognition specificity of 908. In contrast, SC35 selects sequences composed of pyrimidine or purine-rich motifs. Analyses of RNA-protein interactions with purified recombinant 30-kDa SR proteins or in nuclear extracts, by means of UV crosslinking and immunoprecipitation, demonstrate that 908, SC35, and ASF/SF2 recognize their specific RNA targets with high specificity. Interestingly, the RNA sequences selected by the mutated zinc knuckle 908 variant are efficiently recognized by SRp20, in agreement with the fact that the RED of 908 and SRp20 are similar. Finally, we demonstrate the ability of 908 and of its zinc knuckle variant, or SRp20, to act as efficient splicing transactivators through their specific RNA targets. Our results provide the first evidence for cooperation between an RED and a zinc knuckle in defining the specificity of an RNA binding domain.