The WW domain-containing proteins interact with the early spliceosome and participate in pre-mRNA splicing in vivo
The WW domain-containing proteins interact with the early spliceosome and participate in pre-mRNA splicing in vivo
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DOI：
10.1128/mcb.24.20.9176-9185.2004
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发表时间：
2004-10-01
影响因子：
5.3
通讯作者：
Tarn, WY
中科院分区：
文献类型：
--
作者：
Lin, KT;Lu, RM;Tarn, WY
越来越多的证据支持 mRNA 合成及其后续加工事件的协调。含有 WW 和 FF 蛋白相互作用模块的核蛋白与剪接因子以及 RNA 聚合酶 II 结合，可能有助于将转录与剪接连接起来。为了了解 WW 结构域如何协调剪接复合物的组装，我们在 HeLa 细胞核提取物的下拉实验中使用了包含 CA150 或 FBP11 WW 结构域的谷胱甘肽 S 转移酶融合物。 WW 结构域优先与 U2 小核核糖核蛋白以及剪接因子 SF1、U2AF 和 SF3 复合物成分相关。因此，WW 结构域相关因子与前 mRNA 的 3' 部分结合，形成前剪接体样复合物。我们进行了体外和体内剪接测定，以探索包含 WW/FF 结构域的蛋白质在此过程中的作用。然而，尽管CA150与剪接体相关，但它对于体外剪接似乎是可有可无的。然而，体内 CA150 的消耗显着降低了报告基因前体 mRNA 的剪接效率。此外，含有WW和FF结构域的CA150片段的过度表达可能通过促进3'剪接位点识别来激活剪接并调节替代外显子选择。我们的结果表明，包含 WW/FF 结构域的因子在前 mRNA 剪接中发挥着重要作用，这可能与体内转录同时发生。
A growing body of evidence supports the coordination of mRNA synthesis and its subsequent processing events. Nuclear proteins harboring both WW and FF protein interaction modules bind to splicing factors as well as RNA polymerase II and may serve to link transcription with splicing. To understand how WW domains coordinate the assembly of splicing complexes, we used glutathione S-transferase fusions containing WW domains from CA150 or FBP11 in pull-down experiments with HeLa cell nuclear extract. The WW domains associate preferentially with the U2 small nuclear ribonucleoprotein and with splicing factors SF1, U2AF, and components of the SF3 complex. Accordingly, WW domain-associating factors bind to the 3' part of a pre-mRNA to form a pre-spliceosome-like complex. We performed both in vitro and in vivo splicing assays to explore the role of WW/FF domain-containing proteins in this process. However, although CA150 is associated with the spliceosome, it appears to be dispensable for splicing in vitro. Nevertheless, in vivo depletion of CA150 substantially reduced splicing efficiency of a reporter pre-mRNA. Moreover, overexpression of CA150 fragments containing both WW and FF domains activated splicing and modulated alternative exon selection, probably by facilitating 3' splice site recognition. Our results suggest an essential role of WW/FF domain-containing factors in pre-mRNA splicing that likely occurs in concert with transcription in vivo.