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首页> 外文期刊>Molecular and Cellular Biology >RNA Binding Activity of Heterodimeric Splicing Factor U2AF: at Least One RS Domain Is Required for High-Affinity Binding
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RNA Binding Activity of Heterodimeric Splicing Factor U2AF: at Least One RS Domain Is Required for High-Affinity Binding

机译：异二聚体剪接因子U2AF的RNA结合活性：高亲和力结合需要至少一个RS域

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The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3′ splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3′ splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. hU2AF65 contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF35 has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF65. While the requirement for the three RRMs on hU2AF65 is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When theDrosophila small-subunit homolog (dU2AF38) was complexed with the large-subunit (dU2AF50) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF50. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF65. Surprisingly, the RS domain on dU2AF38was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from theDrosophila large-subunit monomer (dU2AF50ΔRS) severely impaired its binding activity. However, if the dU2AF38 RS domain was supplied in a complex with dU2AF50ΔRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.
机译：mRNA前剪接因子U2AF（U2小核糖核蛋白颗粒[snRNP]辅助因子）在3'剪接位点选择中起关键作用。 U2AF在分支点和3'剪接位点之间特异性结合内含嘧啶区域，并在剪接体组装的早期将U2 snRNP靶向分支点。人U2AF是由大（hU2AF 65 ）和小（hU2AF 35 ）亚基组成的异二聚体。 hU2AF 65 包含一个富含精氨酸丝氨酸（RS）的结构域和三个RNA识别基序（RRM）。 hU2AF 35 具有简并的RRM和羧基端RS结构域。最近的遗传学研究表明，果蝇U2AF亚基同系物上的RS结构域都是无关紧要的，可能在体内具有多余的功能。 U2AF异二聚体的位点特异性嘧啶束结合活性先前已被赋予hU2AF 65 。虽然已牢固确立了hU2AF 65 上三个RRM的要求，但大亚基RS结构域在RNA结合中的作用仍未解决。我们已经分析了U2AF异源二聚体的RNA结合活性。当果蝇小亚基同系物（dU2AF 38 ）与大亚基（dU2AF 50 ）嘧啶束复合时，RNA结合活性增加是免费的dU2AF 50 的20倍。比较人类U2AF异二聚体和hU2AF 65 后，我们发现RNA结合活性也有类似的提高。令人惊讶的是，dU2AF 38 上的RS结构域对于增加dU2AF异二聚体的结合活性是必需的。此外，从果蝇大亚基单体（dU2AF 50 ΔRS）中除去RS结构域严重损害了其结合活性。但是，如果以与dU2AF 50 ΔRS的复合物形式提供dU2AF 38 RS结构域，则会恢复高亲和力结合。这些结果表明，无论大亚基还是小亚基，U2AF的一个RS结构域的存在都会促进高亲和力的嘧啶束RNA结合活性，这与体内U2AF RS结构域的冗余作用相一致。

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