Rudner D Z, Breger K S, Kanaar R, Adams M D, Rio D C
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3204, USA.
Mol Cell Biol. 1998 Jul;18(7):4004-11. doi: 10.1128/MCB.18.7.4004.
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 the Drosophila 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 dU2AF38 was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF50DeltaRS) severely impaired its binding activity. However, if the dU2AF38 RS domain was supplied in a complex with dU2AF50DeltaRS, 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.
前体信使核糖核酸（pre-mRNA）剪接因子U2AF（U2小核核糖核蛋白颗粒[snRNP]辅助因子）在3'剪接位点选择中起关键作用。U2AF特异性结合分支点与3'剪接位点之间内含子嘧啶序列，并在剪接体组装的早期步骤将U2 snRNP靶向分支位点。人U2AF是由大亚基（hU2AF65）和小亚基（hU2AF35）组成的异二聚体。hU2AF65包含一个富含精氨酸-丝氨酸的（RS）结构域和三个RNA识别基序（RRMs）。hU2AF35有一个简并的RRM和一个羧基末端RS结构域。遗传学研究最近表明，果蝇U2AF亚基同源物上的RS结构域各自并非必需，且在体内可能具有冗余功能。U2AF异二聚体的位点特异性嘧啶序列结合活性先前已归因于hU2AF65。虽然hU2AF65上三个RRMs的需求已得到明确证实，但大亚基RS结构域在RNA结合中的作用仍未解决。我们在体外分析了U2AF异二聚体的RNA结合活性。当果蝇小亚基同源物（dU2AF38）与大亚基（dU2AF50）嘧啶序列形成复合物时，RNA结合活性比游离的dU2AF50增加了20倍。当我们比较人U2AF异二聚体和hU2AF65时，我们检测到RNA结合活性有类似的增加。令人惊讶的是，dU2AF38上的RS结构域对于dU2AF异二聚体结合活性的增加是必需的。此外，从果蝇大亚基单体（dU2AF50DeltaRS）中去除RS结构域会严重损害其结合活性。然而，如果将dU2AF38 RS结构域与dU2AF50DeltaRS形成复合物提供，高亲和力结合得以恢复。这些结果表明，U2AF的一个RS结构域，无论在大亚基还是小亚基上，都能促进高亲和力嘧啶序列RNA结合活性，这与U2AF RS结构域在体内的冗余作用一致。
