Day Irene S, Golovkin Maxim, Palusa Saiprasad G, Link Alicia, Ali Gul S, Thomas Julie, Richardson Dale N, Reddy Anireddy S N
Department of Biology, Program in Molecular Plant Biology, Program in Cell and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
Plant J. 2012 Sep;71(6):936-47. doi: 10.1111/j.1365-313X.2012.05042.x. Epub 2012 Jun 28.
SR45 is a serine/arginine-rich (SR)-like protein with two arginine/serine-rich (RS) domains. We have previously shown that SR45 regulates alternative splicing (AS) by differential selection of 5' and 3' splice sites. However, it is unknown how SR45 regulates AS. To gain mechanistic insights into the roles of SR45 in splicing, we screened a yeast two-hybrid library with SR45. This screening resulted in the isolation of two spliceosomal proteins, U1-70K and U2AF(35) b that are known to function in 5' and 3' splice site selection, respectively. This screen not only confirmed our prior observation that U1-70K and SR45 interact, but also helped to identify an additional interacting partner (U2AF(35) ). In vitro and in vivo analyses revealed an interaction of SR45 with both paralogs of U2AF(35) . Furthermore, we show that the RS1 and RS2 domains of SR45, and not the RNA recognition motif (RRM) domain, associate independently with both U2AF(35) proteins. Interaction studies among U2AF(35) paralogs and between U2AF(35) and U1-70K revealed that U2AF(35) can form homo- or heterodimers and that U2AF(35) proteins can associate with U1-70K. Using RNA probes from SR30 intron 10, whose splicing is altered in the sr45 mutant, we show that SR45 and U2AF(35) b bind to different parts of the intron, with a binding site for SR45 in the 5' region and two binding regions, each ending with a known 3' splice site, for U2AF(35) b. These results suggest that SR45 recruits U1snRNP and U2AF to 5' and 3' splice sites, respectively, by interacting with pre-mRNA, U1-70K and U2AF(35) and modulates AS.
SR45是一种富含丝氨酸/精氨酸（SR）的类蛋白，具有两个富含精氨酸/丝氨酸（RS）的结构域。我们之前已经表明，SR45通过对5'和3'剪接位点的差异选择来调节可变剪接（AS）。然而，尚不清楚SR45如何调节AS。为了深入了解SR45在剪接中的作用机制，我们用SR45筛选了一个酵母双杂交文库。该筛选结果分离出了两种剪接体蛋白，U1-70K和U2AF(35)b，已知它们分别在5'和3'剪接位点选择中发挥作用。该筛选不仅证实了我们之前观察到的U1-70K和SR45相互作用，还帮助鉴定了另一个相互作用伙伴（U2AF(35)）。体外和体内分析揭示了SR45与U2AF(35)的两个旁系同源物之间的相互作用。此外，我们表明，SR45的RS1和RS2结构域而非RNA识别基序（RRM）结构域，分别独立地与两种U2AF(35)蛋白结合。U2AF(35)旁系同源物之间以及U2AF(35)与U1-70K之间的相互作用研究表明，U2AF(35)可以形成同二聚体或异二聚体，并且U2AF(35)蛋白可以与U1-70K结合。使用来自SR30内含子10的RNA探针（其剪接在sr45突变体中发生改变），我们表明SR45和U2AF(35)b结合到内含子的不同部分，SR45的结合位点在5'区域，而U2AF(35)b有两个结合区域，每个区域都以一个已知的3'剪接位点结束。这些结果表明，SR45通过与前体mRNA、U1-70K和U2AF(35)相互作用，分别将U1snRNP和U2AF募集到5'和3'剪接位点，并调节AS。
