Zhang W J, Wu J Y
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Mol Cell Biol. 1998 Feb;18(2):676-84. doi: 10.1128/MCB.18.2.676.
Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5' and 3' splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.
以往的研究表明，剪接因子之间的蛋白质-蛋白质相互作用可能在mRNA前体剪接中发挥重要作用。我们在此报告一种新的剪接因子Sip1（SC35相互作用蛋白1）的鉴定及功能特征。Sip1最初是因其与SR家族的剪接因子SC35相互作用而被鉴定出来的。Sip1不仅与几种SR蛋白相互作用，还与分别与5'和3'剪接位点相关的U1-70K和U2AF65蛋白相互作用。预测的Sip1序列包含一个富含精氨酸-丝氨酸（RS）的结构域，但没有任何已知的RNA结合基序，这表明它不是SR家族的成员。Sip1还包含一个与果蝇剪接调节因子白杏抑制因子（SWAP）序列相似性较弱的区域。抗Sip1抗体使HeLa细胞核提取物的剪接活性降低，这一观察结果表明Sip1在mRNA前体剪接中起重要作用。纯化的重组Sip1蛋白，而不是其他含RS结构域的蛋白，如SC35、ASF/SF2和U2AF65，恢复了Sip1免疫耗尽提取物的剪接活性。添加U2AF65蛋白进一步增强了Sip1蛋白的剪接重建作用。Sip1耗尽的细胞核提取物中A和B剪接复合物形成的缺陷表明Sip1在剪接体组装中起重要作用。总之，这些结果表明Sip1是一种mRNA前体剪接所需的新型含RS结构域的蛋白，并且Sip1在剪接中的功能作用与已知的含RS结构域的剪接因子不同。
