Phosphorylation of the ASE/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing
Phosphorylation of the ASE/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing
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DOI：
10.1101/gad.11.3.334
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发表时间：
1997-02-01
影响因子：
10.5
通讯作者：
Manley, JL
中科院分区：
文献类型：
--
作者：
Xiao, SH;Manley, JL
ASF/SF 2是已知为SR蛋白的剪接因子的保守家族的成员。这些蛋白质，这是必要的体外剪接，含有一个或两个氨基末端的RNP型RNA结合结构域和广泛磷酸化的羧基末端区域丰富的重复Arg-Ser二肽（RS结构域）。以前的研究表明，RS结构域参与蛋白质-蛋白质相互作用与其他RS结构域的蛋白质。在这里，我们提供的证据表明，RS结构域的非磷酸化的重组ASF/SF 2是必要的，但不是足够的，在体外结合到UI snRNP特异性70 kD蛋白（70 K）。如果不去除污染RNA，则观察到分离的RS结构域与70 K的明显相互作用，这表明基本RS结构域、RNA和70 K之间存在非特异性桥接。在体外磷酸化的重组ASF/SF 2都显着增强结合70 K，也消除了RS结构域RNA的相互作用。ASF/SF 2可以以RS结构域依赖性、磷酸化增强的方式选择性结合U1 snRNP，这为这些相互作用与剪接相关提供了证据。我们还描述了首次揭示ASF/SF 2体外剪接活性的磷酸化要求的条件。
ASF/SF2 is a member of a conserved family of splicing factors known as SR proteins. These proteins, which are necessary for splicing in vitro, contain one or two amino-terminal RNP-type RNA-binding domains and an extensively phosphorylated carboxy-terminal region enriched in repeating Arg-Ser dipeptides (RS domains). Previous studies have suggested that RS domains participate in protein-protein interactions with other RS domain-containing proteins. Here we provide evidence that the RS domain of unphosphorylated recombinant ASF/SF2 is necessary, but not sufficient, for binding to the UI snRNP-specific 70-kD protein (70K) in vitro. An apparent interaction of the isolated RS domain with 70K was observed if contaminating RNA was not removed, suggesting a nonspecific bridging between the basic RS domain, RNA, and 70K. In vitro phosphorylation of recombinant ASF/SF2 both significantly enhanced binding to 70K and also eliminated the RS domain-RNA interaction. Providing evidence that these interactions are relevant to splicing, ASF/SF2 can bind selectively to U1 snRNP in an RS domain-dependent, phosphorylation-enhanced manner. We also describe conditions that reveal for the first time a phosphorylation requirement for ASF/SF2 splicing activity in vitro.