MECHANISMS OF ALTERNATIVE SPLICING OF PRE MRNA

本提案中描述的实验旨在深入了解 前体mRNA剪接的机制和调控。 大部分 工作集中在一个保守的蛋白质家族，称为SR蛋白， 是体外剪接所必需的， 选择性剪接位点 snRNAs在剪接中作用的研究 催化作用也将继续进行。 提出了四个具体目标。 1. SR蛋白功能的体外分析 研究将集中在丰富的 SR蛋白，包括原型ASF/SF 2，以及SC 35、SRp 20和 SRp40。 若干未决问题将得到处理。 一个问题是 对于蛋白质-RNA和蛋白质-蛋白质 交互. 将采用多种分析方法来检测差异 在单个蛋白质的行为中。 磷酸化的影响， 还将检查RNA和蛋白质结合上RS结构域。 2. 体内SR蛋白功能分析 转染测定，涉及 将继续在哺乳动物细胞中过表达SR蛋白， 提供了进一步了解SR蛋白在调节 剪接，包括自动调节，以及其他最近提出的 如核质转运过程。 细胞系生产 使用鸡B细胞系将产生不同量的ASF/SF 2 DT 40，并将确定对细胞生长和剪接的影响。 3. CLK激酶的功能和调节。 Clk/Sty激酶是 含有N-末端RS区域和C-末端 催化域 最近的研究表明Clk/Sty与某些 SR蛋白，磷酸化RS结构域中的丝氨酸，并调节剪接 将进行体外和体内的位点选择。 的可能性 不同的Clk家族成员靶向不同的SR蛋白。 将研究激酶本身的调节。 4.功能 SNRNA在剪接催化中的作用 涉及转染的遗传学研究 将继续进行检测，以进一步研究U2-U6-pre-mRNA 催化剪接所需的相互作用。 相互的要求 将进行涉及U2、U6和前mRNA的排他性碱基配对， 对分子内U6螺旋的研究也是如此， 催化作用 纯化的U2、U6和模型前体RNA之间的相互作用将 在体外进行研究。

The experiments described in this proposal are designed to gain insight into both the mechanism and regulation of pre-mRNA splicing. Much of the work focuses on a family of conserved proteins known as SR proteins that are both essential for splicing in vitro and also able to modulate the use of alternative splice sites. Studies on the role of snRNAs in splicing catalysis will also be pursued. Four Specific Aims are proposed. 1. ANALYSIS OF SR PROTEIN FUNCTION IN VITRO. Studies will focus on abundant SR proteins including the prototype ASF/SF2, as well as SC35, SRp20 and SRp40. Several outstanding issues will be addressed. One is the question of specificity, both with respect to protein-RNA and protein-protein interactions. A number of assays will be employed to detect differences in the behavior of individual proteins. The effects of phosphorylation of the RS domain on RNA and protein binding will also be examined. 2. ANALYSIS OF SR PROTEIN FUNCTION IN VIVO. Transfection assays involving overexpression of SR proteins in mammalian cells will be continued to provide further insight into the role of SR proteins in modulating splicing, including autoregulation, and in other recently suggested processes such as nuclear-cytoplasmic transport. Cell lines producing different amounts of ASF/SF2 will produced using the chicken B cell line DT40, and effects on cell growth and splicing will be determined. 3. FUNCTION AND REGULATION OF CLK KINASES. Clk/Sty kinase is the prototype of a family of kinases containing N-terminal RS regions and C-terminal catalytic domains. Recent studies showing that Clk/Sty binds to certain SR proteins, phosphorylates serines in the RS domain, and modulates splice site selection in vitro and in vivo will be pursued. The possibility that different Clk family members target distinct SR proteins will be examined. Regulation of the kinases themselves will be studies. 4. FUNCTION OF SNRNAs IN SPLICING CATALYSIS. Genetic studies involving transfection assays will be continued to investigate further the U2-U6-pre-mRNA interactions required to catalyze splicing. The requirements of mutually exclusive base pairing involving U2, U6 and the pre-mRNA will be pursued, as will studies of an intramolecular U6 helix that may be critical for catalysis. Interactions between purified U2, U6 and a model pre-RNA will be studied in vitro.