SR PROTEIN REGULATION OF ALTERNATIVE PRE-MRNA SPLICING

本提案的具体目的是了解 内含子从前体信使RNA（pre-mRNA）的剪接受到调节。 剪接是一种高效的过程，在许多情况下， 可变剪接位点的规范使用。拼接是由一个大的 多组分核糖核蛋白复合物，即剪接体， 它的亚基snRNP有序地组装在新生的前体上， mRNA。这个装配定义了内含子边界，剪接将在内含子边界上进行。 发生.我之前已经确认，分离和鉴定了一个家庭 前体mRNA剪接因子，这是所需的最早的步骤， 剪接体组装。我将这些蛋白质命名为丝氨酸SR蛋白质， 富含精氨酸的C末端结构域。至少有六 SR蛋白家族成员，其中每一个都在 后生动物的进化我已经证明，至少有一个 SR蛋白家族成员是前mRNA剪接所需的， 不同的家族成员有不同的能力，以促进剪接， 可变剪接位点。这些选择性剪接因子是必需的 在剪接体组装的第一步，U1 snRNP与 前体mRNA和不同的SR蛋白具有不同的募集能力， U1 snRNP至可变5'剪接位点。 我们对剪接体组装调控机制的理解 将进一步了解SR蛋白如何在 这个规定。为了实现这一目标，我们将研究 SR蛋白与前体mRNA的相互作用，以识别 新生转录本，通过这些转录本，SR蛋白可以指导选择性转录， 剪接位点使用。我们将分析SR的不同域 通过结构域交换实验，了解蛋白质的功能， SR蛋白共有的不同结构域。我们将确定 与不同SR蛋白特异性相互作用的蛋白因子 为了了解SR蛋白促进 剪接体组装以及它们是否在其他步骤中起作用， 可拼接组装和拼接。

The specific aim of this proposal is to understand the mechanisms by which splicing of introns from precursor messenger RNA (pre-mRNA) is regulated. Splicing is a highly efficient process which in many instances involves the regulated use of alternative splice sites. Splicing is done by a large multicomponent ribonucleoprotein complex, the spliceosome, which is formed by an ordered assembly of its subunits, the snRNPs, onto the nascent pre- mRNA. This assembly defines the intron boundaries at which splicing will occur. l have previously identified, isolated and characterized a family of pre-mRNA splicing factors which are required for the earliest step of spliceosome assembly. I named these the SR proteins for the serine and arginine rich C-terminal domain they all contain. There are at least six SR protein family members, each of which has been highly conserved in evolution in metazoans. I have shown that the presence of at least one of the SR protein family members is required for pre-mRNA splicing, and that different family members have distinct abilities to promote splicing at alternative splice sites. These alternative splicing factors are required for their first step of spliceosome assembly, binding of the U1 snRNP to the pre-mRNA, and different SR proteins have distinct abilities to recruit U1 snRNP to alternative 5' splice sites. Our understanding of the mechanisms that regulate spliceosome assembly will be furthered by an understanding of how the SR proteins function in this regulation. In order to accomplish this, we will study the interactions of SR proteins with the pre-mRNA to identify signals on the nascent transcripts through which SR proteins act to direct alternative splice site usage. We will analyze the different domains of the SR proteins through domain swap experiments to understand the functions of the different distinct domains which SR proteins share. We will identify protein factors which interact specifically with the different SR proteins in order to understand the mechanism by which SR proteins promote spliceosome assembly and whether they function at other steps of spliceosome assembly and splicing.