SR PROTEIN REGULATION OF ALTERNATIVE PRE-MRNA SPLICING

这项建议的具体目的是了解通过什么机制 前体信使RNA(Pre-mRNA)内含子的剪接受到调控。 剪接是一种非常高效的过程，在许多情况下涉及 对选择性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体，剪接体，形成 通过其亚基的有序组装，SNRNP，到新生的Pre-RNPs上 MRNA.该组装定义了剪接将在其上的内含子边界 发生。L此前曾确认、隔离并刻画了一个家庭的特征 前信使核糖核酸剪接因子所需的最早步骤 剪接体组装。我把它们命名为丝氨酸的SR蛋白质 它们都含有富含精氨酸的C-末端结构域。至少有六个 SR蛋白家族成员，每个成员都在 后生动物的进化。我已经证明，至少有一名 SR蛋白家族成员是进行前mRNA剪接所必需的，并且 不同的家族成员有不同的能力来促进剪接 备选剪接位点。这些替代剪接因子是必需的 对于剪接体组装的第一步，U1 SnRNP与 Pre-mRNA和不同的SR蛋白具有不同的募集能力 U1 SnRNP连接到替代的5‘剪接位点。 我们对剪接体组装调控机制的理解 将通过对SR蛋白如何在体内发挥作用的了解来进一步 这项规定。为了实现这一目标，我们将研究 SR蛋白与前信使核糖核酸的相互作用识别信号 SR蛋白通过其新生的转录本来指导替代 拼接站点使用情况。我们将分析SR的不同域 通过结构域互换实验了解蛋白质的功能 SR蛋白共享的不同的不同结构域。我们将确定 与不同SR蛋白特异相互作用的蛋白质因子 为了了解SR蛋白促进作用的机制 剪接体组装以及它们是否在其他步骤中起作用 剪接体组装和剪接。

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.