REGULATION OF POLYADENYLATION--THE U1A/U1 SNRNP PARADIGM

真核生物前mRNA加工的转录后调控 （剪接、多聚腺苷酸化、编辑等）是重要的控制点 在真核生物的基因表达中。我们最近发现了一种新的 基因调控，其中U1 A蛋白，U1 snRNP的组分， 参与前体mRNA剪接，负性自动调节其自身的产生 通过结合并抑制其自身前体mRNA（38- 41）。U1 A和U1 A前体mRNA之间的复合物结合并阻断U1 A前体mRNA。 多聚腺苷酸聚合酶（PAP）的活性，该酶催化添加 polyA尾巴U1 A自动调节是此类产品的第一个例子。作为 在最初的建议中，我们描述了另一种系统， U1 snRNA相关的70 K蛋白也通过 居住在PAP。然而，与U1 A的情况不同，70 K抑制PAP， 通过U1 snRNP复合物与前mRNA间接结合， 与前体mRNA碱基配对令人惊讶的是，即使U1 A存在， 在U1 snRNP复合物中，PAP抑制活性完全存在于 70 K蛋白质发现多聚腺苷酸化抑制基序 在U1 A和70 K中是保守的，并且在其他蛋白质中发现， 通过PAP抑制的多聚腺苷酸化调节将更加广泛， 比以前认为的。 该提案的框架是使用U1 A和70 K的示例， 实现以下目标的基础是：1）确定如何 PAP的普遍抑制是一种调节机制， 另外的实施例，2）表征顺式和反式序列 为了定义一个能够控制基因的蛋白质家族， 通过抑制PAP的表达，和3）以确定这种类型的pf 调控可用于控制异源基因在细胞中的表达， vivo.这一提议的实现将促进基础知识的发展 前体mRNA加工的一般性，并表明调节的一般性， U1 snRNA相关蛋白的多聚腺苷酸化。通过阐明 这两种剪接相关蛋白的分子机制 控制多聚腺苷酸化机制，我们将提供深入了解 剪接和多聚腺苷酸化之间的竞争， 前体mRNA的可变加工是如何控制的。

Post-transcriptional regulation of the processing of eukaryotic pre-mRNA (splicing, polyadenylation, editing, etc...) is an important control point in gene expression in eukaryotes. We recently discovered a novel form of gene regulation where the U1A protein, a component of the U1 snRNP involved in pre-mRNA splicing, negatively autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA (38- 41). The complex between U1A and the U1A pre-mRNA binds to and blocks the activity of polyA polymerase (PAP), the enzyme which catalyzes addition of the polyA tail. U1A autoregulation is the first example of its kind. As part of the original proposal we characterized another system in which the U1 snRNA-associated 70K protein inhabits polyadenylation by also inhabiting PAP. Unlike the case of U1A, however, 70K inhibits PAP while being indirectly bound to the pre-mRNA through the U1 snRNP complex which is base-paired to the pre-mRNA. Surprisingly, even though U1A is present in the U1 snRNP complex, the PAP-inhibitory activity resides entirely with the 70K protein. The discovery that the polyadenylation inhibitory motifs in both U1A and 70K are conserved and are found in other proteins suggests that polyadenylation regulation via PAP inhibition will be more widespread than previously thought. The framework for this proposal is to use the examples of U1A and 70K as a basis to achieve the following goals which are: 1) to determine how prevalent inhibition of PAP is as a regulatory mechanism by finding additional examples, 2) to characterize the cis- and transacting sequence motifs in order to define a family of proteins able to control gene expression via inhibition of PAP, and 3) to determine whether this type pf regulation can be used to control expression of heterologous genes in vivo. The achievement of this proposal will advance fundamental knowledge of pre-mRNA processing and indicate the generality of regulation of polyadenylation by U1 snRNA-associated proteins. By elucidating the molecular mechanisms by which these two splicing-associated proteins control the polyadenylation machinery, we will provide insight into the competition between splicing and polyadenylation that in many cases forms the basis for how alterative processing of pre-mRNAs is governed.