Regulation of Polyadenylation: the U1A/U1snRNP Paradigm

描述(申请人提供)：真核细胞前信使核糖核酸加工的转录后调控(剪接、多聚腺苷化、编辑等)是真核生物基因表达的重要控制点。对于几乎所有的mRNAs来说，Polya尾巴对于翻译、稳定性和核出口都是必不可少的。Polya尾巴位置的变化会导致最终mRNA序列的变化，从而导致额外的调控可能性。对Polya站点的监管通常被认为是罕见的，无论是通过替代Polya站点的选择还是通过单一Polya站点的“开关”。值得注意的是，现在估计有30%的人类基因表达为具有不同PolyA位点的mRNAs，这个数字可能适用于其他脊椎动物，因为人类基因组和EST数据库是脊椎动物中最完整和注释最好的。即使这30%中只有一小部分代表受监管的Polya网站，我们可能会看到这种监管形式的频率和重要性出现戏剧性的向上修正。在这笔赠款的第一个资助期，我们：1)鉴定了U1a剪接因子对Polya尾部的一种独特的调控形式；2)发现U1a通过结合非共识的前-mRNA序列，可以调节Polya尾部的添加，而不仅仅是对其自身，并且其他蛋白质也能够以类似的方式调节Polya的尾部添加；以及3)证明了U1 SnRNP可以被用作一种工具，通过抑制Polya的尾部添加来特异性地抑制特定内源基因的表达。该建议的框架建立在第一个资助期所做工作的基础上，以实现以下目标：1)改进/表征U1in技术，从而使我们和其他人能够充分发挥其潜力；2)研究一种新型的Polya尾部调节，包括自然发生的U1 SnRNP抑制内源基因表达的例子；以及3)识别/表征U1a或先前被证明具有调节PolyA尾部添加能力的蛋白质靶向调节Polya尾部的新基因。这些目标的实现将促进对基因表达调控机制的基础知识，充分开发一种有前景的新的基因抑制技术，并阐明剪接机制的组成部分如何与聚腺苷酸化机制相互作用和调节的功能后果。

DESCRIPTION (provided by applicant): Post-transcriptional regulation of the processing of eukaryotic pre-mRNA (splicing, polyadenylation, editing etc.) is an important control point in gene expression in eukaryotes. For nearly all mRNAs, the polyA tail is essential for translation, stability, and nuclear export. Changes in the position of the polyA tail cause changes in the final mRNA sequence leading to additional regulatory possibilities. Regulation of polyA sites, either by alternative polyA site choice or by an "on-off" switch of a single polyA site, was generally considered rare. Remarkably, it is now estimated that 30% of human genes are expressed as mRNAs with different polyA sites, a number likely to be applicable to other vertebrates, as the human genome and EST database are the most complete and best annotated of the vertebrates. Even if only a fraction of this 30% represents regulated polyA sites, we are likely to see a dramatic upward revision in the frequency and importance of this form of regulation. In the first funding period of this grant we: 1) characterized a unique form of polyA tail regulation by the U1A splicing factor, 2) discovered that U1A, via binding non-consensus pre-mRNA sequences, can regulate polyA tail addition to genes other than just to itself and that other proteins are also capable of regulating polyA tail addition in a similar manner, and 3) demonstrated that U1 snRNP can be used as a tool to specifically inhibit expression of specific endogenous genes by inhibiting polyA tail addition. The framework of this proposal builds on work from the first funding period to achieve the following goals: 1) improve/characterize the U1in technique thereby allowing us and others to exploit it to its full potential, 2) investigate a new type of polyA tail regulation involving naturally occurring examples of U1 snRNP inhibiting expression of endogenous genes, and 3) identify/characterize new genes targeted for polyA tail regulation by U1A or by proteins previously shown to have the ability to regulate polyA tail addition. The achievement of these goals will advance fundamental knowledge of mechanisms of regulated gene expression, fully develop a promising new gene inhibitory technique, and elucidate the functional consequences for how components of the splicing machinery nteract with and regulate the polyadenylation machinery.