MOLECULAR RECOGNITION DURING PRE-MRNA SPLICING

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们提出的总体目标是确定3？剪接位点识别大多数人类基因转录物受前体mRNA剪接调控。剪接位点被剪接体的蛋白质和RNA组分顺序识别，剪接体是由100多种蛋白质和5种小的核RNA组成的前体mRNA剪接机器。由必需剪接因子SF 1、U2 AF 65和U2 AF 35组成的复合物识别前mRNA信号并将核心剪接机制募集到靶剪接位点。该建议的具体目标是解决以下关于前体mRNA剪接的关键早期阶段的中心问题：（1）缺乏结构同源物的高度保守的SF 1结构域的结构是什么？(2)通过什么样的结构方式使SF 1结构域的磷酸化增强与U2 AF的结合？(3)U2 AF通过什么结构方式适应不同的剪接位点？(4)SF 1/U2 AF复合物与靶剪接位点的三维结构是什么？同步加速器辐射对于实现这一提议的目标至关重要，原因包括：（一）多波长反常色散实验需要可调谐波长;（二）机器人的能力极大地便利了为目标1确定有用晶体所需的广泛筛选;（三）晶体尺寸小，因此使用传统的内部X射线源衍射非常微弱。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of our proposed is to determine the structural basis for 3? splice site recognition. The majority of human gene transcripts are regulated by pre-mRNA splicing. The splice sites are sequentially recognized by protein and RNA components of the spliceosome, a pre-mRNA splicing machine composed of more than 100 proteins and 5 small nuclear RNAs. A complex composed of essential splicing factors SF1, U2AF65, and U2AF35 recognizes the pre-mRNA signals and recruits the core splicing machinery to a target splice site. Specific aims of this proposal address the following central questions concerning the critical early stages of pre-mRNA splicing: (1) What is the structure of a highly-conserved SF1 domain that lacks structural homologues? (2) By what structural means does phosphorylation of this SF1 domain enhance association with U2AF? (3) By what structural means does U2AF adapt to diverse splice sites? (4) What is the three-dimensional architecture of the SF1 / U2AF complex with the target splice site? Synchrotron radiation is essential to address the aims of this proposal for reasons including: (i) Tunable wavelengths are necessary for multiwavelength anomalous dispersion experiments; (ii) The robotic capability greatly facilitates the extensive screening required to identify useful crystals for Aim 1; (iii) Crystal size is small and consequently diffraction is prohibitively weak using conventional in-house x-ray sources.