Molecular Recognition during pre-mRNA Splicing

选择性前mrna剪接是高等真核生物蛋白质组多样性的主要来源。这一过程需要剪接机制（剪接体）在mrna前序列的数千个核苷酸中选择正确的剪接位点。然而，人们对剪接位点究竟是如何被识别的还知之甚少。本研究计划的总体目标是了解指导3'剪接位点选择和促进剪接体组装的连续三维相互作用。在3'剪接位点识别的关键早期阶段，基本剪接因子U2AF识别聚嘧啶通道（Py-tract）前mrna一致序列。反过来，U2AF/ py通道提供了一个独特的分子表面，促进U2 snRNP的结合，U2 snRNP是活性剪接体的核心成分。该建议的具体目标是：

Alternative pre-mRNA splicing is a major source of proteomic diversity in higher eukaryotes. This process requires that the splicing machinery (spliceosome) select the correct splice sites within thousands of nucleotides of pre-mRNA sequences. However, exactly how the splice sites are recognized is poorly understood. The overall goal of this research proposal is to understand the sequential three-dimensional interactions that guide 3' splice site selection and promote spliceosome assembly. During the critical early stages of 3' splice site recognition, the essential splicing factor U2AF recognizes the poly-pyrimidine tract (Py-tract) pre-mRNA consensus sequence. In turn, the U2AF/Py-tract provides a distinct molecular surface that promotes association of the U2 snRNP, a core component of the active spliceosome. The specific aims of the proposal are: 1. To elucidate the interactions that enable U2AF to recognize a variety of metazoan Py-tract sequences, as opposed to sequence-specific alternative splicing factors. 2. To investigate the sequential interactions with U2AF that recruit the U2 snRNP to the pre-mRNA. 3. To explore the potential protein-interaction targets of other U2AF-homologous motifs (UHM) found in a wide variety of splicing factors. In particular, we focus on the UHM of a medically-relevant target, Tat-SF1, a cellular cofactor for HIV-1 replication. Many serious human diseases are associated with mis-spliced mRNA variants, including CD44 or BRCA1/BRCA2 in cancers, dystrophin in muscular dystrophy, and ATM in ataxia telangiectasia among others. Moreover, disruption of protein-protein interactions mediated by the Tat-SF1 protein presents a potential therapeutic target for treatment of AIDS patients. In the long term, understanding the key interactions that guide normal splice site recognition will provide a basis to target specific molecular strategies, such as peptidomimetics or anti-sense oligonucleotides, against harmful splice variants.