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一个主要来源。这一过程需要剪接机构(剪接体)在数千个核苷酸中选择正确的剪接位置。然而，剪接位点究竟是如何识别的还知之甚少。这项研究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顺序三维相互作用。在3‘剪接位点识别的关键早期阶段，重要的剪接因子U2AF识别多嘧啶链(Py-TRAIL)前-mRNA共有序列。反过来，U2AF/Py-道提供了一个独特的分子表面，促进了活性剪接体的核心成分U2 SnRNP的结合。这项建议的具体目标是： 1.阐明使U2AF识别各种后生动物Py区的相互作用 序列，而不是序列特定的选择性剪接因子。 2.研究与U2AF的序列相互作用，将U2SnRNP募集到Pre-mRNA。 3.探索在多种剪接因子中发现的其他U2AF同源基序(UHM)的潜在蛋白质相互作用靶点。特别是，我们关注的是与医学相关的靶点TAT-SF1的UHM，TAT-SF1是HIV-1复制的细胞辅助因子。 许多严重的人类疾病与错误剪接的mRNA变体有关，包括癌症中的CD44或BRCA1/BRCA2，肌营养不良症中的dystrophin，以及共济失调毛细血管扩张症中的ATM等。此外，TAT-SF1蛋白介导的蛋白质-蛋白质相互作用的中断为艾滋病患者的治疗提供了一个潜在的治疗靶点。从长远来看，了解指导正常剪接位点识别的关键相互作用将为针对有害剪接变体的特定分子策略提供基础，如肽仿制或反义寡核苷酸。

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.