Molecular Recognition during pre-mRNA Splicing

前体mRNA选择性剪接是高等真核生物蛋白质组多样性的主要来源。这个过程需要剪接机器（剪接体）在前mRNA序列的数千个核苷酸内选择正确的剪接位点。然而，剪接位点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顺序三维相互作用。在3'剪接位点识别的关键早期阶段，必需剪接因子U2 AF识别多嘧啶段（Py-tract）前mRNA共有序列。反过来，U2 AF/Py-tract提供了一个独特的分子表面，促进了U2 snRNP（活性剪接体的核心成分）的缔合。该提案的具体目标是： 1.阐明U2 AF识别多种后生动物Py-tract的相互作用 序列，而不是序列特异性选择性剪接因子。 2.研究与U2 AF的顺序相互作用，将U2 snRNP募集到前体mRNA。 3.探索在多种剪接因子中发现的其他U2 AF同源基序（UHM）的潜在蛋白质相互作用靶点。特别是，我们专注于UHM的医学相关的目标，Tat-SF 1，HIV-1复制的细胞辅因子。 许多严重的人类疾病与错误剪接的mRNA变体相关，包括癌症中的CD 44或BRCA 1/BRCA 2，肌营养不良症中的肌营养不良蛋白，以及共济失调毛细血管扩张症中的ATM等。此外，由Tat-SF 1蛋白介导的蛋白质-蛋白质相互作用的破坏为治疗AIDS患者提供了潜在的治疗靶点。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靶向特定分子策略（如肽模拟物或反义寡核苷酸）提供基础，以对抗有害的剪接变体。

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.