Regulation of RNA processing by the novel spliceosomal protein, TTDN1, in development and cancer

摘要 通过选择性剪接将初级转录产物转化为多个不同的成熟mRNA 促进细胞和组织的蛋白质多样性，这是正常细胞功能所必需的。异常 选择性剪接与许多疾病有关，包括扩张型肌萎缩侧索硬化症 心肌病和多种癌症类型，但RNA处理失调的机制 调节选择性剪接的通路还不是很清楚。RNA加工途径的中断是如何 转换为另一种剪接结果是一个正在兴起的感兴趣的领域，最近由 下一代测序技术和全基因组分析的进展。因此，理解 RNA加工蛋白对基因组完整性的贡献对开发治疗方法至关重要。 这些研究将确定TTDN1蛋白作为一种新的RNA加工过程的作用 蛋白。TTDN1基因突变在大多数非光敏性毛发硫代营养不良患者中普遍存在 (NP-TTD)，遗传性发育障碍。NP-TTD属于核苷酸切除修复类-- 尽管有缺陷性疾病，但NP-TTD病例被认为是DNA修复专家。然而，分子缺陷 潜在的NP-TTD尚不清楚。此外，TTDN1在某些癌症中过表达，包括宫颈癌、 前列腺癌和食道癌，这种过度表达预示着更糟糕的预后。我的初步数据 表明TTDN1通过调节内含子套索去分支酶DBR1促进mRNA的加工，以及 这表明这种相互作用对于适当表达可选的转录物亚型是至关重要的。重要的是 与内含子套索加工缺陷同时存在的去调控的DBR1表达，最近被认为与 TTDN1的异常异构体表达和肿瘤发生，但TTDN1在癌症中的功能贡献尚不清楚。 我的初步数据有力地表明了TTDN1和DBR1之间的物理和功能联系，它们联系在一起 前信使核糖核酸的加工和异构体的交替调节。目标1将确定TTDN1-DBR1的影响 内含子套索加工途径分子调控中的相互作用。目标2将确定异构体 在使用TTDN1-/-癌细胞系的恶性和组织特异性环境中TTDN1的调节以及 已经建立了TTDN1缺陷小鼠模型。在这些研究完成后，TTDN1在 将定义调节内含子套索加工，以及对同型表达的后果 TTDN1表达的病理变化。新的RNA连接调控机制的鉴定 对转录完整性的处理对许多遗传疾病和癌症具有广泛的影响 替代剪接中的特征缺陷。

Abstract Conversion of primary mRNA transcripts into multiple distinct mature mRNA’s by alternative splicing promotes cell- and tissue-specific protein diversity that is necessary for normal cellular function. Abnormal alternative splicing is implicated in many diseases, including amyotrophic lateral sclerosis, dilated cardiomyopathy, and multiple cancer types, yet the mechanisms by which dysregulation of RNA processing pathways modulate alternative splicing are not well understood. How the disruption of RNA processing pathways translates to alternative splicing consequences is a rising area of interest that has recently been catalyzed by advances in next-generation sequencing technologies and genome-wide analysis. Therefore, understanding the contribution of RNA processing proteins to genome integrity is paramount to developing therapeutic approaches. These studies will define the role of the largely uncharacterized protein TTDN1 as a novel RNA processing protein. Mutations in TTDN1 are prevalent in the majority of cases of non-photosensitive trichothiodystrophy (NP-TTD), an inherited developmental disorder. NP-TTD belongs to the class of nucleotide excision repair- defective disorders, yet NP-TTD cases are considered DNA repair proficient. However, the molecular defects underlying NP-TTD are unknown. Additionally, TTDN1 is overexpressed in certain cancers, including cervical, prostate, and esophageal cancers, and this overexpression predicts a worse prognosis. My preliminary data indicate TTDN1 promotes mRNA processing by regulating the intron lariat debranching enzyme DBR1, and suggests this interaction is crucial for proper expression of alternative transcript isoforms. Importantly, deregulated DBR1 expression, concurrent with intron lariat processing defects, has been recently linked to aberrant isoform expression and oncogenesis, but the functional contribution of TTDN1 in cancer is unknown. My preliminary data strongly suggests a physical and functional link between TTDN1 and DBR1, tying together pre-mRNA processing and alternative isoform regulation. Aim 1 will determine the influence of the TTDN1-DBR1 interaction on the molecular regulation of the intron lariat processing pathway. Aim 2 will determine isoform regulation by TTDN1 in both malignant and tissue-specific settings using TTDN1-/- cancer cell lines as well as an already established TTDN1-deficient mouse model. Upon completion of these studies, the role of TTDN1 in regulating intron lariat processing will be defined, as will the consequences for isoform expression upon pathological alteration of TTDN1 expression. The identification of novel regulatory mechanisms connecting RNA processing to transcriptional integrity has broad implications for the many genetic disorders and cancers that feature defects in alternative splicing.