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mRNA转录物转化为多种不同的成熟mRNA 促进正常细胞功能所必需的细胞和组织特异性蛋白质多样性。异常 选择性剪接与许多疾病有关，包括肌萎缩性侧索硬化症、扩张型脊髓灰质炎、脊髓灰质炎和脊髓灰质炎。 心肌病和多种癌症类型，但RNA加工失调的机制， 调节选择性剪接的途径还不清楚。RNA加工途径的中断 翻译为选择性剪接的后果是一个不断上升的兴趣领域，最近已经催化， 下一代测序技术和全基因组分析的进展。因此了解 RNA加工蛋白对基因组完整性的贡献对于开发治疗方法是至关重要的。 这些研究将确定TTDN 1作为一种新的RNA加工蛋白的作用， 蛋白TTDN 1突变在大多数非光敏性谷胱甘肽营养不良病例中普遍存在。 （NP-TTD），一种遗传性发育障碍。NP-TTD属于核苷酸切除修复类- 尽管NP-TTD病例被认为是DNA修复熟练的。然而，分子缺陷 潜在的NP-TTD未知。此外，TTDN 1在某些癌症中过表达，包括宫颈癌， 前列腺癌和食管癌，这种过度表达预示着更差的预后。我的初步数据 表明TTDN 1通过调节内含子Ltd 1去分支酶DBR 1促进mRNA加工，和 表明这种相互作用对于选择性转录异构体的正确表达至关重要。重要的是， DBR 1的表达失调，同时伴有内含子的加工缺陷，最近被认为与 TTDN 1在肿瘤中的功能作用尚不清楚。 我的初步数据强烈表明TTDN 1和DBR 1之间存在物理和功能联系， 前mRNA加工和替代亚型调节。目标1将确定TTDN 1-DBR 1的影响 相互作用对内含子lactinase加工途径的分子调控。Aim 2将确定亚型 使用TTDN 1-/-癌细胞系以及肿瘤细胞系， 已经建立了TTDN 1缺陷小鼠模型。在完成这些研究后，TTDN 1在 将定义调节内含子的内含子加工，也将定义 TTDN 1表达的病理改变。新的RNA调控机制的鉴定 转录完整性的加工对许多遗传疾病和癌症具有广泛的影响， 在选择性剪接中的特征缺陷。

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.