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.
The intron lariat pathway is an understudied area of RNA processing, yet has a demonstrated connection to aberrant splicing in cancer. This proposal identifies TTDN1 as a novel regulator of DBR1 and the intron lariat pathway, and characterizes how the pathological alteration of TTDN1 drives aberrant RNA processing and isoform expression in different cancer and organ contexts. The identification of novel regulatory mechanisms connecting RNA processing to transcriptional integrity has far-reaching impacts for the many genetic disorders and cancers that feature defects in alternative splicing.
