Noncoding RNA Mediated Regulation of Gene Expression in Disease and Development

Mueller, Adam, Biochemistry and Molecular Genetics - Graduate School of Arts and Sciences, University of Virginia
Dutta, Anindya, Department of Biochemistry and Molecular Genetics, University of Virginia

It has become clear that proteins are not the sole regulatory molecules in the nucleus, as numerous functional RNAs are being identified and characterized. Noncoding RNAs are broken into two large classes: (1) short RNAs, typified by miRNAs, which bind and inhibit translation of mRNA; (2) long noncoding RNAs, a diverse class of molecules with a variety of regulatory roles. This dissertation examines two instances of ncRNA mediated regulation: firstly the role of microRNAs in the DNA damage response, and secondly, the role of long noncoding RNAs in skeletal myogenesis.
In our first study, we screened for microRNAs whose expression was increased in a radiosensitive breast cancer cells, and whose basal expression was higher as compared to radioresistant breast cancer cells. The miR-99 family was enriched in this population. We found transfection of miR-99 family members to sensitize radioresistant breast and prostate cancer cell lines to DNA damage. miR-99 transfection inhibited both homologous recombination and non-homologous end joining DNA repair by reducing SNF2H expression. Finally we found that induction of miR-99 following DNA damage suppresses the induction of SNF2H after multiple rounds of DNA damage, resulting in decreased DNA repair. This suggests a molecular mechanism contributing to the efficacy of fractionated radiotherapy.
In our second study, we performed a bioinformatics screen to identify long noncoding RNAs upregulated during skeletal myogenesis. Using publicly available ChIP and RNA sequencing data, we found a set of putative lncRNAs upregulated during myogenic differentiation and validated them by qPCR. Among these hits was one lncRNA encoded in the MyoD Distal Regulatory Region, which we termed MyoD Upstream Non-Coding (MUNC). siRNA knockdown of MUNC significantly impaired myogenesis. MyoD recruitment to a number of its genomic targets was reduced by depleting MUNC, which itself associated with MyoD and these genomic loci. Finally, transfection of MUNC stimulated MyoD-dependent luciferase activity. These results suggest that MUNC can facilitate MyoD transcriptional activity, and contributes to MyoD expression through a feed forward loop.

PHD (Doctor of Philosophy)
Molecular Biology, Cancer, Skeletal Muscle, microRNA, long noncodingRNA, ncRNA, Radiation, Radiation Therapy, Radiosensitivity, Prostate Cancer, Breast Cancer, noncoding, miRNA, miR-99, MUNC
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