Student Theses and Dissertations

Date of Award

2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Darnell Robert Laboratory

Abstract

Breast cancer is the most commonly diagnosed and second highest cause of cancer-related mortality in women, with the majority of deaths associated with metastatic spread of tumors to distal organs. The metastatic process involves multiple sequential steps including invasion of the surrounding stroma, intravasation into lymph or blood vessels, survival during transport with successful evasion of the immune system, extravasation and final outgrowth in the new microenvironment. At each step cells acquire new properties through the alteration of gene expression profiles. While a great deal of effort has been made towards understanding the underlying genetic mutations associated with enhanced motility and invasiveness, it is becoming increasingly apparent that post-transcriptional modification of gene expression at the RNA level contributes substantially towards gain of metastatic potential. In eukaryotic cells RNA is subject to multiple layers of regulation that together generate extensive phenotypic and temporal complexity from a relatively small number of genes. RNA regulation is achieved through the concerted action of multiple RNA binding proteins (RBPs) which themselves are subject to tight regulation. Recent studies have implicated the aberrant expression of multiple RBPs in tumor development and metastatic spread, while technological advances have provided methods by which unbiased, transcriptome-wide footprints of RBP-RNA interactions can be mapped. High-throughput cross-linked immunoprecipitation (HITS-CLIP) provides both the identity of regulated transcripts and the location of RBP regulation within a transcript. For the post-transcriptional regulatory protein Argonaute (AGO), which binds mRNA in complex with small non-coding microRNAs (miRNAs), HITSCLIP derived 3’UTR binding sites have been shown to correspond to sites of functional miRNA-mediated target down regulation. In this study, a molecularly characterized cell culture based model system of breast cancer metastasis has been employed to investigate the role of RNA binding proteins in the inhibition of metastatic progression. AGO-mRNA regulation mediated by endogenous miRNAs and exogenous anti-metastatic pre-miR-335 has been interrogated by HITSCLIP to identify on a genome-wide scale differentially regulated targets with respect to metastatic potential. Furthermore, HITS-CLIP has been used to characterize a novel anti-metastatic RNA binding protein, RBM47, uncovering multifunctional regulation of target transcript stability and alternative splicing.

Comments

A Thesis Presented to the Faculty of The Rockefeller University In Partial Fulfillment of the Requirements for The degree of Doctor of Philosophy

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