Dissertations, Theses, and Capstone Projects

Date of Degree

9-2016

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Karen Hubbard

Committee Members

Mark Steinberg

David Calhoun

Frida Kleiman

Reginald Halaby

Subject Categories

Biochemistry

Keywords

HDM2, Gene expression

Abstract

hnRNP A1 is one of the most abundant and ubiquitously expressed member of hnRNPs (Heterogenous Nuclear Ribonucleoproteins) family of proteins that play multiple roles in gene expression by participating in major steps in the processing of nascent RNA transcripts 1. It is involved in mRNA biogenesis mechanisms such as the transcription, splicing, stability, export and translation of cellular and viral transcripts. The functions of hnRNP A1 extend to the processing of microRNAs, telomere maintenance and DNA repair 1. Our previous studies have shown that hnRNPA1 had reduced protein level and increased cytoplasmic accumulation in senescent human diploid fibroblasts 2,10. Our preliminary data showed that HDM2 mRNA associates with hnRNPA1 in IMR-90 cells. We have also observed that alterations of hnRNP A1 expression can modulate HDM2 mRNA levels. HDM2 expression must be regulated in normal and stressed cells to maintain normal p53 levels. Numerous proteins interact with HDM2 to regulate its expression 7. Failure to regulate HDM2 results in aberrant HDM2 expression leading to deregulation of the p53 activity. Consequently, adequate levels of HDM2 are maintained through transcriptional, post-transcriptional and post-translational regulation. In this thesis, we have examined how hnRNPA1 potentially modulates HDM2 gene expression given our preliminary observation that the levels of HDM2 mRNA increased with the down-regulation of hnRNPA1 and decrease with the overexpression of hnRNPA1 (Shimada, N and K, Hubbard.). In this research project, we demonstrated that overexpression of GFP-A1 significantly decreases the pre-HDM2 mRNA levels compared to IMR-90 cells transiently transfected with the empty vector control, GFP-EV. We also observed a decreased in HDM2 and a concomitant increase in p53 protein levels with the overexpression of hnRNP A1. In addition, we also found a significant increased in both p14 mRNA and protein levels with the overexpression of GFP-A1 suggesting that p14 ARF is induced via hnRNP A1. p14 ARF has been shown to directly interact with HDM2 and induced its degradation leading to the stabilization of p53. These results suggest that hnRNPA1 is negatively regulating the expression of HDM2. Our studies also show that hnRNP A1 directly interacts with HDM2 mRNA at a region corresponding to its 3’ UTR. The results from this study demonstrate that hnRNP A1 has a novel role in participating in the regulation of HDM2 gene expression.

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