Author

Shaima Akhlaq

Date of Award

11-2017

Document Type

Thesis

Degree Name

Master of Science in Medical Sciences (MSMS)

Department

Biochemistry

First Advisor

Farah Mustafa

Second Advisor

Tahir A. Rizvi

Third Advisor

Gulfaraz Khan

Abstract

Retroviral genomes have been shown to contain regulatory sequences at the 3’ end of their genomes that have been implicated in critical steps of viral life cycle, including nucleocytoplasmic RNA export, RNA stability, and post nuclear export functions, such as translation. Recently mouse mammary tumor virus (MMTV) has been shown to encode Rem protein and its cis-acting cognate sequence located at the 3’ of the genome, the Rem-responsive element (RmRE). Binding of Rem to RmRE facilitates the export of genomic RNA (gRNA) from the nucleus to the cytoplasm. Using an expression system, our previous results have shown that unspliced MMTV Gag/Pol RNA could be exported out of the nucleus in the absence of a functional Rem/RmRE regulatory system, but its translation required a constitutive transport element (CTE) from Mason-Pfizer monkey virus that can facilitate its expression. Considering that RmRE is located at the junction of env/3’ LTR, a region common to all MMTV mRNAs, 3’ RmRE has the potential to affect all mRNAs. Based on these observations, it was hypothesized that an additional cis-acting control element, a putative 5’ RmRE, could be present at the 5’ end of the MMTV genome, facilitating gRNA nuclear export by distinguishing between the unspliced from the spliced mRNAs, whereas the 3’ RmRE on the other hand, could facilitate translation of all other mRNAs, including the unspliced RNA. To address this hypothesis, a series of deletion and substitution mutations were introduced between the major splice donor (mSD) and Gag ATG, a region that should exclusively be present in the gRNAs. These mutant clones were tested in transient and stable transfections to determine their effect on gRNA nuclear export, stability, and translation. Our results revealed severe defects in the expression of both unspliced gRNA as well as spliced mRNAs, suggesting that a cis-acting element could be present at the 5’ end of the MMTV genome modulating gene expression. Interestingly, nuclear export of the gRNA as well as its stability was not affected by the mutations. Furthermore, a complete abrogation of Gag and Env protein expression was observed, suggesting translational defects in the affected set of mutants. Quantitative real-time PCR analysis revealed that the ix mutants were severely compromised in the expression of genomic as well as the spliced RNAs in the nucleus, suggesting a possible transcriptional defect which compromised the expression of Gag and Env proteins. We next explored the possibility of the cis-acting element to serve as a potential transcription factor binding site. Careful sequence analysis of this region revealed the presence of mirror repeats that could act as potential recruitment sites for transcription factors. Preliminary in silico analysis has predicted multiple transcription factors of the zinc finger family that could interact with this element in a sequence specific manner. Overall, this study suggests that MMTV contains a cis-acting element at the 5’ end of the genome which helps regulate gene expression by facilitating transcription of MMTV mRNAs.

Included in

Biochemistry Commons

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