GSBS Dissertations and Theses

Approval Date

1-5-2016

Document Type

Doctoral Dissertation

Academic Program

Biochemistry and Molecular Pharmacology

Department

RNA Therapeutics Institute

First Thesis Advisor

Melissa J. Moore, PhD

Keywords

Spliceosomes, DEAH-Box ARPase Prp22

Subjects

Dissertations, UMMS; Spliceosomes; DEAD-box RNA Helicases

Abstract

In eukaryotes, the spliceosome is a macromolecular ribonucleoprotein machine that excises introns from pre-mRNAs through two sequential transesterification reactions. The chemistry and fidelity of pre-mRNA splicing are dependent upon a series of spliceosomal rearrangements, which are mediated by trans-acting splicing factors. One key class of these factors is the DEAH-box ATPase subfamily of proteins, whose members couple ATP hydrolysis to promote RNP structural rearrangements within the spliceosome. This is typified by Prp22, which promotes release of the spliced mRNA from the spliceosome and ensures fidelity of the second step of splicing. This role is well documented through classical biochemical and yeast genetics methods. Yet very little is known regarding the comings and goings of Prp22 relative to the spliceosome. My thesis research investigated the dynamics of Prp22 during splicing by using single-molecule fluorescence methods that allowed direct observation of these events. To do this, I helped construct a toolkit that combined yeast genetics, chemical biology and Colocalization Single Molecule Spectroscopy (CoSMoS) with in vitro splicing assays. Specifically, my thesis research consisted of CoSMoS splicing experiments in which fluorescently labeled pre-mRNA, spliceosome components and Prp22 were directly visualized and analyzed. Using these methods, I found that Prp22’s interactions with the spliceosome are highly dynamic and reversible. By simultaneously monitoring Prp22 and individual spliceosome subcomplexes, I was able to frame these Prp22 binding events in context relative to specific steps in spliceosome assembly and splicing. These experiments provide insight into how Prp22 promotes mRNA release from the spliceosome and maintains splicing fidelity.

DOI

10.13028/M29019

Rights and Permissions

Copyright is held by the author, with all rights reserved.

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