GSBS Dissertations and Theses

ORCID ID

0000-0003-0621-8695

Publication Date

2020-01-06

Document Type

Doctoral Dissertation

Academic Program

Biochemistry and Molecular Pharmacology

Department

Biochemistry and Molecular Pharmacology

First Thesis Advisor

Melissa J. Moore

Keywords

Splicing, exon junction complex, transcriptome, transcriptome annotation, mRNPs, pre-mRNA

Abstract

Deep sequencing of mRNAs (RNA-Seq) is now the preferred method for transcriptome-wide quantification of gene expression. Yet many mRNA isoforms, such as those eliminated by nonsense-mediated decay (NMD), are inherently unstable. Thus a significant drawback of steady-state RNA-Seq is that it provides marginal information on the flux through alternative splicing pathways. Measurement of such flux necessitates capture of newly made species prior to mRNA decay. One means to capture nascent mRNAs is affinity purifying either the exon junction complex (EJC) or activated spliceosomes. Late-stage spliceosomes deposit the EJC upstream of exon-exon junctions, where it remains associated until the first round of translation. As most mRNA decay pathways are translation-dependent, these EJC- or spliceosome-associated, pre-translational mRNAs should provide an accurate record of the initial population of alternate mRNA isoforms.

Previous work has analyzed the protein composition and structure of pre- translational mRNPs in detail. While in the Moore lab, my project has focused on exploring the diversity of mRNA isoforms contained within these complexes. As expected, known NMD isoforms are more highly represented in pre-translational mRNPs than in RNA-Seq libraries. To investigate whether pre-translational mRNPs contain novel mRNA isoforms, we created a bioinformatics pipeline that identified thousands of previously unannotated splicing events. Though many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis.

DOI

10.13028/xa68-pv31

Rights and Permissions

Licensed under a Creative Commons license

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.

Available for download on Tuesday, July 28, 2020

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