Covalent modification of mRNAs in S. cerevisiae
- Alexander Fraser Lovejoy.
- Dec. 2013.
- Physical description
- online resource (x, 166 pages) : illustrations (some color)
- Includes bibliographical references (p. 159-166). 119 refs.
- While the presence of modified nucleotides in cellular RNA has been known about for over 60 years, it is only recently that such nucleotides have become studied and mapped in mRNAs. With recent transcriptome-wide maps of N6-methyladenosine and 5-methylcytosine showing widespread mRNA modification and evolutionary conservation of many sites, it is clear that site-specific mRNA modification may represent a new level of post-transcriptional regulation of gene expression. With this in mind, we set out to map other mRNA modifications to begin to get an idea of the role they may play in gene expression. We developed a high-throughput sequencing technique that identified the first known pseudouridines in mRNA. We were able to identify the enzymes that modified a few of the top hits, as well as show that modification of at least one of the top sites is conserved throughout the fungal lineage. We have made progress toward developing an improved high-throughput sequencing technique, which could allow elucidation of a transcriptome-wide map of pseudouridines. Adapting the same technique as was used to identify mRNA pseudouridylation, we also found a few dozen possible 2'-O-methylation sites in mRNA with an interesting functional theme, though these potential modifications still need to be validated. In addition, we attempted to map and find a functional role for N6-methyladenosines in yeast undergoing meiosis, and while this failed, it led to our discovery of a target set of bound mRNAs and role in gene expression of a meiosis-specific RNA binding protein, Rim4p. The work described in this thesis identifies the third known internal, site-specific modified nucleotide in mRNA, suggesting that mRNA modifications may be more common than previously thought and may play an important, under-explored role in post-transcriptional control of gene expression.
- Publication date
- Submitted to the Department of Biochemistry and the Committee on Graduate Studies of Stanford University.
- Thesis (Ph.D.)--Stanford University, 2013.