Date of Completion

2019

Document Type

Honors College Thesis

Department

Microbiology and Molecular Genetics

Thesis Type

Honors College

First Advisor

Stephanie McKay

Second Advisor

Rebecca Guy

Third Advisor

Brenda Tessmann

Keywords

Epigenetics, RFI, RNA, Methylation, COBRA, Epitranscriptomics

Abstract

Nucleic acids are able to undergo covalent modification in a variety of ways resulting in functional regulation at the epigenetic level. Understanding these epigenetic changes may provide insight into gene regulation, which can be applicable to any field within the life sciences that deal with DNA. Methylation of the carbon at the fifth position in the nucleic acid cytosine (5mC) within DNA is one of the most well documented epigenetic modifications. In eukaryotic organisms, 5mC is associated with down-regulating the gene in which the methylated cytosine is associated with. Although cytosine methylation is well documented at the genetic level, little is known about it at a post-transcriptional level. Cytosine methylation within RNA has been observed in mRNA, tRNA, and rRNA; however, demonstrable rationality for this epitranscriptomic modification has yet to be elucidated. There has been documentation of stable and long-lived RNA molecules containing methylated cytosines, possibly insinuating that a role in RNA stability. The dearth of knowledge regarding post-transcriptional modification of RNA can be explained by the fact 5mC is not easily detectable through most sequencing methods and that 5mC does not affect base pair binding, precluding the use of any probing assays. In this study, a survey of mRNA methylation within exon 7 of the bovine PRKAB1 gene is conducted using skeletal muscle of six individual heifer cows in an effort to better understand the biochemical mechanisms driving differences in observed residual feed intake (RFI) among commercial cattle. Skeletal muscle tissue is used since feed efficiency affects muscle growth within cattle. Here we show methylation of cytosines at the post-transcriptional level is detectable through bisulfite treatment of RNA, followed by conversion to cDNA, PCR amplification and finally restriction digest, showing a potential association of differential methylation within animals that have high and low RFIs. These results give preliminary insight into the bovine AMPK epitranscriptome, while also providing more points of data for post-transcriptional 5mC which could possibly help elucidate patterns in a relatively new sub-field of translational regulation. Finally, this study is the first ever survey of epitranscriptomic markers and their documentation conducted within livestock species.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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