RNA epigenetics or Epitranscriptomics has emerged as a new field for understanding the post-transcriptional regulation of gene expression by RNA modifications. Among numerous types of RNA modifications, RNA cytosine-5 methylation (5-mrC) is recognized as an important epitranscriptomic mark that modulates mRNA transportation, stability and translation.
In chapter 1, we summarize the currently available approaches to detect 5-mrC modification at global, transcriptome-wide and locus-specific levels, and compare the corresponding advantages and disadvantages of the techniques. We further focus on the bioinformatics data analysis of RNA bisulfite sequencing datasets by comparing existing packages with respect to key parameters for alignment and methylation calling and filtering of potentially false positive 5-mrC sites.
To investigate the dynamic regulation of 5-mrC modification, as described in chapter 2, we adopt a widely used neuronal activity model, and perform RNA sequencing (RNA-seq) and RNA bisulfite sequencing (RNA BS-seq) to profile gene expression as well as transcriptome-wide 5-mrC modification. We have identified distinct gene expression profiles and differentially methylated 5-mrC sites (DMS) in neurons upon activation, and the genes with DMS sites are enriched with mitochondrial and synaptic functions. Moreover, it reveals a negative correlation between RNA methylation and mRNA expression in mouse cortical neurons during neuronal activity. Thus, these findings identify the dynamic regulation of 5-mrC modification during neuronal activity and reveal a potential link between RNA methylation and mRNA expression.
In chapter 3, we investigate the effect of folate, a methyl-donor, on RNA cytosine-5 methylation (5-mrC) modification in adult mouse neural stem cells (NSCs). Compared to the control, NSCs cultured in folate deficiency or supplementation condition have shown no changes in mRNA expression, but significant changes in mRNA translation efficiency. RNA bisulfite sequencing of both total and polysome poly(A) RNA samples shows distinct 5-mrC profiles in NSCs treated with different concentrations of folic acid. It also shows consistent hypermethylation in polysome mRNAs than that in total mRNAs. This study presents the comprehensive influence of folate deficiency and supplementation on RNA cytosine-5 methylation and mRNA translation. / Doctor of Philosophy / RNA epigenetics, a collection of RNA modifications, has recently emerged as an exciting, new field for understanding post-transcriptional regulation of gene expression. RNA cytosine-5 methylation (5-mrC) is one of the most well-known RNA modifications that modulates mRNA export, stability and translation.
In the first chapter, we summarize the currently available methods for the measurement of 5-mrC modification. We highlight one of the techniques, RNA bisulfite sequencing (RNA BS-seq) and focus on the bioinformatics data analysis of RNA BS-seq datasets. We have compared several existing tools in regard of the key parameters in data analysis.
In the second chapter, we adopt a widely used neuronal activity model to study the dynamic regulation of RNA cytosine-5 methylation (5-mrC). We perform RNA-seq and RNA BS-seq in neurons in response to stimulation. We have identified numerous differentially expressed genes and differentially methylated 5-mrC sites in activated neurons and find that these DMS-related genes are associated with mitochondrial and synaptic functions. Furthermore, we identify a negative correlation between RNA methylation and mRNA expression, indicating a potential role of 5-mrC modification in the regulation of mRNA expression.
In the third chapter, we investigate the influence of a nutrient supplement, folic acid, on 5-mrC modification in adult mouse neural stem cells. Compared to the control, NSCs cultured in folate deficiency or supplementation condition have shown no changes in mRNA expression, but significant changes in mRNA translation efficiency. We perform RNA bisulfite sequencing of both total poly(A) RNA samples and polysome poly(A) RNA samples. We identify distinct 5-mrC profiles in NSCs treated with different concentrations of folic acid. It shows consistent hypermethylation in polysome mRNAs than that in total mRNAs. This study presents the comprehensive influence of folate deficiency and supplementation on RNA cytosine-5 methylation and mRNA translation.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/106805 |
| Date | 09 June 2020 |
| Creators | Xu, Xiguang |
| Contributors | Biological Sciences, Xie, Hehuang David, Li, Liwu, Oestreich, Kenneth, Fox, Michael A. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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