Epitranscriptomics, the study of RNA modifications, has become a hotspot of research over the last decade. Over 170 unique modifications have been discovered with a widespread occurrence in a diverse range of RNAs. 5-methylcytosine, m5C, is an evolutionarily conserved and reversable modification that regulates the stability and export of tRNAs, rRNAs, and mRNAs. m5C has recently been implicated in many biological phenomena including tumorigenesis, embryonic cell expansion and differentiation, brain development, and neuronal functions. While we are just beginning to understand the functions of m5C, a gold standard of m5C detection has yet to be established due to the low signal-to-noise presence of m5C. In this work, we utilize RNA bisulfite sequencing as a transcriptome-wide approach to understand the computational and chemical parameters needed to optimize m5C discovery in the mitochondria and the developing brain. In Chapter 1, we systematically evaluate four preparation conditions of bisulfite sequencing to identify potential presence of m5C-mRNAs localized to the mitochondria in neuronal stem cells. In tandem, we utilize unique molecular identifiers and a consortium of control template transcripts to evaluate sources of false positive m5C sites that may emerge from sequencing errors, PCR amplification, and the inadequate bisulfite conversion of transcripts. While improvements to mitochondrial transcript bisulfite conversion and false positive filtering were observed, no mitochondrial mRNAs were identified to be methylated, indicating no or very few methylated cytosines in mitochondrial mRNAs and the need for improved chemical methods to detect mitochondrial m5C-mRNAs if any. In Chapter 2, we employ the computational approaches established in Chapter 1 to survey the m5C landscape of the developing mammalian brain. We discover a general increase in unique m5C sites in mouse whole brain tissue when compared to neuronal cell cultures. Of these sites, we found the post-natal day 0 and 17 brain time points to undergo significant methylation level changes in comparison to the 6-week-old brain. These differentially methylated sites were significantly enriched for brain development, synaptic development, and transcriptional control gene network pathways. In Chapter 3, we expand on our findings in Chapter 2 to understand the impact of m5C reader FMRP and m5C eraser TET1 loss in the mouse post-natal day 17 brain. Among a set of m5C sites identified in wildtype or knockout samples, few were differentially methylated after protein ablation, suggesting m5C may rely on compensatory enzymes. Using FMRP-RNA pulldown assays to validate FMRP binding positions, we identified Ralbp1 to be hypermethylated and overexpressed in Fmr1-KO brain tissues. RalBP1 is a binding protein responsible for the endocytosis of AMPA receptors, a process critical for neuronal long term depression and brain development. / Doctor of Philosophy / Ribonucleic acid (RNA) is the product of deoxyribonucleic acid (DNA) transcription and the precursor to protein translation. Chemical modifications can be made to the bases of DNA, known as epigenetic modifications, to elicit new functions and responses to the environment. Epitranscriptomics refers to the study of RNA modifications that also serve unique roles and functions depending on the type of modification made. Here, we study the presence of 5-methylcytosine, a methyl group added to the cytosine (C) base of RNA. This modification is found throughout all branches of life and is known to promote the stability and export of many RNA types. Recently, studies have utilized many techniques including RNA bisulfite sequencing to find links between the presence of m5C-RNAs and cancer progression, stem cell development, and brain development. RNA bisulfite sequencing uses chemical applications to convert non-methylated "C"s to the RNA base "U", while retaining a "C" signature on methylated "C"s. However, due to the extremely low presence of RNA-m5C in comparison to DNA-m5C, sources of noise make it difficult to identify a true m5C signal. Because of this discrepancy, established analytical methods based on DNA biology may not be suitable for RNA analysis. To address shortcomings in current detection methods of RNA-m5C, we performed systematic analysis of 1) different preparation methods for improved m5C detection methods and 2) computational approaches for the filtering of false positive m5C sites, as described in Chapter 1. To achieve these goals, we expanded the breadth of analytical methods by including unique molecular identifiers and expanding the set of control RNA sequences to better grasp how false positive sites might be introduced into non-methylated sequences. While noticeable improvements were made to control RNA sequence false positive detection, we found that most mitochondrial RNAs did not carry the same m5C signatures as RNAs from other sources. Because of this difference, we could not conclude that mitochondrial mRNAs were methylated. Therefore, we suggest that future studies may need to develop better or alternative methods for the detection of mitochondrial RNA-m5Cs. In Chapters 2 and 3, we utilize the computational methods developed in Chapter 1 to understand how m5C levels change throughout the development of a mouse's brain. By investigating the m5C profiles of mouse newborn, young child, and juvenile brains, we found significant changes in m5C levels specific to certain RNAs. These RNAs are associated with neuronal growth, development, and maturation, which may have implications for m5C's role in cognitive development, intellectual disabilities, and neurodegenerative disorders. To discover if these RNAs could be affected by the absence of m5C-specific proteins, we created mice deficient in a protein m5C reader, FMRP, and an m5C eraser protein, TET1. Interestingly, we did not find a significant difference in mice deficient in the proteins, indicating m5C may rely on multiple proteins to serve redundant functions. However, one RNA, Ralbp1, was found to be significantly methylated in FMRP deficient models. This RNA is essential for developmental changes in the brain as well as neuronal growth and could be an interesting target for future research.

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.