Virginia Research Day 2021

Influence of Folate on RNA Cytosine Methylation in Neural Stem Cells Xiguang Xu 1,2 , Zachary Johnson 1,3 , Razan Alajoleen 1,4 , Xiaoran Wei 1,4 , Natalie Melville 1,5 , Amanda Wang 1 , Rachel Padget 2,6 , James Smyth 2,6 , Hrubec Terry 4,7 , Hehuang Xie 1,2,3,4,5* 1 Epigenomics and Computational Biology Lab, Fralin Life Sciences Institute of Virginia Tech, VA, 24060; 2 Department of Biological Sciences, College of Science, Virginia Tech, Blacksburg, VA 24061; 3 Genetics, Bioinformatics and Computational Biology program, Virginia Tech, Blacksburg, VA 24061; 4 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine; Virginia Tech, VA, 24060; 5 Translational Biology, Medicine and Health Program, Virginia Tech, VA, 24060; 6 Fralin Biomedical Research Institute at VTC, Virginia Tech, VA, 24060; 7 The Edward Via College of Osteopathic Medicine, VA, 24060 Abstract Distribution of 5-mrC profiles in NSCs

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Influence of folate on polysome mRNA methylation

The study of RNA cytosine methylation (5-mrC) belongs to an emerging field of epitranscriptomics. Recent studies indicated that 5-mrC occurs in brain mRNAs coding for genes involved in central nervous system development and axonogenesis. Folate is a methylation donor and the suboptimal intake of this micronutrient has been linked to neural tube defect at embryonic stage and autism in early childhood. We hypothesize that the intake of folate may have an impact on brain epitranscriptomic profiles during development. In this study, we generated transcriptome- wide landscapes of 5-mrC modification in both total mRNAs and polysome-associated mRNAs for mouse neural stem cells (NSCs) cultured in media with different levels of folate. Distinct 5-mrC modification profiles in both total mRNAs and polysome mRNAs were detected in NSCs cultured with different concentrations of folate. Interestingly, 5-mrC was found to be significantly enriched in polysome mRNAs over total mRNAs; and panel of differentially translated mRNAs carrying hypermethylated 5- mrC was revealed by polysome profiling in combination of bisulfite sequencing. In summary, this study provides the first comprehensive set of epitranscriptomic data to evaluate RNA transcription, RNA methylation, and RNA translation together in NSCs with both low and high folate treatment. Hypothesis and Specific Aims  We hypothesized that the intake of folate may have an impact on the epitranscriptomic profile of neural stem cells (NSCs).  We aimed to :  Examine the influence of folate on RNA cytosine methylation in mouse NSCs.  Examine the influence of folate on mRNA transcription and translation in mouse NSCs.  Examine the relationship between mRNA cytosine methylation and mRNA translation.

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Figure 2. Distribution profile of 5-mrC modification in mouse NSCs. (a) Boxplot showing the methylation levels of 5-mrC sites in total mRNAs in NSCs (I). Bar charts show the numbers of 5-mrC sites (II) and 5-mrC-modified mRNAs (III). (b) Sequence frequency logo for the sequence context proximal to 5-mrC sites in total mRNAs in NSCs. (c) Density plot showing the distribution of 5-mrC sites along mRNA transcripts (5’UTR, CDS, 3’UTR). The moving average of percentages of mRNA 5-mrC sites were shown. (d) GO annotation of 5-mrC- containing mRNAs in the total mRNA methylomes in NSCs. Influence of folate on mitochondrial mRNA methylation

Summary  Folate deficiency and supplementation induce distinct RNA cytosine-5 methylation changes in mitochondrial and non- mitochondrial mRNAs.  Folate deficiency and supplementation induce changes in mRNA translation efficiency in adult mouse neural stem cells.  polysome mRNAs are with increased cytosine methylation in compared to total mRNAs.  Polysome mRNA methylation positively correlates with mRNA translation efficiency. Figure 4. RNA methylation is associated with translation. (a) Schematic representation of sucrose gradient used to segregate ribosome-free and ribosome-bound RNAs, and the representative polysome profile recorded at 254 nm with polysome fraction indicated in red. (b-c) Correlations between biological replicates for total RNA-Seq (b) and Polysome RNA-Seq (c). (d) volcano plot comparing RNA-Seq data .(e-f) Correlations between biological replicates for total RNA-BS-seq (e) and Polysome RNA-BS-Seq (f). (g) Box plot comparing RNA-BS-Seq data . (h) Numbers of methylated transcripts identified with total RNA-BS-seq and Polysome RNA-BS-Seq. (I-k) Integrative analyses of RNA-Seq and RNA-BS-seq show the correlations between the methylation and expression difference between polysome RNA and total RNA. ML stands for methylation level and LFC for log fold change. Acknowledgement This work was supported by the Center for One Health Research at the Vet-Med and The VCOM, NIH grant NS094574, ES031521, and the Fralin Life Sciences Institute fund.

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Material and Methods

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LF, MF, HF folic acid (FA) for 4 days

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Figure 3. Folate deficiency or supplementation influences mitochondrial mRNA methylation in NSCs. (a) Genome browser of reads mapped to mitochondrial genome. (b) Boxplot showing the percentage of reads in each category of mitochondrial genome. (c) Venn diagram showing the overlap of 5-mrC sites in LF, MF and HF groups. (d) Boxplot showing the methylation level in both mitochondrial and non-mitochondrial mRNAs. (e-f) Heatmap showing the methylation level of DMS sites in the comparisons of LF vs MF, and HF vs MF. (g) JC-10 results showing the influence of folate on mitochondrial membrane potentiation. t Student’s t -test was employed . Data are (ns) P>0.05. *P < 0.05, ****P < 0.0001

Figure 1. Experimental design and characterization of mouse neural stem cells. (a) Adult mouse neural stem cells (NSCs) from the subventricular zone (SVZ) of the lateral ventricles are isolated and cultured with media contain different concentration of folate (1.5uM FA as LF),(10uM FA as MF),(80uM FA as HF). (b)Adult mouse NSCs cultured under proliferating conditions were double stained with the neural progenitor markers Nestin (cytoplasmic, green) and Sox2 (nuclear, red; DAPI in blue). Scale bar: 50 μm .

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