Transcriptome-Wide RNA Modification Profiling

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Publication Type dissertation
School or College School of Medicine
Department Oncological Sciences
Author Vishteh, Vahid Khoddami
Title Transcriptome-Wide RNA Modification Profiling
Date 2013-08
Description Post-transcriptional RNA modifications provide new structural and functional features to modified RNA molecules. Extensive research in the past has resulted in isolation of over 100 distinct nucleotide modifications from different organisms and in different RNA species. These modified nucleotides are distributed within the entire transcriptome comprising the cellular epitranscriptome. The ultimate goal of the research in the field is to address what the specific functions of specific modifications are, and also the impact of each on cellular physiology. However, the first question to be addressed is how these > 100 modified nucleotides are distributed within the transcriptome. RNA modification profiling using conventional techniques has provided a great body of knowledge about the distribution of many modifications in RNAs. However, these findings remained limited mostly to tRNAs and rRNAs, the two most abundant and also highly modified RNA species in different organisms. This is partly because of the lower sensitivity of applied classical technologies. Here in this dissertation, in Chapter 2, we are reporting an optimized new RNA bisulfite protocol suitable for high-throughput RNA cytosine methylation profiling. We present the results of application of this technique for 5-methyl-cytosine (m5C) profiling in mouse embryonic fibroblasts (MEFs) RNAs, isolated from wt and dnmt2-/- mice to explore the target specificity of DNA methyltransferase 2 (DNMT2) enzyme. In Chapter 3, we present a substantially novel technique: Aza-IP, for enrichment and identification of the direct targets of RNA cytosine methyltransferases (m5C-RMTs) as well as iv determination of the exact modified bases in the same experiment. We provide the results of the Aza-IP technique for two human m5C-RMTs; DNMT2 and NSUN2, representing their known and novel RNA targets/modified bases. In Chapter 4 we discuss how similar technologies to both of the RNA bisulfite sequencing and Aza-IP techniques as well as other methodologies can be applied and extended for transcriptome-wide profiling of RNA modifications other than m5C. In Chapter 5 we present the future directions of the work focused on cataloguing the direct targets of all human m5C-RMTs in human cultured cells in mouse and fish model systems, to elucidate the functions of cytosine methylation in RNA molecules.
Type Text
Publisher University of Utah
Subject RNA - Analysis; Aza-IP technique - Research
Subject MESH Methylation; RNA, Messenger; RNA, Ribosomal; RNA, Transfer; Methyltransferases; DNA; RNA; Transcriptome; Sulfites; Base Sequence; Nucleotides; Cytosine; Cells, Cultured; 5-Methylcytosine
Dissertation Institution University of Utah
Dissertation Name Doctor of Philosophy
Language eng
Relation is Version of Digital reproduction of Transcriptome-Wide RNA Modification Profiling. Spencer S. Eccles Health Sciences Library. Print version available at J. Willard Marriott Library Special Collections.
Rights Management Copyright © Vahid Khoddami Vishteh 2013
Format Medium application/pdf
Format Extent 16,530,709 bytes
Source Original in Marriott Library Special Collections,
ARK ark:/87278/s6zk8qxw
Setname ir_etd
Date Created 2014-06-26
Date Modified 2018-01-12
ID 196623
Reference URL
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