Genome-wide studies of DNA and RNA with modifications through high-throughput sequencing analysis
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2018
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu1534528759469392021-08-03T07:08:18Z Genome-wide studies of DNA and RNA with modifications through high-throughput sequencing analysis Moreland, Blythe S. Physics Biophysics High-throughput sequencing Methyl-CpG binding domain proteins DNA methylation Thg1 Thg1-like proteins DNA/RNA-protein interactions are crucial to the maintenance, processing, and regulation of the genetic information contained within a cell. Sets of particular DNA/RNA-protein interactions can confer specificity to a protein's binding behavior, which makes the consideration of DNA and RNA with modifications interesting in how they alter the landscape of interactions. High-throughput sequencing (HTS) techniques of DNA and RNA can offer unmatched genome-wide characterizations of where and how proteins interact with DNA and RNA by generating libraries of millions of reads that can capture the whole distribution of these interaction events. In this dissertation we used HTS analysis techniques to probe the location and state of two nucleic acid modifications on a genome-wide scale: The methylation of Cytosines in the CpG dinucleotide (mCpG) in the human genome, and the nucleotide addition by 3'-5' polymerases to the 5'-ends of RNAs. In the case of DNA methylation, the sequencing data represents DNA fragments enriched in mCpGs by way of interaction with the methyl-CpG-binding domain (MBD) protein MBD2. In the case of 5'-end nucleotide addition, the sequencing data is a snapshot of non-coding RNAs whose 5'-ends may have been extended by a Thg1-like protein (TLP). In both sets of studies, the information about the modification in turn conveys information on the relevant DNA-protein and RNA-protein interactions, and what sequences or structures are preferred by the proteins reading or depositing the modifications. In Chapter 2, a deep sample of methylated genomic DNA allows us to characterize the sequence, spacing, and mCpG density biases introduced through MBD2 interaction during a protein pulldown experiment. In Chapter 3, we use the characterizations of those interactions -- the 3 bp minimum separation between mCpGs, the nonlinear relationship between mCpG number and pulldown efficiency -- to build a model to predict expected pulldown from a SssI Control sample, which is then used to increase the accuracy of methylation quantification from MBD pulldown data. We find that the use of our pulldown alignment model of an SssI Control data set is suitable for substitution for observed SssI Control data with use of the empirical Bayes' model BayMeth. In Chapter 4, we pivot to developing a HTS screen for unknown substrates of TLPs, BtTLP and DdiTLP4, by detecting changes in 5'-end composition under different TLP conditions. Importantly, we detect BtTLP's activity on tRNA-His, of U-1 addition, which sufficiently allows for the tRNA's essential recognition by Histidyl-tRNA synthetase (HisRS). HTS techniques and analysis will continue to improve our ability to differentiate the distributions of DNA/RNA-protein interactions between "background" and "modified" states of nucleic acids. 2018 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu153452875946939 http://rave.ohiolink.edu/etdc/view?acc_num=osu153452875946939 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |
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NDLTD |
| language |
English |
| sources |
NDLTD |
| topic |
Physics Biophysics High-throughput sequencing Methyl-CpG binding domain proteins DNA methylation Thg1 Thg1-like proteins |
| spellingShingle |
Physics Biophysics High-throughput sequencing Methyl-CpG binding domain proteins DNA methylation Thg1 Thg1-like proteins Moreland, Blythe S. Genome-wide studies of DNA and RNA with modifications through high-throughput sequencing analysis |
| author |
Moreland, Blythe S. |
| author_facet |
Moreland, Blythe S. |
| author_sort |
Moreland, Blythe S. |
| title |
Genome-wide studies of DNA and RNA with modifications through high-throughput sequencing analysis |
| title_short |
Genome-wide studies of DNA and RNA with modifications through high-throughput sequencing analysis |
| title_full |
Genome-wide studies of DNA and RNA with modifications through high-throughput sequencing analysis |
| title_fullStr |
Genome-wide studies of DNA and RNA with modifications through high-throughput sequencing analysis |
| title_full_unstemmed |
Genome-wide studies of DNA and RNA with modifications through high-throughput sequencing analysis |
| title_sort |
genome-wide studies of dna and rna with modifications through high-throughput sequencing analysis |
| publisher |
The Ohio State University / OhioLINK |
| publishDate |
2018 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu153452875946939 |
| work_keys_str_mv |
AT morelandblythes genomewidestudiesofdnaandrnawithmodificationsthroughhighthroughputsequencinganalysis |
| _version_ |
1719454537301884928 |