Authors:	Tserovski, Lyudmil Aleksandrov
Title:	Chemical labeling and next-generation sequencing for detection of RNA-modifications
Online publication date:	9-Jan-2017
Year of first publication:	2017
Language:	english
Abstract:	In the field of life sciences RNA modifications start to reveal their important role in the dynamic regulation of gene expression which can be influenced by stress response, immunity reactions or other environmental factors. These modifications are found in archaea, bacteria and eukaryota where they decorate RNA molecules and thus expand the nucleotide repertoire. Although methods were developed in the past ten years that allow the detection of numerous modified RNA nucleosides, these techniques still lack sufficient sensitivity and specificity. The present PhD thesis addresses several aspects on detection of naturally occurring RNA modifications. First, a library preparation protocol was adapted and optimized to capture reverse transcriptase (RT) events during synthesis of a complementary DNA sequence. Therefore, not only abortive products, but also misincorporations could be traced and later used in a bioinformatic approach for detection and prediction of modified sites. Second, N-1-methyladenosine (m1A), a well described and highly conserved modification, was used as a model to test this approach. Two major events were observed: (i) the methyl group at N-1 of adenosine leads to a substantial number of RT-stops and (ii) a certain amount of read-through is possible, leading to misincorporations at the modified positions. It was demonstrated that RT leaves a specific signature at m1A sites depending not only on the underlying modification but also on the 3’-neighboring nucleoside. Together, these results led to the discovery of new m1A positions. Furthermore, the applicability of osmium tetroxide-bipyridine (os-bipy) as labeling agent for 5-methylcytidine and 5-methyluridine in RNA was evaluated. On the nucleoside level, a five- and ten-fold preference, respectively, over the corresponding unmodified nucleoside was observed. In a short pentanucleotide, however, this preference was strongly reduced. It was demonstrated that the sterical environment in the short oligonucleotide has a strong hindering impact on the reaction rate. Importantly, this effect could be linked to an altered diastereoselectivity which was due to an impediment of the attack of os-bipy toward the preferred si side of the diastereotopic 5,6 double bond of the nucleobase. As a result, in the pentanucleotide context preference toward 5-methylcytidine over cytidine was almost lost, whereas for 5-methyluridine it remained about eight times higher than for uridine. Finally, labeling with osmium tetroxide-bipyridine was used in combination with high-throughput sequencing on a total transfer RNA population of S. cerevisiae. Bioinformatic analysis revealed a discrimination between 5-methylcytidine and cytidine. On the contrary, 5-methyluridine containing sites remained undetectable upon reaction with os-bipy. Nevertheless, results obtained from reaction of osmium tetroxide and bipyridine with 5-methylpyrimidines are promising and provide an important basis for the development of appropriate labeling agents for a transcriptomewide detection of these modifications.
DDC:	500 Naturwissenschaften 500 Natural sciences and mathematics
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 09 Chemie, Pharmazie u. Geowissensch.
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-2779
URN:	urn:nbn:de:hebis:77-diss-1000009302
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	https://rightsstatements.org/vocab/InC/1.0/
Extent:	130 Seiten
Appears in collections:	JGU-Publikationen