Chemical labeling and next-generation sequencing for detection of RNA-modifications
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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.