Year: 2024
Type: dissertation
Abstract: Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications, which can affect the function and structure of RNAs but do not entail any changes to the ribonucleotide sequence. RNA modifications are found on all forms of RNA including messenger RNA (mRNA) with methylations being the most common forms of mRNA modifications. Two such mRNA methylations are N6-methyladenosine (m6A) and N1-methyladenosine (m1A) which are known to be reversible. Studies have demonstrated that mRNA methylations are involved in and influence numerous biological processes including the DNA damage response. The DNA damage response is vital for the maintenance of genomic integrity and cell survival. DNA double strand breaks (DSB) are thought the most cytotoxic form of DNA damage as unrepaired or incorrectly repaired DSBs can result in cell death or lead to increased genomic instability and increase risk of cancer development. The ability to detect and correctly repair DNA DSB is vital for cells to maintain genome stability and to transfer correct genetic information from one generation to the next. ALKBH3 and FTO (ALKBH9) are dioxygenases belonging to the AlkB protein family. ALKBH3 has a well-established role in DNA alkylation repair but can also act as an mRNA demethylase, a function shared with other members of the AlkB family. Specifically, ALKBH3 removes m1A from mRNA, whereas FTO is primarily recognized for demethylating m6A from mRNA. In a previously published article, the Sigurdsson laboratory revealed that ALKBH3 is promoter hyper-methylated in ~20% of breast cancers. This form of epigenetic regulation reduced ALKBH3 expression and was correlated with reduced survival. Exploring the function of silencing ALKBH3 and other members of the AlkB family revealed that knockdown of ALKBH3 and FTO caused protein downregulation of RNF168, a pivotal protein in DNA DSB repair. The first aim of this project involved elucidating the regulatory role ALKBH3 and FTO have on RNF168. Further exploration into silencing ALKBH3 and FTO revealed that loss of ALKBH3 and FTO lead to increased m1A and m6A methylation marks on the RNF168 transcript, resulting in impaired mRNA export and decreased protein expression. These results point towards a novel form of epitranscriptomic regulation of RNF168. The secondary aim of this project was to explore the potential influence of ALKBH3 and FTO on the DNA DSB response, attributed to their regulation of RNF168. Cells depleted of ALKBH3 and FTO demonstrate strong signs of RNF168 dysfunction, including impaired 53BP1 recruitment to DNA DSB and altered DNA DSB repair dynamics. Subsequent investigations revealed heightened genome instability and increased sensitivity to genotoxic agents upon the loss of ALKBH3 and FTO. The findings presented in this thesis present a novel regulatory mechanism for RNF168 and unveil the involvement of mRNA modification in DNA DSB repair signaling. Furthermore, providing evidence of interplay between alkylation repair and DNA DSB repair. The removal of mRNA modification from the DNA repair factor RNF168, presents a new regulatory mechanism into the DNA DSB repair signaling, making it a unique addition to the rapidly evolving field of epitranscriptomics. The fact that demethylases play a part in genomic maintenance and cell survival in response to genotoxic agents is likely to have impact on the DNA repair field and could prove to be a vital factor to cancer development. Lack of ALKBH3 and FTO might be used as a potential marker for cancer treatment response due to both lack of alkylation repair and possibly because of DNA double strand repair deficiency, highlighting the possible clinical benefits of this project.
Source: Opin vísindi (Opin vísindi)
Author: Kristjánsdóttir, Karen
Language: English
Topic: RNA modifications and cancer
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funder: Háskóli Íslands
OpenAlex ID: W7037658928