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Identifier 000460185
Title Epitranscriptomic control of DNA damage induced R-loops
Alternative Title Επιμεταγραφικός έλεγχος των επαγωμένων από βλάβες στο DNA δομών R-loops
Author Πατεράκη, Νικολέτα Ι.
Thesis advisor Ντίνη, Ευγενία
Reviewer Βιδάκη, Μαρίνα
Γαρίνης, Γεώργιος
Abstract The “epitranscriptome” refers to the total pool of biochemical modifications of RNA within a cell that do not alter the sequence itself. The most abundant RNA modification on RNA transcripts is N6-methyladenosine (m6A), which plays a regulatory role in many biological processes, including transcription, splicing and RNA metabolism. Recent studies have focused on unraveling potential roles of m6A in response to various stress factors, including those that induce DNA damage. However, the mechanisms underlying the connection between m6A, and DNA damage response have not been fully elucidated. In this project, I aim to characterize the potential role of m6A in response to DNA damage and its involvement in regulating R-loop formation and/or resolution. To resolve this question, my primary focus is on the nuclear m6A reader YTHDC1, which has been shown to localize to double-strand DNA breaks. It is worth noting that this m6A reader also regulates responses to heat stress, suggesting a potential role in DNA damage response as well. In this Master’s thesis, bioinformatic, molecular and biochemical methods were used to approach this question. Firstly, reanalysis of published RNA-seq data upon YTHDC1 knockdown in HeLa cells was performed, in which several genes that take part in the DNA damage response were identified. Consequently, experiments using DNA damage induced methods (UV radiation) were performed to evaluate the expression levels as well as the localization of YTHDC1. Additionally, in order to better dissect the role of YTHDC1 in response to DNA damage, a specific protein degradation tag (dTAG) system against YTHDC1 was designed and incorporated in HCT116 colon cancer cells. This tool enables the targeted and controlled depletion of the protein of interest. Last but not least, the RChIP protocol was established in order to be able to map R-loops specifically by using dRNase H fused with V5 tag. The dRNase H has a mutation on its catalytic domain but not on the RNA:DNA binding domain, therefore, it can recognize the R-loops without cleaving the RNA moiety. These tools will be used in future experiments to unravel the role of YTHDC1 in stress responses.
Language English
Subject DNA damage
Epitranscriptome
RNA methylation
RNA μεθυλίωση
Βλάβη DNA
Επιμεταγράφωμα
Issue date 2023-11-24
Collection   School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses
  Type of Work--Post-graduate theses
Permanent Link https://elocus.lib.uoc.gr//dlib/f/d/7/metadata-dlib-1699261074-626038-24378.tkl Bookmark and Share
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