Authors:	Körtel, Nadine
Advisor:	König, Julian
Title:	The role of m6A RNA modification in post-transcriptional regulation of gene expression
Online publication date:	5-Oct-2023
Year of first publication:	2023
Language:	english
Abstract:	The epitranscriptome is increasingly recognized as a crucial player in post-transcriptional gene expression regulation. As the most abundant internal mRNA modification, m6A is an essential regulator in almost all aspects of mRNA metabolism. Global mapping has only recently been enabled by developing RIP-seq and iCLIP-seq based methods. iCLIP is a state-of-the-art method to map RNA interactions with RNA-binding proteins in an transcriptome-wide manner. The m6A-antibody based method miCLIP allowed to map m6A in single-nucleotide resolution. This paved the way to further study the biological consequences of m6A. Despite the important insights we gained from using these methods, they inherit several limitations and subsequent computational analysis remained challenging. Broad antibody reactivity results in high-background signal and hindered computational analysis. The high required input material made global m6A-mapping exclusionary for rare samples e.g. for clinical samples or in vivo tissues, which are restricted in material. In this study, we overcome these limitations and provide an improved m6A-detection method combined with enhanced extensive computational analysis. First, we improved the iCLIP procedure for efficient library preparation and developed iCLIP2. We combined miCLIP with our recently improved iCLIP2 protocol, which we subsequently termed miCLIP2. Our improved protocol results in high-complexity libraries and allows to perform global mapping of m6A in low-input samples. We combined the protocol with a robust computational pipeline and a machine learning classifier. Calibrating our machine learner with mESC WT and Mettl3 KO miCLIP2 data helped to understand the characteristics of m6A in miCLIP2 data. The now so-called m6Aboost predictor allows the identification of genuine m6A sites without the need for a Mettl3 KO or DRACH filtering steps. Importantly, we were able to identify m6A sites outside of DRACH motifs and can apply m6Aboost for miCLIP2 data across species. Using miCLIP2 in combination with m6Aboost enables further dissection of the biological role of m6A. Therefore, we found that m6A accumulated towards the 5’ splice site and introns are retained upon m6A methylation. The m6A modification was found to act as an important player in gene expression regulation by promoting mRNA degradation. Overall, balancing gene expression is crucial and disturbance by aneuploidy can have detrimental consequences. Due to sex-chromosome evolution from an ancestral pair of autosomes, mammalian females possess two X chromosomes while males have one X and one Y chromosome. The genetic imbalance between both sexes is balanced by X-chromosome inactivation. The resulting imbalance between the single remaining X chromosome and the two active copies of autosomes is balanced by dosage compensation mechanisms. However, the existence and the mechanism behind dosage compensation is still actively debated. This work unravelled a novel role of m6A in dosage compensation. We find that m6A modifications are less abundant on X-chromosomal transcripts. Due to its degrading nature, more m6A results in higher degradation of autosomal transcript compared to their X-chromosomal counterparts. Hence, X-chromosomal transcripts are more stable, which is mediated by differential m6A methylation numbers. Consequently, X-chromosomal transcripts are differentially affected by acute m6A depletion. Importantly, we find that lower numbers of m6A is internally hardcoded by reduced GGACH motifs on X-chromosomal transcripts. Taken together, we find that mammalian dosage compensation is accomplished by higher RNA stabilities of X-chromosomal transcripts, which is mediated by an epitranscriptomic RNA regulatory mechanism.
DDC:	500 Naturwissenschaften 500 Natural sciences and mathematics 570 Biowissenschaften 570 Life sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 10 Biologie
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-9022
URN:	urn:nbn:de:hebis:77-openscience-3e2b8f4e-833f-4583-b5a9-24fef0a8411f1
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	http://rightsstatements.org/vocab/InC/1.0/
Extent:	192 Blätter ; Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen