title: The Impact of Adenosine mRNA Modifications on Macrophage Function
creator: Griesche, Valerie
subject: ddc-500
subject: 500 Natural sciences and mathematics
subject: ddc-570
subject: 570 Life sciences
description: Macrophages are innate immune cells characterized by a high level of plasticity, which enables their quick adaptation to a changing environment required for initiating, promoting, and resolving inflammatory processes to protect from external threats and maintain tissue homeostasis. Such reprogramming cannot be achieved via transcriptional changes alone, and therefore RNA modifications on macrophages’ transcripts play a crucial role in allowing such versatility needed for a fast and adaptive response. Utilizing the mouse macrophage cell line RAW 264.7 (RAW) as a model system, I investigated the impact of METTL3-mediated m6A and ADAR1-mediated adenosine-to-inosine (A-to-I) editing on macrophage pro- inflammatory activation and immune function. Mapping A-to-I editing sites using short-read Illumina sequencing and m6A using single molecule Nanopore sequencing in RAW macrophages with wild-type genotype or genetic defects in Adar1 or Mettl3, I identified RNA modification sites on more than half of the genes that are involved in macrophages’ immunological functions. Gene expression analysis revealed that loss of m6A induced innate immune sensing, whereas loss of A-to-I editing dampened innate immune activation in macrophages. Additionally, transcriptomic analysis identified ADAR1’s and METTL3’s crucial role in macrophage activation in response to pro-inflammatory stimuli. Using functional assays, I confirmed METTL3’s and ADAR1’s impact on macrophage activation such as the upregulation and presentation of immunostimulatory cell surface markers and phagocytosis activity. While m6A levels remained mostly stable upon ADAR1 depletion, loss of METTL3 globally decreased A-to-I editing levels after pro-inflammatory stimulation. Besides the regulatory effect of some m6A sites on the Adar1 transcript that impact Adar1’s splicing and translation into protein, I found a positive association between A-to-I and m6A sites in transcripts where the modifications were placed at a distance above 139 nucleotides, suggesting a role of m6A in supporting ADAR1-mediated editing. Using a reporter assay for targeted ADAR1 editing, I observed increased ADAR1 recruitment and editing when ADAR1- engaging guide RNAs contained m6A modifications as compared to unmodified guide RNAs. Collectively, the demonstrated interdependency between m6A and A-to-I RNA modifications holds potential to advance therapeutic RNA editing strategies in the future.
date: 2025
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserver/37323/1/Valerie_Griesche_PhD_thesis.pdf
identifier: DOI:10.11588/heidok.00037323
identifier: urn:nbn:de:bsz:16-heidok-373236
identifier:   Griesche, Valerie  (2025) The Impact of Adenosine mRNA Modifications on Macrophage Function.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/37323/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng