Authors:	Fradera-Sola, Albert
Advisor:	Butter, Falk Andrade, Miguel
Title:	Mass spectrometry-based quantitative proteomics to investigate RNA-protein interactions
Online publication date:	21-Mar-2024
Year of first publication:	2024
Language:	english
Abstract:	Mass spectrometry-based proteomics is a versatile tool, offering a global and unbiased approach to analyse proteins and their interacting partners. Within the realm of molecular biology, RNA-protein interactions stand as fundamental and intricate components that oversee vital processes in the cell. These interactions, often mediated by specific RNA-binding proteins (RBPs), orchestrate a wide array of cellular functions. From the regulation of gene expression to the maintenance of genomic stability, the post-transcriptional processing of RNA molecules, and even the spatial organisation of the cell nucleus, RNA-protein interactions play a central role in shaping the intricate web of cellular activities. In this thesis, the interaction between RNA and proteins is investigated using state-of-the-art MS. Article Ⅰ delved into the characterization of Telomeric repeat-containing RNA (TERRA) molecules in M. musculus, examining their genomic origins and comparing their interactomes to their H. sapiens counterparts. RNA-FISH analysis revealed disparities in behaviour, with M. musculus TERRA foci primarily located outside of telomeres, in contrast to H. sapiens TERRA foci, which recurrently resided at telomeres. As a result, a distinct genomic origin for M. musculus TERRA molecules outside telomeres was hypothesised. Through a comprehensive genomic analysis, four major chromosomal regions, including known Telo 18q, PAR-Xq/Yq, and ChrX Tsix locus regions, and a novel Chr2 region, were identified as potential sources of TERRA molecules. Conservation of TERRA-associated functions was evaluated with an affinity purification-mass spectrometry (AP-MS) approach. A comparison of the enriched proteins with publicly available H. sapiens TERRA-interacting protein datasets revealed that, despite having a distinct genomic origin, functions are conserved between M. musculus and H. sapiens. Article Ⅱ centred on the functional assignment of RBPs in S. cerevisiae. An AP-MS screen was designed to elucidate the interaction partners of 40 selected RBPs, which were chosen based on their involvement in various stages of mRNA processing. Functional analysis of the collected data highlighted the overrepresentation of canonical RNA-binding domains (RBDs) and RNA binding-related GO molecular function terms among the RBPs' interaction partners. KEGG pathway analysis demonstrated the enrichment of RNA pathways, consistent with the RBP selection criteria, as well as involvement in metabolic and synthesis pathways. Finally, network-based function assignment of RBPs was facilitated by concurrent binding patterns within the network.
DDC:	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-10153
URN:	urn:nbn:de:hebis:77-openscience-d69fe09a-fa24-4f6c-ac23-99204b9dc5ab8
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	http://rightsstatements.org/vocab/InC/1.0/
Extent:	ix, 108 Seiten ; Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen