Mass spectrometry-based quantitative proteomics to investigate RNA-protein interactions
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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.