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Abstract

From the onset of their transcription in the nucleus until their degradation in the cytoplasm, Ribonucleic acid (RNA) transcripts associate with RNA-binding proteins (RBPs) to form ribonucleoprotein (RNP) complexes. RNPs are dynamic macromolecular assemblies that regulate the fate of RNA molecules by coordinating all aspects of their post transcriptional maturation and regulation such as splicing, modification, transport, translation and decay. Conversely, recent studies pointed to a more RNA centric view of RNA-protein interactions, termed “riboregulation”, where RNA modulates RBP localization, conformation, interactions and function. RBPs can bind their RNA targets with a wide range of affinities, modulated by e.g. post-translational modifications, interacting partners and structure. Due to their central role in various key cellular processes, dysregulation of RBP functions is implicated in the initiation and development of diseases such as neurological disorders, cancer and muscular atrophies. RBPs represent one of the largest protein groups and >1000 RBPs are associated with diseases. Therefore, RBPs have attracted increased interest in the past years, leading to the development of multiple strategies and tools to establish a comprehensive catalogue of RBPs in human and other various species. Complementary methods such as the R-DeeP screen have been developed to identify RNA dependent proteins (proteins that rely on RNA to form larger RNPs and directly or indirectly bind to RNA). R-DeeP is based on sucrose density gradient ultracentrifugation and fractionation, followed by mass spectrometry analysis. It provides quantitative information on the fraction of a protein being RNA dependent as well as allows the reconstruction of protein complexes based on co-segregation. Hence, to obtain a comprehensive view on RBPs in lung cancer, in the first part of my PhD project, I took advantage of the adaptability of the R-DeeP screen and identified RNA-dependent proteins in A549 lung adenocarcinoma cells. With this proteome-wide technique, I identified 1189 RNA-dependent proteins which includes 170 proteins which had never been linked to RNA before. Out of the 170 novel RNA-dependent proteins, I validated the RNA-dependence of three newly identified shifting proteins: DOCK5, ELMO2, and ABRAXAS1 using western blot analysis. Further, the direct RNA interaction of the cell migration-related protein DOCK5 and BRCA1-associated protein ABRAXAS1 was verified using iCLIP2. The R-DeeP 2.0 database https://R-DeeP2.dkfz.de provides proteome-wide and cell line-specific information on proteins and their RNA dependence from A549 and HeLa S3 cell lines. This study contributes to increase our understanding of the functional role of RNA and RNA-binding proteins in cancer cells. Given the role of RBPs in a wide range of cellular processes, and particularly in the context of cell division, several studies have reported the presence of RNA, i.e. protein-coding (mRNAs), non-coding RNAs (ncRNAs) and various RBPs within structures of the mitotic spindle apparatus such as the centrosomes and the spindle MTs. Collectively, these works pointed to the importance of RNA and RBPs for the structural and functional integrity of the mitotic spindle. However, the underlying mechanisms remain unknown. A recent R-DeeP screen in HeLa cells synchronized in prometaphase generated a huge resource on RNA-dependent proteins with cell cycle-specific information. In the second part of my PhD project, I took advantage of this new resource and identified the RNA dependence of several key mitotic factors: AURKA, KIFC1 and TPX2. Further using immunoprecipitation coupled with mass spectrometry analysis, I uncovered new interacting partners of AURKA including KIFC1. KIFC1 interacted with AURKA and TPX2 in an RNA-dependent manner. Importantly, I discovered that TPX2 also interacted with AURKA in an RNA-dependent fashion. Though the interaction between AURKA and TPX2 has been well characterized, their RNA-mediated interaction was unknown. To uncover the functional significance of the interaction between AURKA and KIFC1, an in vitro kinase assay was performed and it revealed that AURKA phosphorylated KIFC1 in an RNA-dependent manner at S349 and T359 amino acid residues. With the aim to identify the RNA targets mediating their interactions, iCLIP2-sequencing of KIFC1-bound RNAs was performed in HeLa cells synchronized in prometaphase. The sequencing results demonstrated that, KIFC1 predominantly bound to ribosomal RNAs (rRNAs) and protein-coding RNAs (mRNAs) in prometaphase without sequence specificity. Though, KIFC1 lacked sequence specificity in binding to RNA, I identified that these RNA targets also bound AURKA and TPX2 in prometaphase cell lysates. These data suggest that these three key mitotic factors interact with each other and exist within the same complex. Notably, RNA played a crucial role in mediating their interaction, which is crucial for spindle assembly and faithful cell division, indicating the riboregulation of mitotic protein-protein interactions during spindle assembly. This offers new perspectives on the control of cell division processes by RNA-protein complexes.