<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "RNA-dependent proteins in lung cancer and their riboregulation in mitosis"^^ . "From the onset of their transcription in the nucleus until their degradation in the cytoplasm, \r\nRibonucleic acid (RNA) transcripts associate with RNA-binding proteins (RBPs) to form \r\nribonucleoprotein (RNP) complexes. RNPs are dynamic macromolecular assemblies that \r\nregulate the fate of RNA molecules by coordinating all aspects of their post transcriptional \r\nmaturation and regulation such as splicing, modification, transport, translation and decay. \r\nConversely, recent studies pointed to a more RNA centric view of RNA-protein interactions, \r\ntermed “riboregulation”, where RNA modulates RBP localization, conformation, interactions\r\nand function. RBPs can bind their RNA targets with a wide range of affinities, modulated by \r\ne.g. post-translational modifications, interacting partners and structure. Due to their central role \r\nin various key cellular processes, dysregulation of RBP functions is implicated in the initiation \r\nand development of diseases such as neurological disorders, cancer and muscular atrophies. \r\nRBPs represent one of the largest protein groups and >1000 RBPs are associated with diseases. \r\nTherefore, RBPs have attracted increased interest in the past years, leading to the development \r\nof multiple strategies and tools to establish a comprehensive catalogue of RBPs in human and \r\nother various species. \r\nComplementary 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 \r\nbind to RNA). R-DeeP is based on sucrose density gradient ultracentrifugation and \r\nfractionation, followed by mass spectrometry analysis. It provides quantitative information on \r\nthe fraction of a protein being RNA dependent as well as allows the reconstruction of protein \r\ncomplexes based on co-segregation. Hence, to obtain a comprehensive view on RBPs in lung \r\ncancer, in the first part of my PhD project, I took advantage of the adaptability of the R-DeeP \r\nscreen and identified RNA-dependent proteins in A549 lung adenocarcinoma cells. With this \r\nproteome-wide technique, I identified 1189 RNA-dependent proteins which includes 170 \r\nproteins which had never been linked to RNA before. Out of the 170 novel RNA-dependent \r\nproteins, I validated the RNA-dependence of three newly identified shifting proteins: DOCK5, \r\nELMO2, and ABRAXAS1 using western blot analysis. Further, the direct RNA interaction of \r\nthe cell migration-related protein DOCK5 and BRCA1-associated protein ABRAXAS1 was \r\nverified 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 \r\nHeLa S3 cell lines. This study contributes to increase our understanding of the functional role \r\nof RNA and RNA-binding proteins in cancer cells.\r\nGiven the role of RBPs in a wide range of cellular processes, and particularly in the context of \r\ncell division, several studies have reported the presence of RNA, i.e. protein-coding (mRNAs), \r\nnon-coding RNAs (ncRNAs) and various RBPs within structures of the mitotic spindle \r\napparatus such as the centrosomes and the spindle MTs. Collectively, these works pointed to \r\nthe importance of RNA and RBPs for the structural and functional integrity of the mitotic \r\nspindle. However, the underlying mechanisms remain unknown. A recent R-DeeP screen in \r\nHeLa cells synchronized in prometaphase generated a huge resource on RNA-dependent \r\nproteins with cell cycle-specific information.\r\nIn the second part of my PhD project, I took advantage of this new resource and identified the \r\nRNA dependence of several key mitotic factors: AURKA, KIFC1 and TPX2. Further using \r\nimmunoprecipitation coupled with mass spectrometry analysis, I uncovered new interacting \r\npartners 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 \r\nRNA-dependent fashion. Though the interaction between AURKA and TPX2 has been well \r\ncharacterized, their RNA-mediated interaction was unknown. To uncover the functional \r\nsignificance of the interaction between AURKA and KIFC1, an in vitro kinase assay was \r\nperformed and it revealed that AURKA phosphorylated KIFC1 in an RNA-dependent manner \r\nat S349 and T359 amino acid residues. With the aim to identify the RNA targets mediating their \r\ninteractions, iCLIP2-sequencing of KIFC1-bound RNAs was performed in HeLa cells \r\nsynchronized in prometaphase. The sequencing results demonstrated that, KIFC1 \r\npredominantly bound to ribosomal RNAs (rRNAs) and protein-coding RNAs (mRNAs) in \r\nprometaphase without sequence specificity. Though, KIFC1 lacked sequence specificity in \r\nbinding to RNA, I identified that these RNA targets also bound AURKA and TPX2 in \r\nprometaphase cell lysates. These data suggest that these three key mitotic factors interact with \r\neach other and exist within the same complex. Notably, RNA played a crucial role in mediating \r\ntheir interaction, which is crucial for spindle assembly and faithful cell division, indicating the \r\nriboregulation of mitotic protein-protein interactions during spindle assembly. This offers new \r\nperspectives on the control of cell division processes by RNA-protein complexes."^^ . "2025" . . . . . . . "Varshni"^^ . "Rajagopal"^^ . "Varshni Rajagopal"^^ . . . . . . "RNA-dependent proteins in lung cancer and their riboregulation in mitosis (PDF)"^^ . . . "PhD_Thesis_VarshniRajagopal.pdf"^^ . . . "RNA-dependent proteins in lung cancer and their riboregulation in mitosis (Other)"^^ . . . . . . "medium.jpg"^^ . . . "RNA-dependent proteins in lung cancer and their riboregulation in mitosis (Other)"^^ . . . . . . "preview.jpg"^^ . . . "RNA-dependent proteins in lung cancer and their riboregulation in mitosis (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "RNA-dependent proteins in lung cancer and their riboregulation in mitosis (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "RNA-dependent proteins in lung cancer and their riboregulation in mitosis (Other)"^^ . . . . . . "small.jpg"^^ . . "HTML Summary of #36200 \n\nRNA-dependent proteins in lung cancer and their riboregulation in mitosis\n\n" . "text/html" . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .