The role of HN1 in mTOR-mediated centrosome and ribosome maintenance
Project no. 23342 · Status: Kapanmış · 2022
Summary
<p>In this thesis study, functional aspects, and characterization of HN1 was performed. As an oncogene for some cancers, HN1 was investigated in terms of centrosome stability as well as cell cycle dynamics, nuclear movement, and the protein synthesis.</p><p>To describe the role of HN1 in the cell cycle, inducible and stabilized overexpression systems were established in cells to perform acquired function of HN1. Therefore, it was characterized in prostate and breast cancer cell lines using kinase inhibitor’s experiments, western blotting, flow cytometry, immunofluorescence, and cellular fractionation approaches. According to these results, HN1 is phosphorylated by Cdk1 in the mitotic phase, and in cell cycle synchronizations. As it was determined that cells slow-down in the S phase in the case of high HN1 expression before mitosis, and cells exit mitosis early in the case of high expression after G2. As a result, HN1 was found to be a new cell cycle regulatory protein with a potential dual role in the modulation of cell cycle progression in the S phase and mitotic exit.</p><p>The roles of HN1 in centrosome maturation/growing during cell division in the PC3 cell line, the centrosome component plus end kinesin protein Eg5 and Aurora A-Plk1 interactions including pericentriolar matrix protein (PCM1) were examined. Also, the role of HN1 in centrosome nucleation and maturation that accompanies Plk1-Eg5 axis, as well as in the mitotic spindle formation were defined.</p><p>In cells with suppressed (KD) HN1 expression, proliferation, migration, invasion and colony formation were found to be reduced in comparison to control cells, and flow cytometry analyses showed that the nuclei of these cells were enlarged. All these findings and the recent report suggesting that HN1 activates the mTOR signaling pathway led us to study components of protein synthesis.</p><p>When investigating the role of HN1 in the mTOR signaling pathway using various gain- and loss-of-function experiments in different cell lines, <strong>we have shown first time that the HN1 is required for the integrity and function of the Nucleolar Organizing Region (NOR), and protein synthesis.</strong> Immunoprecipitation-based interaction and colocalization studies also confirmed that <strong>HN1 is an important component of the mTOR-RPS6 axis</strong> and its suppression results in reduced mRNA translation in mammalian cancer cell lines. <strong>This study shows that suppression of HN1 causes irregular distribution of nucleolar structures, potentially leading to cell cycle perturbation.</strong>Accordingly, components of the translation machinery assemble into a distinct speckled pattern in nucleoli, lose their essential interactions, and consequently negatively affect mRNA translation efficiency when HN1 expression is suppressed.</p><p><strong>These results suggest that HN1 is an essential component required for ribosome biosynthesis and global mRNA translation, conserved throughout metazoan evolution.</strong></p><p><strong> </strong></p><p><strong>Keywords:</strong> HN1, Centrosome, Nucleolus, NOR, Ribosome biogenesis, Protein Synthesis</p>