Browsing by Author "Atbinek, Melis"

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    Article
    Citation - WoS: 4
    Citation - Scopus: 4
    Epitranscriptomics M6a Analyses Reveal Distinct M6a Marks Under Tumor Necrosis Factor Α (tnf-Α) Apoptotic Conditions in Hela Cells
    (Wiley, 2024) Akçaöz Alasar, Azime; Tuncel, Özge; Sağlam, Buket; Gazaloğlu, Yasemin; Atbinek, Melis; Çağıral, Umut; İşcan, Evin; Özhan, Güneş; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of Technology
    Tumor necrosis factor-alpha (TNF-alpha) is a ligand that induces both intrinsic and extrinsic apoptotic pathways in HeLa cells by modulating complex gene regulatory mechanisms. However, the full spectrum of TNF-alpha-modulated epitranscriptomic m(6)A marks is unknown. We employed a genomewide approach to examine the extent of m(6)A RNA modifications under TNF-alpha-modulated apoptotic conditions in HeLa cells. miCLIP-seq analyses revealed a plethora of m(6)A marks on 632 target mRNAs with an enrichment on 99 mRNAs associated with apoptosis. Interestingly, the m(6)A RNA modification patterns were quite different under cisplatin- and TNF-alpha-mediated apoptotic conditions. We then examined the abundance and translational efficiencies of several mRNAs under METTL3 knockdown and/or TNF-alpha treatment conditions. Our analyses showed changes in the translational efficiency of TP53INP1 mRNA based on the polysome profile analyses. Additionally, TP53INP1 protein amount was modulated by METTL3 knockdown upon TNF-alpha treatment but not CP treatment, suggesting the existence of a pathway-specific METTL3-TP53INP1 axis. Congruently, METLL3 knockdown sensitized HeLa cells to TNF-alpha-mediated apoptosis, which was also validated in a zebrafish larval xenograft model. These results suggest that apoptotic pathway-specific m(6)A methylation marks exist in cells and TNF-alpha-METTL3-TP53INP1 axis modulates TNF-alpha-mediated apoptosis in HeLa cells.
  • Thumbnail Image
    Article
    Citation - WoS: 4
    Epitranscriptomics M6a Analyses Reveal Distinct M6a Marks Under Tumor Necrosis Factor Α (tnf-Α) Apoptotic Conditions in Hela Cells
    (Wiley, 2024) Akçaöz Alasar, Azime; Tüncel, Özge; Sağlam, Buket; Gazaloğlu, Yasemin; Atbinek, Melis; Çağıral, Umut; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of Technology
    Tumor necrosis factor-alpha (TNF-alpha) is a ligand that induces both intrinsic and extrinsic apoptotic pathways in HeLa cells by modulating complex gene regulatory mechanisms. However, the full spectrum of TNF-alpha-modulated epitranscriptomic m(6)A marks is unknown. We employed a genomewide approach to examine the extent of m(6)A RNA modifications under TNF-alpha-modulated apoptotic conditions in HeLa cells. miCLIP-seq analyses revealed a plethora of m(6)A marks on 632 target mRNAs with an enrichment on 99 mRNAs associated with apoptosis. Interestingly, the m(6)A RNA modification patterns were quite different under cisplatin- and TNF-alpha-mediated apoptotic conditions. We then examined the abundance and translational efficiencies of several mRNAs under METTL3 knockdown and/or TNF-alpha treatment conditions. Our analyses showed changes in the translational efficiency of TP53INP1 mRNA based on the polysome profile analyses. Additionally, TP53INP1 protein amount was modulated by METTL3 knockdown upon TNF-alpha treatment but not CP treatment, suggesting the existence of a pathway-specific METTL3-TP53INP1 axis. Congruently, METLL3 knockdown sensitized HeLa cells to TNF-alpha-mediated apoptosis, which was also validated in a zebrafish larval xenograft model. These results suggest that apoptotic pathway-specific m(6)A methylation marks exist in cells and TNF-alpha-METTL3-TP53INP1 axis modulates TNF-alpha-mediated apoptosis in HeLa cells.
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    Master Thesis
    Investigation of Long Non-Coding Rna and Chromatin Interactions in Hela Cells
    (Izmir Institute of Technology, 2022-07) Atbinek, Melis; Akgül, Bünyamin; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of Technology
    The DNA in the cells is surrounding histone proteins to form nucleosomes. The structure is packed further into chromatin. The chromatin structure is dynamic and flexible. It is regulated by many factors including long non-coding RNAs (lncRNAs). LncRNAs are a class of non-coding RNAs, transcripts that do not encode protein. They are longer than 200 nucleotides and might contain a polyA tail and a 5’ cap. Thus, they are localized in the nucleus. lncRNAs interact with chromatin in two ways, indirect and direct. Direct interaction occurs via two mechanisms: R-loop and triplex formation. These interactions affect the folding of chromatin inducing gene expression under various cellular conditions. LncRNAs interacting with chromatin regulating genes are found in HEK cells. Thus, it is hypothesized that lncRNA – chromatin interactions may differ in cancerous cells as well. In this study, the iMARGI method is optimized to be used in adenocarcinoma HeLa cells. The chromatin digestion and incubation conditions are adjusted to give optimal results for HeLa cells. iMARGI is a recently developed method employed to investigate such interactions in a genome-wide manner. iMARGI allows the isolation of all lncRNAs interacting with the whole genome. The interacting RNA – DNA molecules are pulled down with streptavidin conjugated beads after linker ligation. The chimeric molecules are amplified with PCR forming lncRNA – chromatin libraries of HeLa cells. In the future, new libraries can be formed after inducing apoptosis in HeLa cells. Identification of lncRNAs involved in chromatin remodeling in apoptotic conditions can facilitate new therapeutic methods for fighting tumor initiation and development.