ABSTRACT
BACKGROUND: The Ccr4-Not complex (CNOT complex in mammals) is a unique and highly conserved complex with numerous cellular functions. Until now, there has been relatively little known about the importance of the CNOT complex subunits in the DNA damage response (DDR) in mammalian cells. CNOT4 is a subunit of the complex with E3 ubiquitin ligase activity that interacts transiently with the CNOT1 subunit. Here, we attempt to investigate the role of human CNOT4 subunit in the DDR in human cells. MATERIAL AND METHODS: In this study, cell viability in the absence of CNOT4 was assessed using a Cell Titer-Glo Luminescence assay up to 4 days post siRNA transfection. In a further experiment, CNOT4-depleted HeLa cells were exposed to 3Gy ionizing radiation (IR). Ataxia telangiectasia-mutated (ATM) and ATM Rad3-related (ATR) signaling pathways were then investigated by western blotting for phosphorylated substrates. In addition, foci formation of histone 2A family member X (γH2AX), replication protein A (RPA), TP53 binding protein 1 (53BP1), and DNA repair protein RAD51 homolog 1 was also determined by immunofluorescence microscopy comparing control and CNOT4-depleted HeLa cells 0, 8, and 24 h post IR treatment. RESULTS: Our results from cell viability assays showed a significant reduction of cell growth activity at 24 (P value 0.02) and 48 h (P value 0.002) post siRNA. Western blot analysis showed slightly reduced or slightly delayed DDR signaling in CNOT4-depleted HeLa cells after IR. More significantly, we observed increased formation of γH2AX, RPA, 53BP1, and RAD51 foci after IR in CNOT4-depleted cells compared with the control cells. CONCLUSION: We conclude that depletion of CNOT4 affects various aspects of the cellular response to DNA damage.
Subject(s)
Cell Cycle Proteins , Radiation, Ionizing , Animals , Humans , HeLa Cells , Ataxia Telangiectasia Mutated Proteins/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA Damage , DNA Repair , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Phosphorylation , Mammals/genetics , Mammals/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
BACKGROUND: The Ccr4-Not protein complex (CNOT complex) is a key regulator of gene expression in eukaryotic cells. Ccr4-Not Complex is composed of at least nine conserved subunits in mammalian cells with two main enzymatic activities. CNOT8 is a subunit of the complex with deadenylase activity that interacts transiently with the CNOT6 or CNOT6L subunits. Here, we focused on the role of the human CNOT8 subunit in the DNA damage response (DDR). METHODS: Cell viability was assessed to measure ATP level using a Cell Titer-Glo Luminescence reagent up to 4 days' post CNOT8 siRNA transfection. In addition, expression level of phosphorylated proteins in signalling pathways were detected by western blotting and immunofluorescence microscopy. CNOT8- depleted Hela cells post- 3 Gy ionizing radiation (IR) treatment were considered as a control. RESULTS: Our results from cell viability assays indicated a significant reduction at 72-hour post CNOT8 siRNA transfection (p= 0.04). Western blot analysis showed slightly alteration in the phosphorylation of DNA damage response (DDR) proteins in CNOT8-depleted HeLa cells following treatment with ionizing radiation (IR). Increased foci formation of γH2AX, RPA, 53BP1, and RAD51 foci was observed after IR in CNOT8-depleted cells compared to the control cells. CONCLUSION: We conclude that CNOT8 deadenylase subunit is involved in the cellular response to DNA damage.
ABSTRACT
Long non-coding RNAs (lncRNAs) are a class of cellular transcripts, which are involved in various biological processes. There is conflicting data regarding to the origin of these non-coding molecules and lncRNAs are thought to be the origin of viral genome. Here we sought to find the homology between human lncRNAs and viruses. For this purpose, the lncRNAdb database was searched for human lncRNAs. The lncRNAs' sequences were aligned with virus taxa using NCBI's BLAST tool. The phylogenic study was performed with maximum-likelihood based algorithm. The database contains 152 human lncRNAs. As a result, 63 (41.44%) of the lncRNAs have homologies with viruses. Of which, 50 (79.36%) have homology with Stealth virus. Other viruses with homology to lncRNAs were nuclear integrating DNA/RNA viruses. Moreover, 35 of 64 (23.03%) of cancer-associated lncRNAs have sequence homology with the same viruses. In phylogenetic analyses, lncRNAs with no homology to viruses were found to be the ancestor of those with homology to viruses and cancer-irrelevant lncRNAs were found to be the ancestor of cancer-related transcripts. In conclusion, lncRNAs could be the origin of nuclear integrating viruses and the nuclear integrating viruses may evolved from the non-coding regions. The results imply the role of lncRNAs with homology to viruses in human cancers.
