| Objective: The cell cycle is an essential process by which a single cell divides to two daughter cells. During this process, the cell duplicates its contents and then divides into two cells with high fidelity. The cell cycle is the most important mechanism by which all living things develop and renew. Three key classes of regulatory proteins,cyclins, cyclin-dependent kinases(Cdks) and Cdk inhibitory protein(CKIs) are involved in the cell cycle regulation. Tudor-SN(Tudor staphylococcal nuclease)protein, which is a conserved and multi-functional protein, implicates in many cellular biological processes. In the previous studies, we found that Tudor-SN protein was high-expressed in the proliferating-cells while barely expressed in the terminal differentiation cells, which suggested that the expression level of Tudor-SN is related to the proliferation ability of cells. Further studies showed that Tudor-SN, as a new regulator of cell cycle, promoted the transition of He La cells from G1 phase to S phase. Knockdown the expression level of Tudor-SN protein could arrest the He La cells in G1 phase, and prevent the synchronized G1 phase cells releasing to S phase.This project contains two parts. Part I aims to figure out the mechanism of Tudor-SN protein on cell cycle regulation, and part II will investigate the regulation mechanism of low expression of Tudor-SN in terminal differentiation cells.Methods: Part I is to investigate the mechanism of Tudor-SN protein on cell cycle regulation. Firstly, we will perform the Co-Immunoprecipitation(Co IP) and cell cycle synchronization to discover the potential kinase of Tudor-SN during cell cycle progress. Then, bioinformatics prediction, point mutation, flow cytometry and dual luciferase report system will be used to detect the key phosphorylation site of Tudor-SN in the cell cycle regulation. At last, Co IP and Chromatin Immunoprecipitation(Ch IP) will be performed to illustrate how Tudor-SN regulates the transcription activity of S phase genes. Part Ⅱ is to study the biological significance of Tudor-SN in cell cycle regulation. WB, Realtime PCR and Polysome profile analysis will be used to detect the regulation mechanism of low expression of Tudor-SN in terminal differentiation cells. Then dual luciferase report gene analysiswill be performed to ensure the regulation of PI3 K / AKT / m TOR signaling pathway on Tudor-SN in H9c2 and He La cell lines. Flow cytometry will be used to detect the role of Tudor-SN in the regulation of PI3 K / AKT / m TOR signaling pathway on cell cycle.Result: Part I: The phosphorylation level of Tudor-SN is varied in different cell cycle phases, and Cdks are the upstream kinases of Tudor-SN. Serine 426/ Threonine 429 are the important phosphorylation amino acid residues of Tudor-SN in cell cycle regulation. Tudor-SN could enhance the interaction of E2F-1 with histone acetyltransferase GCN5, and increases the binding ability of transcription factor E2F-1 to the promoter of cyclin A and E2F-1 genes, as a result, improves the transcriptional activaty of these two genes.Part Ⅱ : In the terminal differentiation cardiac muscle cells, the protein level of Tudor-SN is down-expressed while the m RNA level was constant. The regulation of Tudor-SN expression in the terminal differentiation cardiac muscle cells is occurred in the protein translation level. Tudor-SN m RNA is a new TOP m RNA which contains the 5’TOP motif. PI3 K / AKT / m TOR/4EBP signaling pathway participates in the regulation of Tudor-SN expression. Tudor-SN influences the regulation of PI3K/ AKT / m TOR pathway on the cell cycle.Conclusion: Part I: Tudor-SN protein could be phosphorylated by the Cdks at Serine426/ Threonine 429 residues, meanwhile, Tudor-SN protein could promote G1/S phases transition through acting as a new co-activator of E2F-1.Part Ⅱ: Tudor-SN is a new TOP m RNA. PI3 K / AKT / m TOR pathway regulates the expression of Tudor-SN, and Tudor-SN influences the regulation of PI3 K / AKT /m TOR pathway on the cell cycle. |
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