The Functional Analysis Of Ribosomal Protein L11 From Euplotes Octocarinatus

Protein synthesis is one of the most important processes in organism, and thus is essential for cell growth, proliferation and differentiation. Ribosome is the site of protein translation in all living cells. In eukaryotes, a mature ribosome consists of 79 ribosomal proteins and four rRNA species. The best-known function shared by ribosomal proteins is their role in the assembly of ribosomal subunits, and as a result, their role in translation. Many ribosomal proteins are also believed to have important functions in various other cellular processes.Ribosomal protein L11 (RPL11) is part of the large 60S ribosomal subunit in eukaryotes. RPL11 is highly conserved during evolution. RPL11 participates in ribosomal assembly. In addition, RPL11 was reported to activate p53 which then inhibits oncoprotein HDM2 and subsequently the inhibition of cell cycle progression and regulate c-Myc-enhanced ribosomal biogenesis as a feedback inhibitor of c-Myc in mammalian cells.Euplotes octocarinatus, a free-living unicellular protozoan, was studied as the experimental material in our lab. The gene encoding RPL11 protein from the macronuclear DNA of Euplotes octocarinatus had been cloned previously. In the present study, the subcellular localization of the Euplotes ribosomal protein L11 (EoRPL11) and its regulatory function in protein synthesis process were investigated in the heterologous system. The results obtained are as follows.The coding region of cDNA for EoRPL11 was fused in frame to the green fluorescent protein (GFP). When GFP-EoRPL11 was expressed in HEK293T cells, green fluorescence clearly accumulated within the nucleus, particularly in the nucleolus and no fluorescence could be detected within the cytoplasm. As a control, expression of GFP alone resulted in a homogeneous distribution of fluorescence throughout the cytoplasm and nucleus. To further confirm the localization of EoRPL11, we carried out similar experiment in Hela cells. The same results were obtained. To investigate its function in the process of protein synthesis, EoRPL11 was cloned into the expression vector pCMV-Myc. Both pCMV-Myc-EoRPL11 and pCMV-Myc-GST (as a control) were transiently cotransfected with renilla luciferase reporter plasmid pRL-TK into the HEK293T cells.The expression of Myc-EoRPL11 in HEK293T cells was confirmed by RT-PCR and Western blot. Data from luciferase reporter gene analysis demonstrated that the luciferase activity was decreased significantly in cells transfected with EoRPL11 in a dose dependent manner, while quantitative real-time PCR showed that the reporter gene was not affected at the transcriptional level by the introduction of Myc-EoRPL11. Taken together, these results indicated that EoRPL11 down-regulated the cellular protein synthesis in vivo at the translational level.The decreased cellular protein synthesis of HEK293T cells upon EoRPL11 overexpression prompted us to evaluate the effect on the proliferation and cell cycle of the transfected cells. The cell proliferation was determined by MTT assay. The growth rate of the cells transfected EoRPL11 was slightly lower than that of cells transfected with empty plasmid pCMV-Myc after two days. The effect of EoRPL11 overexpression on the cell cycle was also carried out in HEK293T cells by transfecting with EoRPL11 plasmid DNA. There was clearly no difference between the number of cells in different phases of the cell cycle in the control and EoRPL11-overexpressed cells. Therefore, EoRPL11 did not affect the distribution of cells in the distinct phases of the cells.The RPL11 can interact with HDM2 and inhibit HDM2 function, thus leading to the stabilization and activation of p53. Through RT-PCR analysis of the expression about the HDM2 protein in Hela cells, the wild type gene of HDM2 and a new HDM2 splice variant were cloned. The new splice variant possessed 1401 bp reading frame which was predicted to code 466 amino acids, generating an amino acidic loss from residue R29 to V53 and two point mutations (S395F and S407C). This splice variant, which is partially deleted upstream the p53 binding site, may be responsible for the inactivation of p53 and tumor cells transforming pathway.