Rrp14 Protein Affects RRNA Transcription Via Facilitating The Translocation Of Pol5 Into Thenucleolus In Schizosaccharomyces Pombe

Objective:Ribosomes are essential for protein synthesis in every cell.Recent studies have shown that nucleolar proteins that link cell proliferation to ribosome biogenesis are potential oncogenes,the human SURF6 protein is one of the evolutionarily conserved nucleolar proteins and has an unknown function.The research on this protein may provide new ideas for tumor therapy.Recent studies have confirmed that SURF6 gene is highly expressed in tumor cells,and this high expression may provide ribosomal material for tumors to achieve high protein expression,but the specific mechanism of participation remains unclear.In Schizosaccharomyces pombe,The molecular biology operation is simple and rapid,and the protein encoded by rrp14 gene is highly homologous to the amino terminus of human SURF6 protein.Therefore,this experiment chose fission yeast as a research object to study the function of protein.In Schizosaccharomyces Pombe,the SURF6 gene is divided into two different genes,rrp14 and rrp1402.The proteins they encode correspond to the N-terminus and C-terminus of SURF6,respectively.This provides a natural model organism for our study of the N-terminus of SURF6.In this experiment,we will investigate the effect of the rrp14 gene on nucleolar transport and ribosomal RNA.Methods:The rrp14 gene knockout yeast was prepared by homologous recombination of the gene,and the growth state of the yeast was observed.The relationship between in situ labeled Rrp14 protein and Pol5 protein in yeast was studied by mass spectrometry,immunoprecipitation and immunofluorescence microscopy.The desired plasmid was constructed by molecular cloning and transferred into yeast.The Pil1 co-anchoring experiment was used to find the region where Rrp14 protein interacted with Pol5 protein.Apply to the CRISPR/Cas9-mediated genome editing technique to screen out the desired yeast and observe the growth of the yeast.The effect of rrp14 gene on ribosomal RNA was observed by RNA extraction and real-time quantitative PCR.Results:Growth experiments indicate that rrp14 knockdown affects cell growth.The results of mass spectrometry,co-immunoprecipitation and immunofluorescence microscopy showed that Rrp14 protein interacted with Pol5 protein and colocalized in nucleolus,and Rrp14 was likely to have the function of assisting Pol5 nucleolar localization.Pil1 co-anchoring experiments indicated that Rrp14 protein may interact with Pol5 protein through amino acids 7-12.Moreover,the interaction of Rrp14 protein with Pol5 protein may affect yeast growth.The total RNA content of the S.Pombe that knocked out Rrp14 amino acids 7-12 and knocked out rrp14 was continuously reduced compared to wild-type.Real-time quantitative PCR showed that the levels of 28 S,18S,and ITS1 were decreasing,indicating that the rrp14 gene affects the transcription of ribosomal RNA.Conclusion:1.Rrp14 and Pol5 proteins interact physically in the nucleolus.2.Rrp14 interacts with Pol5 through the 7-RINAWN-12 domain.3.Rrp14 has the function of assisting the transport of Pol5 nucleoli.4.Rrp14 affects the transcription of rRNA by affecting the transport of Pol5 nucleoli.