Identification Of The Interaction Region Of Pol5 With Rrp14

Objective:Ribosomes are the Organelles that produce all cellular proteins by a complex and highly regulated biochemical process termed translation.The synthesis of ribosomes initiated in the nucleolus,pre-r RNAs(35S pre-r RNA)are transcribed,processed and transported to the cytoplasm to be mature ribosomes with ribosomal proteins.Both Pol5 and Rrp14 are expressed in the nucleolus,Studies have shown that Pol5 is the assembly factor of the ribosomes and involved in the pre-process of ribosome assembly.The maturation of large ribosomal subunits(LSU)and small ribosomal subunits(SSU)requires Pol5.In addition,studies have shown that Pol5 plays an important role in r DNA transcription,early embryonic development,and tumorigenesis.Rrp14 is a member of the SURF-6 family of nucleolar proteins,and is involved in the interaction between proteins and proteins and between proteins and RNA in the nucleolus.The previous experiment results of the research group have shown that Pol5 interacts with Rrp14.In this study,we intend to further identify the interaction region of Pol5 and Rrp14 in fission yeast which is a classical model organism,and study the implication of the region in Pol5’s nucleolar localization.Methods:Construct the plasmid with a molecular cloning method based on homologous recombination,The functional domain of the interaction between Pol5 and Rrp14 was identified by the Yeast two-hybrid assay.The CRISPR-Cas9 system was used to knock out the interaction domain of Pol5 with Rrp14.Observe the expression and distribution of Pol5 in the cell after knocking out the Pol5 interaction region through a fluorescence microscope to clarify the implication of the interaction region in the function of Pol5.Results:1.Pol5 protein does not interact with Rrp14 protein after knocking out the amino acid sequence from 1 to 20 of the N-terminus of Pol5 protein2.Knockout of lysine at position 18 and alanine at position 19 on the N-terminal of Pol5 protein affects the localization of Pol5 protein in the nucleolus.Conclusion:The amino acid sequence at positions 1 to 20 of the N-terminal of the Pol5 protein is an important region that determines the interaction between the Pol5 protein and the Rrp14 protein.The amino acid sequence at positions 1 to 20 of the N-terminal of Pol5 protein affects the localization of Pol5 protein in the nucleolus.