Construction Of Auxin–inducible NPM1 Degradation System And Analysis Of Early Phenotypes

Objective:Nucleophosmin1(NPM1)is an abundant and conserved multifunctional protein in cells.In addition to participating in ribosome formation in nucleoli,NPM1 can also be dynamically distributed in the nucleoplasm and cytoplasm,and participate in important biological processes such as cell stress response and maintenance of cell homeostasis.Early studies on knockout mouse models showed that NPM1 is an essential gene for embryonic development.NPM1 knockout MEF cells showed genomic instability such as multipolarization and aneuploidy.The cumulative effects of NPM1 deletion eventually lead to embryo death,the mechanism of which is still unknown.After knockdown of NPM1 by RNAi technique in human cervical cancer cell line He La,genomic instability was also observed,such as chromosomal disorder and frequent occurrence of multinucleated and micronucleated cells.However,the inherent defects of RNAi techniques,such as off-target and knockout efficiency,limit the observation of phenotypes and the interpretation of mechanisms.In order to overcome the problems in the above studies and further elucidate the molecular mechanism of NPM1 maintaining genomic stability,In this study,the auxininducible degron(AID)system for endogenous NPM1 gene degradation was first established in human Jurkate6-1 cells to achieve rapid and reversible degradation of endogenous NPM1 protein.We expect that this novel functional loss assay system can reveal the most direct initial biological effects of NPM1 deletion and become a powerful tool for the future study of multifunctional protein NPM1.Methods:1.Establishment of auxin-inducible NPM1 degradation systemIn this study,we combined CRISPR/Cas9 gene editing with homologous recombination mediated gene knockin to construct an auxin induced degradation system targeting endogenous NPM1 protein in human T-cell lymphoma Jurkate6-1 cells.The homozygous knockin cell lines were screened by genomic PCR and western blot,and the degradation efficiency and reversible recovery expression of NPM1 were evaluated by western blot and fluorescence microscopy,and an ideal cell line was finally obtained as the cell model in this study.2.Early phenotypic analysis of cells after NPM1 degradationIn this study,the effects of rapid degradation of NPM1 on cell proliferation and cell cycle were firstly observed.Secondly,the cell morphology and subcellular structure were observed in detail,and the expression level and intracellular localization of NPM1 interacting protein were also detected.3.Proteomic analysisMass spectrometer-quantitative proteomics was used to search for differentially expressed proteins after rapid degradation of NPM1.Combined with the early phenotypic changes observed,the functional enrichment analysis of the differential proteins was conducted to preliminarily predict the pathways and key factors involved in NPM1.Results:1.The Jurkat E6-1 cell line targeting the degradation of endogenous NPM1 was successfully established.2.G1-S phase arrest occurred after NPM1 degradation,and cell proliferation was inhibited.3.The proportion of heteromorphic nucleated cells increased significantly after NPM1 degradation.4.After NPM1 degradation,nucleolar protein NCL was transferred from nucleolus to nucleoplasm.5.Abnormal nucleolar morphology was observed after NPM1 degradation.6.161 proteins were found to be differentially expressed after NPM1 degradation.Conclusions:1.NPM1 plays an important role in maintaining cell cycle progression and cell proliferation.2.NPM1 plays an important role in maintaining the normal morphology of the nucleus and nucleoli.3.NPM1 deletion causes extensive intracellular proteomic changes.