八肋游仆虫中无义介导的mRNA降解途径因子的细胞共定位研究  

Co-localization Studies of Nonsense-mediated mRNA Decay Pathway Factors in Euplotes octocarinatus

作　　者：周宝春 王软林 柴宝峰 ZHOU Bao-Chun;WANG Ruan-Lin;CHAI Bao-Feng(Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education,Institute of Biotechnology,Shanxi University,Taiyuan 030006,China)

机构地区：[1]化学生物学与分子工程教育部重点实验室,山西大学生物技术研究所,太原030006

出　　处：《中国生物化学与分子生物学报》2020年第6期646-655,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.31772450,81700182)资助。

摘　　要：无义介导的mRNA降解途径(nonsense-mediated mRNA decay, NMD)是一种mRNA质量监控机制,识别和降解含有提前终止密码子(premature termination codons, PTCs)的异常转录本,以保障基因的准确表达。到目前为止,有报道的从高等哺乳动物到果蝇、线虫、酵母和原生动物中,均有NMD调控途径,但其机制模型存在一定的差异。由于原生动物在生物的起源与进化上的特殊地位,以及所含的调控因子相对简单,在各种分子机制的研究领域成为热点材料。本文以八肋游仆虫(Euplotes octocarinatus)作为研究对象,从八肋游仆虫大核基因组数据库中,经过同源序列比对,分析鉴定到参与NMD途径的相关因子,包括无义mRNA识别因子poly(A)结合蛋白(poly(A) binding protein, PABP);启动NMD途径的核心因子上游移码蛋白1(up-frameshift 1, UPF1)和上游移码蛋白2(up-frameshift 2, UPF2);外显子连接复合体(exon junction complex, EJC)组分因子MAGO(Mago nashi)、Y14(Tsunagi或RMB8)、eIF4AIII(eukaryotic initiation factor 4A3)和UAP56(U2AF56 associated protein 56);降解无义mRNA的相关因子外切体(exosome)、脱帽酶(decapping mRNA 2, DCP2)、外切酶(5′-3′exoribonuclease 1, XRN1)和去腺苷酸化酶(PGK promoter directed over production, POP2)。其中,后3种蛋白质是mRNA加工小体(processing body, P-Body)的组分。通过荧光共定位分析,分别依次证实UPF1与UPF2之间、EJC组分因子之间和P-body组分因子之间的相互作用关系。随后,通过UPF2分别与MAGO和Y14的荧光共定位结果,推测八肋游仆虫依赖于EJC的NMD途径模型。通过UPF1分别与DCP2和外切体(exosome)的荧光共定位结果,推测了八肋游仆虫无义mRNA的两种降解方式:一种是无义mRNA被介导到P-body中分别被DCP2和POP2脱去5′端帽和3′端Poly(A)尾,随后在XRN1的作用下,沿着5′→3′的方向降解;另一种是在胞质中,无义mRNA直接通过招募POP2去腺苷酸化,随后又在招募来的外切体作用下,沿�Nonsense-mediated mRNA decay(NMD)is one of mRNA quality control systems that identifies and decays aberrant transcripts containing premature termination codons(PTCs)and ensures accurate expression of genes.NMD pathway exists in higher mammals,D.melanogaster,C.elegans,S.cerevisiae,and protozoan,however,the detailed mechanisms are different in these organisms.Because of protozoa’s special position in evolution and its relatively simple regulatory factors,it has become a focus of research.Herein we analyzed the NMD pathway factors in Euplotes octocarinatus.By homologous sequence alignment from the Euplotes octocarinatus genome database,we obtained the protein factors involved in the NMD pathway,such as poly(A)binding protein(PABP),as a factor for distinguishing nonsense mRNA;NMD triggering protein factors Up-frameshift 1(UPF1)and Up-frameshift 2(UPF2);component protein factors of exon-exon junction complex(EJC)-MAGO(Mago nashi),Y14(Tsunagi or RMB8),eIF4AIII(eukaryotic initiation factor 4A3),and UAP56(U2AF56 Associated Protein 56);protein factors involved in degradation of nonsense mRNA—exosome and processing body(P-body),protein factors DCP2(decapping mRNA 2),XRN1(5'-3'exoribonuclease 1)and POP2(PGK promoter directed over production).By using the fluorescence co-localization approach,we confirmed the interaction between UPF1 and UPF2,among protein factors of EJC components,and among protein factors of P-body components,respectively.Then,the EJC-dependent NMD model was confirmed by fluorescence co-localization of UPF2 with MAGO and Y14,respectively.According to the fluorescence co-localization results of UPF1 with DCP2 and exosomes,we inferred the degradation of nonsense mRNA via two modes:one was that nonsense mRNA was mediated into P-body,and then the 5'-Cap and 3'-poly(A)tail was removed by DCP2 and POP2,respectively.The other was that marked nonsense mRNA directly recruited POP2 and deadenylation occured in the cytoplasm.After that,the nonsense mRNA was degraded by exosome in 3'→5'direction.Our study thus u

关 键 词：八肋游仆虫 无义介导的mRNA降解途径 外切体 加工小体 外显子连接复合体 

分 类 号：Q786[生物学—分子生物学]