【作者】 刘刚；

【导师】 万由衷；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2024， 硕士

【摘要】 背景:作为基因表达的两个重要步骤,pre-mRNA剪接与mRNA核输出是紧密相连的。新生的pre-mRNA经过剪接形成成熟的mRNA后才能被转运出核,进入细胞质从而表达成能够行使生物学功能的蛋白质。mRNA核输出的平衡至关重要,过多的核输出可能会导致未加工完全的pre-mRNA泄露到细胞质中;核输出过少则会导致mRNA在细胞核内的积累,最终被降解。SF3B1是剪接体的重要组成部分,有研究表明,SF3B1的异常表达或突变会影响mRNA的核输出。当SF3B1被敲低或发生K700E突变时,mRNA的核输出被抑制,这表明SF3B1除了在剪接中发挥重要作用外,还参与mRNA的核输出。但SF3B1突变抑制mRNA核输出的机制尚未见报道。目的:探究SF3B1突变导致mRNA核输出抑制的分子机制。方法:通过RNA测序、核质分离和FISH探究SF3B1 K700E突变影响了哪些mRNA的核输出;通过免疫共沉淀探究SF3B1 K700E突变是否影响与mRNA核输出因子的结合;通过RIP、免疫共沉淀以及核质分离探究SF3B1 K700E突变影响mRNA核输出的机制;转染多种SF3B1突变表达载体,探究其他癌症相关SF3B1突变是否会造成mRNA核输出抑制及其机制;通过Western Blot验证mRNA核输出被抑制的基因的蛋白表达水平。结果:本研究通过RNA测序、qPCR以及FISH结果表明SF3B1K700E突变造成了BCHE、ENDOD1、MAT2A和DDX5的mRNA核输出抑制;免疫共沉淀证明SF3B1 K700E突变会造成与THOC5的结合减弱,并且突变不影响SF3B1与THOC5的核质分布;RIP、免疫共沉淀以及FISH等实验表明SF3B1 K700E突变可能是通过减弱SF3B1对THOC5的mRNA呈递作用导致THOC5结合到的mRNA减少,从而抑制mRNA的核输出;其他癌症相关SF3B1突变通过与K700E突变相似的机制调节mRNA核输出;mRNA核输出的抑制会导致蛋白表达水平的下降。结论:SF3B1突变通过减弱与THOC5的结合,减少mRNA的呈递,抑制mRNA的核输出,并最终导致蛋白表达水平的降低。本研究为明确SF3B1如何参与mRNA核输出提供了见解,为研究mRNA剪接与核输出的联系提供了一条新的思路。更多还原

【Abstract】 Background:As two important steps of gene expression,pre-mRNA splicing is closely related to mRNA nuclear export.After splicing to form mature mRNA,the newborn pre-mRNA can be transported out of the nucleus and into the cytoplasm,which can be expressed into proteins that can perform biological functions.The balance of mRNA nuclear export is critical.Excessive nuclear export may lead to leakage of unprocessed pre-mRNA into the cytoplasm;Too little nuclear export leads to mRNA accumulation in the nucleus,which eventually degrades.SF3B1 is an important part of the spliceosome,and studies have shown that abnormal expression or mutation of SF3B1 can affect the nuclear export of mRNA.When SF3B1 is knocked down or has a K700 E mutation,the nuclear export of mRNA is inhibited,suggesting that SF3B1 is involved in the nuclear output of mRNA in addition to its important role in splicing.However,the mechanism by which SF3B1 mutation inhibits the nuclear export of mRNA has not been reported.Objective:To explore the molecular mechanism of SF3B1 mutation leading to inhibition of mRNA nuclear export.Method:RNA sequencing,nucleoplasmic separation,and FISH were then used to investigate which mRNAs’ nuclear export were affected by the SF3B1 K700 E mutation.Immunoprecipitation was used to investigate whether the SF3B1 K700 E mutation affected the binding of mRNA nuclear export factors.To explore the mechanism behind the inhibition of mRNA nuclear export caused by the SF3B1 K700 E mutation,RIP,immunoprecipitation,and nucleoplasmic separation were performed.Multiple SF3B1 expression vectors were transfected to investigate whether other cancer-associated SF3B1 mutations would also cause mRNA nuclear export inhibition and to ascertain the underlying mechanisms.Finally,the protein expression levels of genes whose mRNA nuclear export was inhibited were verified using Western Blot.Results:In this study,the results of RNA sequencing,q PCR and FISH showed that SF3B1 K700 E mutation caused the inhibition of nuclear export of BCHE,ENDOD1,MAT2 A and DDX5 mRNA.Coimmunoprecipitation showed that SF3B1 K700 E mutation could weaken the binding of SF3B1 to THOC5,and the mutation did not affect the nuclear and cytoplasmic distribution of SF3B1 and THOC5.RIP,co-immunoprecipitation and FISH experiments showed that SF3B1 K700 E mutation may reduce the mRNA binding of THOC5 by weakening the mRNA presentation of SF3B1 to THOC5,thereby inhibiting the nuclear export of mRNA.Other cancer-associated SF3B1 mutations regulate mRNA nuclear export similarly to the K700 E mutation.Inhibition of mRNA nuclear export results in a decrease in protein expression level.Conclusion:SF3B1 mutations reduce mRNA presentation,inhibit mRNA nuclear export,and ultimately lead to reduced protein expression levels by weakening binding to THOC5.This study provides insights into how SF3B1 participates in mRNA nuclear export and provides a new way to study the relationship between mRNA splicing and nuclear export.更多还原