CREBBP-m^6A报告系统的建立和验证

The Construction and Verification of CREBBP-m^6A Report System

N^6-甲基腺嘌呤(N6-methyladenosine,m^6A)是真核生物信使RNA(Messenger RNA,mRNA)上最丰富的一种化学修饰,受到甲基转移酶和去甲基化酶的调控而呈现动态变化的状态。m^6A识别蛋白可识别并特异性结合到m^6A修饰位点,调控mRNA的剪接、降解和翻译进程,从而调节基因的表达。选择包含3个m^6A修饰位点的CREBBP基因3'UTR序列连接到红色荧光mCherry序列的下游,将得到的mCherry-CREBBP 3'UTR片段插入到pCD513B-CMV载体的EcoRⅠ和BamHⅠ酶切位点之间,得到CREBPP-m^6A报告质粒pCMV-mCherry-CREBBP,并在GC-1细胞和293T细胞中验证了CREBBP-m^6A报告质粒的功能,发现红色荧光强度和m^6A水平呈负相关。研究结果提示,可将pCMV-mCherry-CREBBP质粒转入靶细胞,通过红色荧光的强度来检测m^6A修饰的变化。

N6-methyladenosine(m^6A)is the most abundant post-transcription modificational in eukaryotic mRNAs.This dynamic RNA modification has been demonstrated to be regulated by methyltransferase complex and demethylases.M6 A could be recognized by m^6A readers which regulates cellular RNA metabolism including splicing,degradation and translation of target mRNAs.In this study,we constructed CREBBP-m^6A reporter by inserting fragment of CREBBP 3’UTR containing three m^6A motif into downstream of mCherry in pCD513 B-CMV vector,then construct the acquired mCherry-CREBBP 3’UTR fragment in to the pCD513 B-CMV vector between EcoRI and BamHI enzyme cutting cites,and validate the expression of mCherry in GC-1 and 293 Tcells.It was found red fluorescence intensity was negatively related to m^6A level.Results show that CREBBP 3’UTR could regulate target mRNA expression,and CREBBPm^6A reporter could be used to real-timely the change of m^6A modification in cells through expression of mCherry.