大鼠Mocs2基因跳跃型外显子可变剪接保留率的检测

Detection of Alternative Splicing Exon Inclusive Ratio of Exon 2 of Mocs2 Gene in Rat

可变剪接是真核生物基因表达调控的一种重要方式,它通过剪接位点调控ORFs(Open reading frames)区域外显子或内含子的表达,产生不同的mRNA剪接体,进一步翻译成具有相同或不同功能的蛋白质,从而对诸如发育、疾病、环境响应等生物学过程产生重要影响。已有文献报道,人类Mocs2基因exon 1a与exon 1b外显子发生互斥型可变剪接,产生的两个剪接体分别编码钼喋呤合酶的大小亚基,参与钼辅因子生物的合成过程。基于本课题组前期大鼠肺部组织RNA-seq测序的结果,发现Mocs2基因2号外显子转录后可被剪接或保留,产生两个不同的剪接体。为验证测序结果的准确性,本研究利用半定量PCR与实时荧光定量PCR两种方法检测大鼠脑、海马、肺组织中Mocs2基因2号外显子的保留率。半定量PCR方法检测大鼠肺、海马、大脑组织中Mocs2基因的保留率分别为(88.28±3.09)%、(99.44±0.24)%、(99.54±0.19)%。实时荧光定量PCR检测的保留率分别为(66.76±20.47)%、(75.60±12.44)%、(87.28±16.4)%。实验结果表明,Mocs2基因2号外显子在大鼠中存在可变剪接现象,且不同组织具有一定差异。本工作进一步验证了两种PCR方法检测前体mRNA可变剪接保留率的可行性。

Alternative splicing is an essential step to regulate gene expression in eukaryotes.It can regulate the expression of exons or introns in ORFs regions via different splicing ways,and produce a variety of mRNA isoforms.mRNAs are further translated into different protein isoforms,with the same or different functions to biological processes such as development,diseases,environmental response.It has been reported that exon 1a and 1b of Mocs2 genes are mutually exclusive and produce two splice isoforms which are translated into the small and large subunit of molybdopterin synthase,respectively.And molybdopterin synthase participates in the biosynthesis process of molybdenum cofactor.Based on the previous results of RNA-seq data from lung tissue of rats,it was found that the Mocs2 gene has two splice isoforms as exon2 can be spliced or retained in transpription level.In order to validate the alternative splicing event,the rate of exon2 inclusion was detected by semi-quantitative PCR and RT-qPCR in brain tissue,hippocampus tissue and lung tissue of rat.The percentage of exon2 inclusion was(88.28±3.09)%in lung tissue,(99.44±0.24)%in hippocampus tissue and(99.54±0.19)%in brain tissue,which was calcu lated by using semi-quantitative PCR.The percentage of exon2 inclusion was(66.76±20.47)%in lung tissue,(75.60±12.44)%in hippocampus tissue and(87.28±16.4)%in brain tissue,respectively,while the RT-qPCR method was employed to detect the events of alternative splicing.The result suggested that exon2 of Mocs2 gene had alternative splicing phenomenon in rat and the inclusion rate of exon 2 was different in various tissues.And the work also validated the feasibility of two PCR methods to detect the alternative splicing of pre-mRNA.