中文摘要：

无义突变介导的mRNA降解（nonsense-mediated mRNA decay,NMD）途径是真核生物体内一种重要的mRNA监督质控机制,它降解含有由无义突变、错误剪接、移码突变等产生的提前终止翻译密码子（premature translation termination codon,PTC）的mRNA,从而防止这种mRNA翻译产生的截短型蛋白质对机体造成的伤害.研究发现,一些含有PTC的mRNA发生了NMD途径逃逸,但具体机制仍不清楚.本研究将成视网膜细胞瘤基因1（retinoblastoma gene 1,RB1）作为NMD途径的靶基因,构建mini-RB1基因,包括外显子1-14（cDNA）、内含子14-外显子15-内含子15和外显子16-27（cDNA）的三部分序列,将其构建到真核表达载体pcDNA 3.1（-）中.根据人类基因组突变数据库选择3个突变位点W99X、G310X和R467X,构建相应无义突变体.分别将mini-RB1基因野生型和无义突变体转入HeLa细胞进行表达.用qRT-PCR检测发现,W99X无义突变体与野生型相比mRNA的水平无显著差异.为了进一步证实mini-RB1（W99X）发生了NMD逃逸,利用NMD途径抑制剂放线菌酮和转录抑制剂放线菌素D,分别处理转入野生型的mini-RB1基因及其无义突变体mini-RB1（W99X）的哺乳动物细胞,发现mini-RB1基因无义突变体的mRNA水平与野生型无明显差异,说明含有W99X无义突变的mini-RB1基因的mRNA发生了NMD逃逸.Western印迹检测mini-RB1基因的蛋白质表达发现,在无义突变位点W99X下游重新起始了蛋白质的翻译,因此,PTC下游蛋白质翻译的重新起始可能是导致无义mRNA逃逸NMD途径监控的主要原因.

英文摘要：

Nonsense-mediated mRNA decay（ NMD） acts as an important supervision and quality control mechanism regulating the elimination of mRNA containing premature translation termination codon（ PTC）. PTC is produced by nonsense,miss-splicing,frame-shift mutations. NMD pathway-mediated mRNA degradation will prevent the deleterious damage caused by improperly truncated proteins. Manyaberrant mRNAs containing PTC could escape from the NMD pathway,but the mechanism remains unclear. In this study,we constructed a mini-RB1（ retinoblastoma gene 1,RB1） fragment,which included cDNA of exon 1 - 14,DNA of intron 14-exon 15-intron 15 and cDNA of exon 16 - 27,and inserted it into mammalian expression vector pcDNA3. 1（-）. Three nonsense mutants of mini-RB1 were generated by introducing W99 X,G310X and R467 X into mini-RB1 gene,individually,based on Human Genome Mutation Database（ HGMD）. The real-time q PCR was used to analyze the mRNA level of the mini-RB1 before and after treatment with actinomycin D,an inhibitor of transcription,or cycloheximide,an inhibitor of NMD pathway. No significant difference of mRNA levels of mini-RB1（ W99X） mutants was observed with treatment of the either of the two drugs. Also,comparable mRNA levels were observed between the wild-type mini-RB1 and mini-RB1 mutants. All these facts suggest that the RB1（ W99X）might be capable of escaping from the NMD pathway. Furthermore,Western blotting indicated that the translation of mini-RB1（ W99X） was reinitiated downstream of W99 X and the resultant N-terminus truncated proteins of RB1 might lead to the escape of the nonsense mRNA from NMD. Our results highlighted the possibility that translation reinitiation of the nonsense W99 X mutation down-stream might be the major mechanism responsible for the NMD escape of RB1 mutants.