Translational Control And Readthrough Of Tumor Suppress Gene RB1 And MEN1 With Nonsense Mutation

The nonsense mutation of genes resulted in aberrant mRNAs containing premature termination code in cells. And these PTC-mRNAs were recognized and degraded by nonsense-mediated mRNA decay pathway during translation to protect the cells from the possible accumulation of aberrant mRNA and potentially toxic truncate proteins. NMD is an important post-transcriptional mRNA surveillance, and because of the recognition and degradation of aberrant mRNA dependent on the pioneer round of translation, NMD was a type of translation control. Many studies revealed that cause of some tumors or cancers were the low content of tumor suppressor proteins in cells resulted from nonsense mutation. The study on the mechanism of translation control on tumor suppressor genes with nonsense mutation will make for elucidating pathogenesis of many cancers and tumors.Retinoblastoma is one of most common intraocular malignant tumors in infants. In 1971, Kundson et al. identified the virulence RBI gene. This tumor is caused by deficiency or inactivation of both the allele located in the 13th chromosome (13q14). All of these showed that this gene could inhibit tumorigenesis. So, the RBI gene was firstly defined as suppressor gene. According to the data of RJBGMdb (http://rbl-lsdb.d-lohmann.de/), nonsense mutation accounted for 42.4% of the retinoblastoma cases caused by mutation of RB1 gene.The first part of this study focused on the translational regulatory mechanism of RBI gene with nonsense mutation. We constructed a mini-RB1 gene, 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 pCMV-MH. Three nonsense mutants of mini-RB1 were generated by introducing W99X, G310X and R467X into mini-RBI gene, individually, based on Human Genome Mutation Database (HGMD). Then, using the Real-Time qPCR we analyzed the RBI mRNA level of HeLa cells transfected with the mini-RB1 wild-type or 3 mutants, and no significance could be found. Furthermore, to confirm whether these mutants escaped from NMD, the mRNA levels of the mini-RBI from HeLa cells were analyzed before and after treatment with actinomycin D, an inhibitor of transcription, or Cycloheximide, an inhibitor of NMD pathway by using the Real-Time qPCR. No significant difference of mRNA levels of 3 mini-RBI mutants was observed with treatment of the either of the two drugs. All these facts suggested that the mutants of RBI gene (W99X and G310X) might be capable of escaping from the NMD pathway. Western blot experiments indicated that the translation of mini-RB 1 (W99X and G310X) was re-initiated downstream of W99X and G310X, and the resultant N-terminus truncated proteins of RBI might lead from the escape of the nonsense mRNA from NMD. Consequently, our results highlighted the possibility that translational re-initiation down-stream of the nonsense W99X or G310X mutation might be the major mechanism responsible for the NMD escape of RBI mutants.The therapy investigation of genetic diseases, e.g. Cystic fibrosis, hemophilia, et. al and cancers caused from nonsense mutation focus on the readthrough drugs, for example aminoglycosides, and small molecules PTC 124, and clinical drugs Amlexanox, and so on. After treatment with these drugs, some full-length protein could be detected and clinical symptoms could be relieved in some degree. However, the amount of readthrough resultant of full-length protein was limited from low levels of substrate mRNA degraded by NMD.In the second part of this study, we focused on the cooperation between NMD inhibition and readthrough of nonsense codon to produce more full-length menin protein from MEN1 tumor suppress gene with nonsense mutation, which was confirmed to be subjected to NMD pathway previously, and the mechanism of the cooperation was further investigated. Firstly, we transfected the wild-type or mutants M1 (Q1 41X), M2 (Y312X), M3(Y312X) and M4 (Y417X)) of mini-MEN1 gene into HeLa cells, and then, the cells were treated by using two type of readthrough drugs, PTC 124 (0,2,10,20μM) and Amlexanox (0,5,10,100μM) to obtain full-length menin proteins. The results indicated that the optimum concentration of readthrough drugs of PTC 124 and Amlexanox were 20μM and 1000μM, respectively, for 4 mutants. The methods RNAi (pSIR-UPF1) and microRNA (miR125a) were used to inhibit the NMD pathway, which were confirmed to work on NMD pathway previously. We treated the transfected cells with combination of NMD inhibitor and readthrough drugs and analyzed by Western blot. The results indicated that expression of full-length menin proteins increased by 0-10 folds compared with contrasts. To investigate the mechanism of cooperation, the substrate levels of target mRNA in treated cells were analyzed by using qRT-PCR. The results displayed that both inhibitors could significantly increase the substrate mRNA, meanwhile, PTC 124 could decrease the level of target mRNA and Amlexanox increased the levels of ones. In general, the combinant treatment could increase the levels of full-length menin protein in cells, where NMD inhibition keep more PTC-mRNA in cells to provid more substrate for readthrough drugs. The results of this study will provid important data for research and considerable therapy of the diseases caused by gene mutation.