Identification of mutated genes containing premature termination codon and their translational suppression after nonsense-mediated mRNA decay block

Abstract

Frameshift mutations at coding mononucleotide repeats (cMNR) are frequent in cancers of high-microsatellite instability (MSI-H). Frameshift mutations in cMNR result in the formation of a premature termination codon (PTC) in the transcribed mRNA, and these abnormal mRNAs are generally degraded by nonsense mediated mRNA decay (NMD). In this study, 12 novel genes were identified that are frequently mutated at their cMNR by blocking NMD in two MSI-H cancer cell lines. After blocking NMD, differentially-expressed genes were screened using DNA microarrays, and then database analysis was used to select 28 candidate genes containing cMNR with more than 9 nucleotide repeats. Among them, mutations at cMNR of 15 genes have not been previously reported in cancers. Mutations at cMNR of each of the 15 genes in 10 MSI-H cell lines and 21 MSI-H cancers were analyzed, and frequent mutations of 12 genes in MSI-H cell lines and cancers were found, but not in microsatellite stable (MSS) cancers. In addition, these mutated genes are degraded by NMD and protein expressions are down-regulated in MSI-H cancers. Although NMD is an efficient mechanism that down-regulate the PTC-containing mRNA at post-transcription level, there are several stressors to inhibit NMD in vivo. Thus, down-regulated transcripts by NMD can be recovered and might produce truncated proteins. To clarify whether truncated proteins are produced or not, β-globin modified vector system was generated which has been used as a conventional model construct to study NMD mechanism. When NMD was blocked using siRNA of hUPF1, three different drugs, and hypoxic condition, mutant proteins were rarely detected, although PTC-containing transcripts were sufficiently recovered. To verify this discrepancy, several possibilities including translation repression, rapid degradation, and insufficiency of recovered transcript, were checked

by using polysome analysis, proteasomal inhibition by MG132, and quantitation of their mRNA expressions, respectively. These experiments indicated that the translations are repressed from PTC-containing transcripts which are recovered by NMD inhibition. In addition, eIF4A3 was involved in this translation repression of PTC-containing mRNAs. All of these results demonstrated that PTC-containing mRNA derived from frameshift mutation is inhibited in the generation of mutant protein production via NMD. Although mutant mRNA expression is recovered by NMD inhibition through cellular stressors, translation repression is developed, and therefore harmful cellular changes by truncated proteins are prevented.