Selective translational regulation of premature termination codon containing mutant mRNAs

Abstract

Abnormal mRNAs containing premature termination codons (PTCs) are normally degraded through nonsense-mediated mRNA decay (NMD) at the posttranscriptional level, thus preventing the production of potentially harmful truncated mutant proteins. However, NMD is known to be imperfect. The mRNAs containing PTCs in the last exon (NMD-irrelevant) are not recognized by NMD, so they are not degraded. About 10%-30% of NMD-competent PTC-containing mRNAs are resistant to NMD (NMD-resistant) and exist as stably as their PTC-free counterparts. Moreover, if NMD is inhibited by endogenous or exogenous ways, PTC-containing mRNAs to be degraded are expected to be rescued from NMD (rescued PTC-containing mRNAs). These NMD-irrelevant PTC-containing mRNAs, NMD-resistant PTC-containing mRNAs and rescued PTC-containing mRNAs from NMD are the potential sources for the generation of mutant proteins. Therefore, it has been urged to clarify whether mutant proteins are generated from the PTC-containing mRNAs, but detailed molecular mechanisms have been poorly understood.

In this study, to clarify whether mutant proteins from the NMD-irrelevant PTC-containing mRNAs are produced, I identified 29 genes with frequent frameshift mutations in the last exon in colon cancers with high microsatellite instability (MSI-H) and selected three genes (TTK, TCF7L2, and MARCKS) for NMD-irrelevant PTC-containing mRNAs and protein expression analysis. The NMD-irrelevant PTC-containing mRNAs from these mutated genes were not degraded by NMD. However, only faint amounts of endogenous mutant TTK and TCF7L2 were detected, and I failed to detect endogenous mutant MARCKS. By polysome analysis, I demonstrated that NMD-irrelevant PTC-containing mRNAs are actively translated and inhibition of the proteasomal degradation facilitated the rescue of endogenous mutant TTK, TCF7L2, and MARCKS. By comparing the expression of neopeptide-containing or neopeptide-lacking truncated mutant proteins derived from genomic MARCKS constructs containing nonsense or frameshift mutations, I demonstrated that the enhanced degradation of these mutant proteins was driven by neopeptides. To demonstrate the generation of mutant proteins from NMD-resistant PTC-containing mRNAs and rescued PTC-containing mRNAs from NMD, human genomic β-globin expression constructs with nonsense mutations were used in order to avoid enhanced degradation of neopeptide-containing mutant proteins derived from frameshift mutations and I found that about 30 % of PTC-containing mRNAs are resistant to NMD in steady-state and these NMD-resistant PTC-containing mRNAs showed similar stability with their PTC-free counterparts. I demonstrated that the NMD-resistant PTC-containing mRNAs were translationally repressed and therefore, only trace amount of detected mutant proteins was generated from the pioneer round of translation step. Moreover, I found that PTC-containing mRNAs were significantly rescued when NMD was inhibited by down-regulating hUPF1, a key NMD factor and a large amount of mutant proteins from the rescued PTC-containing mRNAs was generated from the bulky translation step. I further demonstrated that down-regulation of UPF1 plays key roles to relieve the translational repression of PTC-containing mRNAs.

In conclusion, I found 1) NMD-irrelevant mutant mRNAs containing PTC in the last exon are not degraded by NMD in cells and translated efficiently. 2) Truncated proteins containing neopeptides are rarely detected because of extensively degradation by the ubiquitin？proteasome system which is caused by neopeptides. 3) About 30% of PTC-containing mRNAs from NMD-competent β-globin expression constructs are resistant to NMD and exist as stably as their PTC free counterparts. 4) NMD-resistant PTC-containing mRNAs are translationally repressed and trace amount of mutant proteins from them is mainly generated from the pioneer round of translation step. 5) Mutant proteins from the rescued PTC-containing mRNAs from NMD by UPF1 down-regulation are mostly generated from bulky translation. 6) UPF1 plays key roles in the selective translational regulation of PTC-containing mRNAs depending on NMD status. These findings suggest that PTC-containing mRNAs are the pool of the sources for the mutant protein generation and are subject to differential translational and posttranslational regulations depending on NMD status.