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Selective translational repression of truncated proteins from frameshift mutation-derived mRNAs in tumors.

ABSTRACT: Frameshift and nonsense mutations are common in tumors with microsatellite instability, and mRNAs from these mutated genes have premature termination codons (PTCs). Abnormal mRNAs containing PTCs are normally degraded by the nonsense-mediated mRNA decay (NMD) system. However, PTCs located within 50-55 nucleotides of the last exon-exon junction are not recognized by NMD (NMD-irrelevant), and some PTC-containing mRNAs can escape from the NMD system (NMD-escape). We investigated protein expression from NMD-irrelevant and NMD-escape PTC-containing mRNAs by Western blotting and transfection assays. We demonstrated that transfection of NMD-irrelevant PTC-containing genomic DNA of MARCKS generates truncated protein. In contrast, NMD-escape PTC-containing versions of hMSH3 and TGFBR2 generate normal levels of mRNA, but do not generate detectable levels of protein. Transfection of NMD-escape mutant TGFBR2 genomic DNA failed to generate expression of truncated proteins, whereas transfection of wild-type TGFBR2 genomic DNA or mutant PTC-containing TGFBR2 cDNA generated expression of wild-type protein and truncated protein, respectively. Our findings suggest a novel mechanism of gene expression regulation for PTC-containing mRNAs in which the deleterious transcripts are regulated either by NMD or translational repression.

SUBMITTER: You KT

PROVIDER: S-EPMC1854916 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundTDP-43 proteinopathy is a pathological hallmark of many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). So far, there is no therapy available for these neurodegenerative diseases. In addition, the impact of TDP-43 proteinopathy on neuronal translational profile also remains unknown.MethodsBiochemical, immunohistology and assay-based studies were done with cell cultures and transgenic mice models. We also used Ribotag with microarray and proteomic analysis to determine the neuronal translational profile in the mice model of ALS/FTD.ResultsHere, we report that oral administration of a novel analog (IMS-088) of withaferin-A, an antagonist of nuclear factor kappa-B (NF-?B) essential modulator (NEMO), induced autophagy and reduced TDP-43 proteinopathy in the brain and spinal cord of transgenic mice expressing human TDP-43 mutants, models of ALS/FTD. Treatment with IMS-088 ameliorated cognitive impairment, reduced gliosis in the brain of ALS/FTD mouse models. With the Ribotrap method, we investigated the impact of TDP-43 proteinopathy and IMS-088 treatment on the translation profile of neurons of one-year old hTDP-43A315T mice. TDP-43 proteinopathy caused translational dysregulation of specific mRNAs including translational suppression of neurofilament mRNAs resulting in 3 to 4-fold decrease in levels type IV neurofilament proteins. Oral administration of IMS-088 rescued the translational defects associated with TDP-43 proteinopathy and restored the synthesis of neurofilament proteins, which are essential for axon integrity and synaptic function.ConclusionsOur study revealed that induction of autophagy reduces TDP-43 pathology and ameliorates the translational defect seen in mice models of ALS/FTD. Based on these results, we suggest IMS-088 and perhaps other inducers of autophagy should be considered as potential therapeutics for neurodegenerative disorders with TDP-43 proteinopathies.

Project description:Purpose: To identify genes and molecular mechanisms associated with disease progression during (7 weeks of age) and after (16 weeks) the peak of photoreceptor death in dogs affected with XLPRA2, a canine model of early-onset XLRP caused by a microdeletion in RPGR exon ORF15. Methods: Expression profiles of diseased and normal dog retinas at both ages were compared using a canine retinal custom cDNA microarray. Data normalization and filtering were performed with GeneSpring, statistical analysis with Significance Analysis of Microarrays. Real-time PCR using Taqman probes, western blot and immunohistochemistry (IHC) were applied on selected genes to confirm and expand the microarray results. Results: At 7 and 16 weeks, respectively, 56 and 18 transcripts were down-regulated in mutant retinas compared to normals. None of the transcripts was differentially expressed (DE) at both ages, suggesting the involvement of temporally distinct molecular pathways. Down-regulated genes included PAX6, CHML, and RDH11 at 7 weeks and CRX and SAG at 16 weeks, which are necessary for visual system development and function. Genes directly or indirectly active in apoptotic processes were altered at 7 weeks (CAMK2G, NTRK2, PRKCB, RALA, RBBP6, RNF41, SEPT5, SMYD3, SPP1, and TUBB2C) and 16 weeks (SLC25A5 and NKAP). Furthermore, DE genes at 7 weeks (ELOVL6, GLOD4, NDUFS4, and REEP1) and 16 weeks (SLC25A5 and TARS2) are related to mitochondrial functions. Real-time PCR of 11 genes confirmed the microarray results and showed differential expression for additional genes not on the array, such as GFAP, RHO, OPN1SW, CNGB3 and the mutated RPGR. Western blotting and IHC analysis also confirmed the high reliability of the presented transcriptomic data. Conclusions: A list of mutated genes in RPGRORF15 diseased retinas, which are likely candidates to further study their role in age-related photoreceptor degeneration diseases, is reported at different crucial ages. The results indicate that at 7 weeks a combination of non-classical anti- and pro-apoptotic genes appears to be involved in photoreceptor degeneration, whereas at both 7 and 16 weeks expression of mitochondria related genes indicates they may play a relevant role in the disease process.

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Selective translational repression of truncated proteins from frameshift mutation-derived mRNAs in tumors.

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