Nonsense-mediated mRNA Decay System Research Articles

Targeting nonsense-mediated mRNA decay in colorectal cancers with microsatellite instability

Nonsense-mediated mRNA decay (NMD) is responsible for the degradation of mRNAs with a premature termination codon (PTC). The role of this system in cancer is still quite poorly understood. In the present study, we evaluated the functional consequences of NMD activity in a subgroup of colorectal cancers (CRC) characterized by high levels of mRNAs with a PTC due to widespread instability in microsatellite sequences (MSI). In comparison to microsatellite stable (MSS) CRC, MSI CRC expressed increased levels of two critical activators of the NMD system, UPF1/2 and SMG1/6/7. Suppression of NMD activity led to the re-expression of dozens of PTC mRNAs. Amongst these, several encoded mutant proteins with putative deleterious activity against MSI tumorigenesis (e.g., HSP110DE9 chaperone mutant). Inhibition of NMD in vivo using amlexanox reduced MSI tumor growth, but not that of MSS tumors. These results suggest that inhibition of the oncogenic activity of NMD may be an effective strategy for the personalized treatment of MSI CRC.

Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation

We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis.

Abstract 2416: Protein expression analysis of premature termination codon containing mutant proteins in colon cancers with high microsatellite instability

Abstract Frameshift mutations at coding mononucleotide repeats (cMNR) are common in the tumors with high microsatellite instability (MSI-H). These mutations invariably generate mRNAs containing abnormal coding sequences and premature termination codons (PTC). Normally, these mRNAs containing PTC are degraded by nonsense mediated mRNA decay (NMD) system. However, mRNAs containing PTC located in the last exon are not subject to be degraded by NMD system, and thus expected to generate truncated mutant proteins. We identified 29 novel mutations at coding region mononucleotide repeats (cMNR) genes by a PCR based method and sequencing. The frequency was varying (10% to 90%) in the 10 MSI-H colon cancer cell lines. To confirm the mRNA level, we analyzed the mutant mRNAs from identified genes in colon cancer cell lines and found that the mutant mRNAs were not degraded in cells. For mutant protein detection, we adopted both immunobloting and a target mass method to detect endogenous mutant proteins in cells. By immunobloting analysis, we found rare level of endogenous mutant proteins in cells. Using a target mass method, however, we could detect potential mutant protein candidates and confirmed one of the mutant candidates (VPS37B mutant proteins) is stably expressed in cells by in vitro overexpression experiments. When mutated, VPS37B mutant proteins came to acquire very short neopeptides (only two novel amino acids) which mainly disturb the stable expression of mutant proteins and this might explain the intact expression of VPS37B mutant proteins. Taken together, our findings suggest that frameshift mutations at cMNR of genes usually lead to the rare expression of the mutant proteins in tumor cells but, some mutant proteins with short neopeptides could be stably detected by a highly sensitive target mass method. Citation Format: SeongJu Yoon, Won Kyu Kim, Jeon Han Park, Hoguen Kim. Protein expression analysis of premature termination codon containing mutant proteins in colon cancers with high microsatellite instability. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2416. doi:10.1158/1538-7445.AM2014-2416

Nonsense-mediated mRNA decay was demonstrated in two hypofibrinogenemias caused by heterozygous nonsense mutations of FGG, Shizuoka III and Kanazawa II

We report two novel hypofibrinogenemias, Shizuoka III and Kanazawa II, which are caused by heterozygous mutations in FGG. Shizuoka III showed c.147delT and 147_149insACA in FGG exon 3 and a subsequent frameshift mutation, resulting in mature protein γ23X (native protein: γ49X), and Kanazawa II showed c.1205G>A in FGG exon 9, resulting in γ376X (native protein: γ402X). To determine whether the truncated γ-chains, γ23X and γ376X, were synthesized and participated in the assembly of fibrinogen, mutant-type cDNA vectors were transfected into Chinese hamster ovary (CHO) cells. Significant levels of mutant fibrinogen were not detected by ELISA in the culture media and cell lysates. Immunoblot analysis of cell lysates revealed that the mutant γ-chain of γ376X was observed but intact fibrinogen was not. On the other hand, mutant γ-chain was not observed in γ23X-expressing cells. To demonstrate the involvement of the mechanisms of nonsense-mediated mRNA decay (NMD), we cloned wild- and mutant-type mini-genes containing γ23 or γ376 codon and transfected these into CHO cell lines in the absence or presence of cycloheximide as an NMD inhibitor. mRNA levels were determined using real-time quantitative RT-PCR in CHO cells. In the absence of cycloheximide, levels of mRNAs transcribed from the mutant gene were lower than from the wild-type gene whereas, in the presence of cycloheximide, levels of mRNAs transcribed from the mutant gene increased dose-dependently. Finally, these results demonstrated that mRNAs containing γ23X or γ376X are degraded by the NMD system and translation of the truncated γ-chain polypeptide decrease in patients' hepatocytes, resulting in hypofibrinogenemias.

Truncated serine/arginine-rich splicing factor 3 accelerates cell growth through up-regulating c-Jun expression

Serine/arginine-rich splicing factor 3 (SRSF3), a member of the SRSF family, plays a wide-ranging role in gene expression. The human SRSF3 gene generates a major mRNA isoform encoding a functional, full-length protein and a PTC-containing isoform (SRSF3-PTC). The latter is expected to be degraded through the nonsense-mediated mRNA decay system. However, it was reported that SRSF3-PTC mRNA was produced under stressful conditions and translated into a truncated SRSF3 protein (SRSF3-TR). To disclose unknown functions of SRSF3-TR, we established Flp-In-293 cells stably expressing SRSF3-TR. The SRSF3-TR-expressing cells increased mRNA and protein levels of positive regulators for G1 to S phase transition (cyclin D1, cyclin D3, CDC25A, and E2F1) and accelerated their growth. c-Jun is required for progression through the G1 phase, the mechanism by which involves transcriptional control of the cyclin D1 gene. We also found that the JUN promoter activity was significantly increased in the Flp-In-293 cells stably expressing SRSF3-TR, compared with mock-transfected control cells. The SRSF3-TR-expressing cells increased c-Jun and Sp-1 levels, which are important for the positive autoregulation and basal transcription of JUN, respectively. Our results suggest that stress-inducible SRSF3-TR may participate in the acceleration of cell growth through facilitating c-Jun-mediated G1 progression under stressful conditions.

JAK2 exon 14 Skipping in Patients with Primary Myelofibrosis (PMF),

Abstract 3844In recent years the diagnosis and treatment of patients with myeloproliferative disorders has focused on the JAK2 gene. Recently, an alternative splicing of JAK2 transcript lacking the entire exon 14 (88bp), has been described (Ma et al. (2010) PLoS ONE, 5, e12165). This mRNA has a stop codon in exon 15 and is translated into a truncated protein that has a deletion within the JH2 pseudokinase domain and complete deletion of the kinase domain (JH1). The alternative transcript was detected in plasma of patients with myeloproliferative neoplasms (MPNs) (58% of patients positive for the V617F mutation and 33% of negative) in proportions ranging from 2% to 26% compared to the normal transcript, but never in healthy subjects. However, quantification of the transcript was performed by quantitative fragment length analysis, which is not the most appropriate method for such determination especially if, as in this case, the PCR is not limited to the exponential phase (Cirigliano, V. et al. (2001) Mol. Hum. Reprod., 7, 1001–1006).We investigate the mRNA levels of JAK2 and its splicing variant lacking exon 14, in granulocytes isolated from peripheral blood of 9 healthy individuals and 41 patients diagnosed with primary myelofibrosis (19 negative and 22 positive for the JAK2 V617F mutation). To overcome the limitations of quantitative fragment length analysis, the levels of the splice variant were assessed by qPCR, using a boundary-spanning primer designed on the junction between exons 13 and 15 (Vandenbroucke II, et al. (2001) Nucleic Acids Res., 29:E68). The JAK2 V617F allele burden was measured as previously described (Lippert, E. et al. (2006) Blood, 108, 1865–1867).Low levels of the alternative-spliced product was found in all RNA samples from patients and from all healthy individuals, in proportions ranging from 0.3 % to 3.5% compared to the normal transcript. The wild-type patients (95% C.I.: 0.3–0.8%) did not differ from controls (95% C.I.: 0.4–0.7%) while, in patients with the JAK2 V617F mutation, the levels correlates with allele burden (R^2= 0.513; p= 0.0002) (Figure 1). [Display omitted] To explain this observation we performed a bioinformatic analysis of the exon 14 sequence, using the software Human Splice Finder 2.4.1 (Desmet, F.O. et al. (2009) Nucleic Acids Res37 (e67) and ESE finder 3.0 (Cartegni, L. et al. (2003) Nucleic Acids Res31 (13)). The G/T transversion causing the aminoacidic substitution V617F also breaks an high-score, putative, Exon Splicing Enhancer (ESE) sequence that potentially binds the splicing regulator protein SRP55 (TGTGTC; positions 70 to 75 of exon 14). The higher level of the splice variant found in patients with the mutation could be due to an alteration of the binding of molecular factors that determine the inclusion of exon 14 in mature transcripts. The low levels of alternative transcripts observed, could be due to the presence of a stop codon in exon 15 as a result of the frameshift caused by the exclusion of exon 14. We know that a stop codon at more than 55 bases upstream of the last exon junction (in this case between exons 24 and 25) determines the activation of the Nonsense Mediated mRNA Decay (NMD) system and the consequent destruction of the messenger. In order to knowhow a small percentage of mRNA, subjected to degradation by NMD system, can produce the predicted 70 KDa protein that has been detected after immunoprecipitation and western blot analysis using JAK2 N-terminal antibodies, we are characterizing the 3' end of the alternative transcript.We show that the somatic mutation in exon 14 of JAK2 gene, in addition to the effects caused by the V617F amino acid change, has a moderate influence on splicing, increasing the levels of a splicing variant that is produced in small amounts also in healthy subjects. Our study demonstrates the efficacy of qPCR technique and the predictive power of bioinformatics tools used here. Further studies are needed to understand how this isoform is processed at the post-transcriptional level and whether there are significant effects on clinical phenotype. Disclosures:Barosi:Novartis: Membership on an entity's Board of Directors or advisory committees.

Incomplete nonsense-mediated mRNA decay in Giardia lamblia

Messenger RNAs containing premature translation stop codons are degraded by a nonsense-mediated mRNA decay (NMD) system. The NMD pathway is present in yeast, plants and mammals and is thought to protect cells from production of nonfunctional proteins by rapidly degrading mutant mRNAs. There is little understanding of the biology of the origins of eukaryotes, particularly of the NMD pathway. Searches using the BLAST program revealed that the protozoan Giardia lamblia has only some of the components of the NMD pathway. We developed a luciferase reporter system with a nonsense mutation to monitor NMD in Giardia. The nonsense mutation triggered a decrease in luciferase mRNA levels and stability, suggesting that the NMD phenomenon could be present in Giardia. We also found a significant reduction of the mRNA levels of another system containing Giardia its own cyst wall protein 3 gene with a nonsense mutation. However, the reduction levels observed in these two systems are lower than that in late-branching eukaryotes, suggesting that the NMD system in Giardia may be less functional. Interestingly, the effect of G418 in promoting read-through of the nonsense mutation and inhibiting NMD in Giardia is similar to that in late-branching eukaryotes. We also characterised the giardial homologue of a conserved NMD factor, UPF1. Immunofluorescence assays revealed that giardial UPF1, like yeast UPF1, is expressed in the cytoplasm, but not in the nucleus. In addition, overexpression of UPF1 resulted in a reduction of the levels of nonsense-containing transcripts and enhanced translation termination at a nonsense codon. These results suggest that Giardia may have an incomplete NMD pathway and giardial UPF1 may be functionally conserved, involved in NMD and in preventing nonsense suppression.

Context Analysis of Termination Codons in mRNA that are Recognized by Plant NMD

The nonsense-mediated mRNA decay (NMD) system is an RNA surveillance system that degrades mRNAs possessing premature translation termination codons (PTCs). Although NMD factors are well conserved in eukaryotes, it is speculated that the contexts of those termination codons that are subject to NMD are different depending on the organism. Context analysis of termination codons that are recognized by the plant NMD system would clarify NMD target mRNAs in plants, and contribute to our understanding of its biological relevance in plants. In the present study we analyzed the positions of termination codons that were recognized as PTCs using an Agrobacterium transient expression assay, i.e. the accumulation of a series of plant mRNAs with nonsense mutations in different contexts was tested in plants. The results indicated that termination codons that are located distant from the mRNA 3' termini or >50 nucleotides upstream of the 3'-most exon-exon junction are recognized as substrates for NMD.

Selective Translational Repression of Truncated Proteins from Frameshift Mutation-Derived mRNAs in Tumors

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.

Both introns and long 3′-UTRs operate as cis-acting elements to trigger nonsense-mediated decay in plants

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control mechanism that identifies and eliminates aberrant mRNAs containing a premature termination codon (PTC). Although, key trans-acting NMD factors, UPF1, UPF2 and UPF3 are conserved in yeast and mammals, the cis-acting NMD elements are different. In yeast, short specific sequences or long 3′-untranslated regions (3′-UTRs) render an mRNA subject to NMD, while in mammals' 3′-UTR located introns trigger NMD. Plants also possess an NMD system, although little is known about how it functions. We have elaborated an agroinfiltration-based transient NMD assay system and defined the cis-acting elements that mediate plant NMD. We show that unusually long 3′-UTRs or the presence of introns in the 3′-UTR can subject mRNAs to NMD. These data suggest that both long 3′-UTR-based and intron-based PTC definition operated in the common ancestors of extant eukaryotes (stem eukaryotes) and support the theory that intron-based NMD facilitated the spreading of introns in stem eukaryotes. We have also identified plant UPF1 and showed that tethering of UPF1 to either the 5′- or 3′-UTR of an mRNA results in reduced transcript accumulation. Thus, plant UPF1 might bind to mRNA in a late, irreversible phase of NMD.

UPF3 suppresses aberrant spliced mRNA in Arabidopsis

It has been reported that eukaryotic organisms have a nonsense-mediated mRNA decay (NMD) system to exclude aberrant mRNAs that produce truncated proteins. NMD is an RNA surveillance pathway that degrades mRNAs possessing premature translation termination codons (PTCs), thus avoiding production of possibly toxic truncated proteins. Three interacting proteins, UPF1, UPF2 and UPF3, are required for NMD in mammals and yeasts, and their amino acid sequences are well conserved among most eukaryotes, including plants. In this study, 'The Arabidopsis Information Resource' database was searched for mRNAs with premature termination codons. We selected five of these mRNAs and checked for the presence of PTCs in these mRNAs when translated in vivo. As a result we identified aberrant mRNAs produced by alternative splicing for each gene. These genes produced at least one alternative splicing variant including a PTC (PTC+) and another variant without a PTC (PTC-). We analyzed their PTC+/PTC- ratios in wild-type Arabidopsis and upf3 mutant plants and showed that the PTC+/PTC- ratios were higher in atupf3 mutant plants than wild-type plants and that the atupf3 mutant was less able to degrade mRNAs with premature termination codons than wild-type plants. This indicated that the AtUPF3 gene is required by the plant NMD system to obviate aberrantly spliced mRNA.

MRNA surveillance mitigates genetic dominance in Caenorhabditis elegans.

Nonsense mutant mRNAs are unstable in all eucaryotes tested, a phenomenon termed nonsense-mediated mRNA decay (NMD) or mRNA surveillance. Functions of the seven smg genes are required for mRNA surveillance in Caenorhabditis elegans. In Smg(+) genetic backgrounds, nonsense-mutant mRNAs are unstable, while in Smg(-) backgrounds such mRNAs are stable. Previous work has demonstrated that the elevated level of nonsense-mutant mRNAs in Smg(-) animals can influence the phenotypic effects of heterozygous nonsense mutations. Certain nonsense alleles of a muscle myosin heavy chain gene are recessive in Smg(+) backgrounds but strongly dominant in Smg(-) backgrounds. Such alleles probably express disruptive myosin polypeptide fragments whose abundance is elevated in smg mutants due to elevation of mRNA levels. We report here that mutations in a variety of C. elegans genes are strongly dominant in Smg(-), but recessive or only weakly dominant in Smg(+) backgrounds. We isolated 32 dominant visible mutations in a Smg(-) genetic background and tested whether their dominance requires a functional NMD system. The dominance of 21 of these mutations is influenced by NMD. We demonstrate, furthermore, that in the case of myosin, the dominant-negative effects of nonsense alleles are likely to be due to expression of N-terminal nonsense-fragment polypeptides, not to mistranslation of the nonsense codons. mRNA surveillance, therefore, may mitigate potentially deleterious effects of many heterozygous germline and somatic nonsense or frame-shift mutations. We also provide evidence that smg-6, a gene previously identified as being required for NMD, performs essential function(s) in addition to its role in NMD.