Nonsense-mediated mRNA Decay Pathway Research Articles

Genome-wide suppression of aberrant mRNA-like noncoding RNAs by NMD in Arabidopsis

The nonsense-mediated mRNA decay (NMD) pathway is a well-known eukaryotic surveillance mechanism that eliminates aberrant mRNAs that contain a premature termination codon (PTC). The UP-Frameshift (UPF) proteins, UPF1, UPF2, and UPF3, are essential for normal NMD function. Several NMD substrates have been identified, but detailed information on NMD substrates is lacking. Here, we noticed that, in Arabidopsis, most of the mRNA-like nonprotein-coding RNAs (ncRNAs) have the features of an NMD substrate. We examined the expression profiles of 2 Arabidopsis mutants, upf1-1 and upf3-1, using a whole-genome tiling array. The results showed that expression of not only protein-coding transcripts but also many mRNA-like ncRNAs (mlncRNAs), including natural antisense transcript RNAs (nat-RNAs) transcribed from the opposite strands of the coding strands, were up-regulated in both mutants. The percentage of the up-regulated mlncRNAs to all expressed mlncRNAs was much higher than that of the up-regulated protein-coding transcripts to all expressed protein- coding transcripts. This finding demonstrates that one of the most important roles of NMD is the genome-wide suppression of the aberrant mlncRNAs including nat-RNAs.

The mammalian nonsense-mediated mRNA decay pathway: To decay or not to decay! Which players make the decision?

Nonsense-mediated mRNA decay (NMD) degrades mRNAs carrying premature translation termination codons (PTCs). Although the core process and several NMD effectors are conserved among species, the involvement of a splicing-dependent signal seems to be specific for mammalian PTC definition. Still, recent data shed new light on physical parameters and mechanistic pathways involved in NMD. Here, we examine these findings, updating the roles for potential NMD players, such as the exon junction complex and the cytoplasmic poly(A)-binding protein 1 – the former acting as enhancer rather than an essential factor and the latter functioning as NMD repressor.

VHL Frameshift Mutation as Target of Nonsense‐Mediated mRNA Decay in Drosophila melanogaster and Human HEK293 Cell Line

There are many well-studied examples of human phenotypes resulting from nonsense or frameshift mutations that are modulated by Nonsense-Mediated mRNA Decay (NMD), a process that typically degrades transcripts containing premature termination codons (PTCs) in order to prevent translation of unnecessary or aberrant transcripts. Different types of germline mutations in the VHL gene cause the von Hippel-Lindau disease, a dominantly inherited familial cancer syndrome with a marked phenotypic variability and age-dependent penetrance. By generating the Drosophila UAS:Upf1D45B line we showed the possible involvement of NMD mechanism in the modulation of the c.172delG frameshift mutation located in the exon 1 of Vhl gene. Further, by Quantitative Real-time PCR (QPCR) we demonstrated that the corresponding c.163delG human mutation is targeted by NMD in human HEK 293 cells. The UAS:Upf1D45B line represents a useful system to identify novel substrates of NMD pathway in Drosophila melanogaster. Finally, we suggest the possible role of NMD on the regulation of VHL mutations.

SMD and NMD are competitive pathways that contribute to myogenesis: effects on PAX3 and myogenin mRNAs

UPF1 functions in both Staufen 1 (STAU1)-mediated mRNA decay (SMD) and nonsense-mediated mRNA decay (NMD), which we show here are competitive pathways. STAU1- and UPF2-binding sites within UPF1 overlap so that STAU1 and UPF2 binding to UPF1 appear to be mutually exclusive. Furthermore, down-regulating the cellular abundance of STAU1, which inhibits SMD, increases the efficiency of NMD, whereas down-regulating the cellular abundance of UPF2, which inhibits NMD, increases the efficiency of SMD. Competition under physiological conditions is exemplified during the differentiation of C2C12 myoblasts to myotubes: The efficiency of SMD increases and the efficiency of NMD decreases, consistent with our finding that more STAU1 but less UPF2 bind UPF1 in myotubes compared with myoblasts. Moreover, an increase in the cellular level of UPF3X during myogenesis results in an increase in the efficiency of an alternative NMD pathway that, unlike classical NMD, is largely insensitive to UPF2 down-regulation. We discuss the remarkable balance between SMD and the two types of NMD in view of data indicating that PAX3 mRNA is an SMD target whose decay promotes myogenesis whereas myogenin mRNA is a classical NMD target encoding a protein required for myogenesis.

Structural Characterization of the Rous Sarcoma Virus RNA Stability Element

In eukaryotic cells, an mRNA bearing a premature termination codon (PTC) or an abnormally long 3' untranslated region (UTR) is often degraded by the nonsense-mediated mRNA decay (NMD) pathway. Despite the presence of a 5- to 7-kb 3' UTR, unspliced retroviral RNA escapes this degradation. We previously identified the Rous sarcoma virus (RSV) stability element (RSE), an RNA element downstream of the gag natural translation termination codon that prevents degradation of the unspliced viral RNA. Insertion of this element downstream of a PTC in the RSV gag gene also inhibits NMD. Using partial RNase digestion and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry, we determined the secondary structure of this element. Incorporating RNase and SHAPE data into structural prediction programs definitively shows that the RSE contains an AU-rich stretch of about 30 single-stranded nucleotides near the 5' end and two substantial stem-loop structures. The overall secondary structure of the RSE appears to be conserved among 20 different avian retroviruses. The structural aspects of this element will serve as a tool in the future design of cis mutants in addressing the mechanism of stabilization.

Macronuclear genome structure of the ciliate Nyctotherus ovalis: Single-gene chromosomes and tiny introns

BackgroundNyctotherus ovalis is a single-celled eukaryote that has hydrogen-producing mitochondria and lives in the hindgut of cockroaches. Like all members of the ciliate taxon, it has two types of nuclei, a micronucleus and a macronucleus. N. ovalis generates its macronuclear chromosomes by forming polytene chromosomes that subsequently develop into macronuclear chromosomes by DNA elimination and rearrangement.ResultsWe examined the structure of these gene-sized macronuclear chromosomes in N. ovalis. We determined the telomeres, subtelomeric regions, UTRs, coding regions and introns by sequencing a large set of macronuclear DNA sequences (4,242) and cDNAs (5,484) and comparing them with each other. The telomeres consist of repeats CCC(AAAACCCC)n, similar to those in spirotrichous ciliates such as Euplotes, Sterkiella (Oxytricha) and Stylonychia. Per sequenced chromosome we found evidence for either a single protein-coding gene, a single tRNA, or the complete ribosomal RNAs cluster. Hence the chromosomes appear to encode single transcripts. In the short subtelomeric regions we identified a few overrepresented motifs that could be involved in gene regulation, but there is no consensus polyadenylation site. The introns are short (21–29 nucleotides), and a significant fraction (1/3) of the tiny introns is conserved in the distantly related ciliate Paramecium tetraurelia. As has been observed in P. tetraurelia, the N. ovalis introns tend to contain in-frame stop codons or have a length that is not dividable by three. This pattern causes premature termination of mRNA translation in the event of intron retention, and potentially degradation of unspliced mRNAs by the nonsense-mediated mRNA decay pathway.ConclusionThe combination of short leaders, tiny introns and single genes leads to very minimal macronuclear chromosomes. The smallest we identified contained only 150 nucleotides.

Comparison of nonsense-mediated mRNA decay efficiency in various murine tissues

BackgroundThe Nonsense-Mediated mRNA Decay (NMD) pathway detects and degrades mRNAs containing premature termination codons, thereby preventing the accumulation of potentially detrimental truncated proteins. Intertissue variation in the efficiency of this mechanism has been suggested, which could have important implications for the understanding of genotype-phenotype correlations in various genetic disorders. However, compelling evidence in favour of this hypothesis is lacking. Here, we have explored this question by measuring the ratio of mutant versus wild-type Men1 transcripts in thirteen tissues from mice carrying a heterozygous truncating mutation in the ubiquitously expressed Men1 gene.ResultsSignificant differences were found between two groups of tissues. The first group, which includes testis, ovary, brain and heart, displays a strong decrease of the nonsense transcript (average ratio of 18% of mutant versus wild-type Men1 transcripts, identical to the value measured in murine embryonic fibroblasts). The second group, comprising lung, intestine and thymus, shows much less pronounced NMD (average ratio of 35%). Importantly, the extent of degradation by NMD does not correlate with the expression level of eleven genes encoding proteins involved in NMD or with the expression level of the Men1 gene.ConclusionMouse models are an attractive option to evaluate the efficiency of NMD in multiple mammalian tissues and organs, given that it is much easier to obtain these from a mouse than from a single individual carrying a germline truncating mutation. In this study, we have uncovered in the thirteen different murine tissues that we examined up to a two-fold difference in NMD efficiency.

Expression levels of Protocadherin-α transcripts are decreased by nonsense-mediated mRNA decay with frameshift mutations and by high DNA methylation in their promoter regions

The mouse protocadherin ( Pcdh) clusters, Pcdh-α, -β, and -γ, are located on chromosome 18. Many polymorphic variations are found in the Pcdh-α genes in wild-derived and laboratory mouse strains. In comparing the expression levels of Pcdh-α isoforms among several strains, we observed lower expression levels of Pcdh-α9 in BLG2 and BFM/2, and of Pcdh-α8 in C57BL/6 (B6) than in the other strains. For Pcdh-α8, high DNA methylation (72.7%) in the promoter region was found only in B6, whereas 36.4–44.3% methylation was seen in the other strains. On the other hand, the Pcdh-α9 DNA-methylation levels were similar (23.6–36.3%) among the strains regardless of the difference in expression levels. Interestingly, however, the Pcdh-α9 variable exon in both BLG2 and BFM/2 included a premature termination codon (PTC) generated by a nucleotide deletion or insertion. Treatment with emetine, a potent inhibitor of nonsense-mediated mRNA decay (NMD), increased the expression level of Pcdh-α9 from the BLG2- Pcdh-α locus. These data indicate that the transcription levels of mature Pcdh-α mRNAs are decreased by the DNA-methylation state of the Pcdh-α promoter regions and by the NMD pathway during RNA maturation. And we correct some previous data on Sugino, H., Toyama, T., Taguchi, Y., Esumi, S., Miyazaki, M., Yagi, T., (2004) Negative and positive effects of an IAP-LTR on nearby Pcdaalpha gene expression in the central nervous system and neuroblastoma cell lines, Gene 337 91–103.

UPF1, a Conserved Nonsense-Mediated mRNA Decay Factor, Regulates Cyst Wall Protein Transcripts in Giardia lamblia

The Giardia lamblia cyst wall is required for survival outside the host and infection. Three cyst wall protein (cwp) genes identified to date are highly up-regulated during encystation. However, little is known of the molecular mechanisms governing their gene regulation. Messenger RNAs containing premature stop codons are rapidly degraded by a nonsense-mediated mRNA decay (NMD) system to avoid production of non-functional proteins. In addition to RNA surveillance, NMD also regulates thousands of naturally occurring transcripts through a variety of mechanisms. It is interesting to know the NMD pathway in the primitive eukaryotes. Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression. In this study, we tested the hypothesis that NMD factors can regulate some naturally occurring transcripts in G. lamblia. We found that overexpression of UPF1 resulted in a significant decrease of the levels of CWP1 and cyst formation and of the endogenous cwp1-3, and myb2 mRNA levels and stability. This indicates that NMD could contribute to the regulation of the cwp1-3 and myb2 transcripts, which are key to G. lamblia differentiation into cyst. Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD. Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia.

The mammalian gene ZNF268 is regulated by hUpf1

Nonsense-mediated mRNA decay (NMD), also called RNA surveillance, is a process that degrades mRNAs with premature translation termination codons. In Saccharomyces cerevisiae, it has also been shown that NMD can regulate gene expression at the transcriptional level. To date, there has been no example where promoters are regulated by the NMD pathway in higher eukaryotes. Taking advantage of our previous research on ZNF268 transcription control, we studied the relationship between the ZNF268 promoter and the NMD pathway. We showed by transient transfection that the ZNF268 promoter activity was influenced by hUpf1, not hSmg6, in HeLa cells. This result was confirmed by the analysis of the steady state mRNA of ZNF268 after depletion of endogenous hUpf1 or hSmg6 in HeLa cells. Direct mutational analysis revealed that the C/EBP site in the promoter region is important for hUpf1 function on ZNF268 promoter. Together our results demonstrated that the mammalian gene ZNF268 is regulated by hUpf1 via its promoter.

Upf1 potentially serves as a RING-related E3 ubiquitin ligase via its association with Upf3 in yeast

Three Upf proteins are essential to the nonsense-mediated mRNA decay (NMD) pathway. Although these proteins assemble on polysomes for recognition of aberrant mRNAs containing premature termination codons, the significance of this assembly remains to be elucidated. The Cys- and His-rich repeated N terminus (CH domain) of Upf1 has been implicated in its binding to Upf2. Here, we show that CH domain also plays a RING-related role for Upf1 to exhibit E3 ubiquitin ligase activity in yeast. Despite the sequence divergence from typical E3-RING fingers, the CH domain of yeast Upf1 specifically and directly interacted with the yeast E2 Ubc3. Interestingly, Upf1 served as a substrate for the in vitro self-ubiquitination, and the modification required its association with Upf3 rather than Upf2. Substitution of the coordinated Cys and His residues in the CH domain impaired not only self-ubiquitination of Upf1 but also rapid decay of aberrant mRNAs. These results suggest that Upf1 may serve as an E3 ubiquitin ligase upon its association with Upf3 and play an important role in signaling to the NMD pathway.

Identification of ARHGEF17, DENND2D, FGFR3, and RB1 mutations in melanoma by inhibition of nonsense‐mediated mRNA decay

Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven to be a strategy for genome-wide discovery of genes containing inactivating mutations in colon and prostate cancers. Here, we present the first study of inhibition of the nonsense-mediated mRNA decay (NMD) pathway in melanoma. We used a combination of emetine and actinomycin D treatment to stabilize mRNAs containing premature termination codons (PTCs), followed by microarray analysis and sequencing to identify novel tumor suppressor genes (TSGs) in a panel of 12 melanoma cell lines. Stringent analysis of the array data was used to select 35 candidate genes for sequencing. Of these, 4 (11%) were found to carry PTCs, including ARHGEF17, DENND2D, FGFR3, and RB1. While RB1 mutations have previously been described in melanoma, the other three genes represent potentially novel melanoma; TSGs. ARHGEF17 showed a G1865A mutation leading to W622X in a cell line derived from a mucosal melanoma; in RB1 a C1411T base change resulting in Q471X was discovered in a cell line derived from an acral melanoma; and the FGFR3 and DENND2D genes had intronic insertions leading to PTCs in cell lines derived from superficially spreading melanomas. We conclude that although the false positive rate is high, most likely due to the lack of DNA mismatch repair gene defects, the GINI protocol is one approach to discover novel TSGs in melanoma.

Regulation of multiple core spliceosomal proteins by alternative splicing-coupled nonsense-mediated mRNA decay.

Alternative splicing (AS) can regulate gene expression by introducing premature termination codons (PTCs) into spliced mRNA that subsequently elicit transcript degradation by the nonsense-mediated mRNA decay (NMD) pathway. However, the range of cellular functions controlled by this process and the factors required are poorly understood. By quantitative AS microarray profiling, we find that there are significant overlaps among the sets of PTC-introducing AS events affected by individual knockdown of the three core human NMD factors, Up-Frameshift 1 (UPF1), UPF2, and UPF3X/B. However, the levels of some PTC-containing splice variants are less or not detectably affected by the knockdown of UPF2 and/or UPF3X, compared with the knockdown of UPF1. The intron sequences flanking the affected alternative exons are often highly conserved, suggesting important regulatory roles for these AS events. The corresponding genes represent diverse cellular functions, and surprisingly, many encode core spliceosomal proteins and assembly factors. We further show that conserved, PTC-introducing AS events are enriched in genes that encode core spliceosomal proteins. Where tested, altering the expression levels of these core spliceosomal components affects the regulation of PTC-containing splice variants from the corresponding genes. Together, our results show that AS-coupled NMD can have different UPF factor requirements and is likely to regulate many general components of the spliceosome. The results further implicate general spliceosomal components in AS regulation.

Splicing and dicing with a SERRATE d edge

The maturation and quality control of mRNA in eukaryotes is a tightly regulated, multistep process that begins on nascent transcripts. A 7-methyl guanosine (7MeG) cap structure is added to the 5′ end of pre-mRNA as it emerges from the exit channel of RNA Polymerase II. The multifunctional nuclear cap-binding complex (CBC), consisting of two protein subunits (CBP80 and CBP20), assembles at the pre-mRNA cap early during transcript formation and helps recruit the splicesome machinery to the cap-proximal intron (1, 2). Termination of transcription involves cleavage and polyadenylation at the 3′ end, and the mature mRNA is retained in the nucleus or exported to the cytoplasm. In either case, the mRNA undergoes a pioneer round of translation and surveillance by the nonsense-mediated mRNA decay (NMD) pathway to eliminate defective or misspliced transcripts. Although the CBC localizes primarily to the nucleus, it remains associated with mRNAs during export to the cytoplasm and during the pioneer round of translation and mRNA surveillance. After the first round of translation, the CBC is replaced by the eukaryotic initiation complex eIF4F, and the mRNA steady-state translation initiation complex is formed (3). But not all RNA Pol II transcripts are predestined for translation. Primary transcripts for microRNA (pri-miRNA) are retained in the nucleus, where they are processed into ≈21- to 22-nt miRNA that generally function as posttranscriptional regulators of mRNA expression. Although it is known that pri-miRNA transcripts form by RNA Pol II transcription, the extent to which pri-miRNA and pre-mRNA transcripts share common processing components is far from settled. In this issue of PNAS, Laubinger et al. (4) identify an important relationship between mRNA maturation and miRNA primary transcript processing in plants.

A Protective Role for the Human SMG-1 Kinase against Tumor Necrosis Factor-α-induced Apoptosis

The human suppressor of morphogenesis in genitalia-1 (hSMG-1) protein kinase plays dual roles in mRNA surveillance and genotoxic stress response pathways in human cells. Here, we report that small interfering RNA-mediated depletion of hSMG-1, but not ATM, ATR, hUpf1, or hUpf2, in human U2OS osteosarcoma cells markedly increases the magnitude and accelerates the rate of apoptosis induced by tumor necrosis factor-alpha (TNFalpha) stimulation. The increase in TNFalpha-mediated cell killing observed in hSMG-1-depleted cells is not related to the suppression of nonsense-mediated mRNA decay or to the inhibition of TNFalpha-induced NF-kappaB activation. Rather, we observed that loss of hSMG-1 accelerates the degradation of the long form of the FLICE-inhibitory protein (FLIP(L)), an inhibitor of death-inducing signaling complex-mediated caspase-8 activation, in TNFalpha-treated cells. These results suggest that hSMG-1 plays an important role in cell survival during TNFalpha-induced stress.

Inter-kingdom conservation of mechanism of nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a quality control system that degrades mRNAs containing premature termination codons. Although NMD is well characterized in yeast and mammals, plant NMD is poorly understood. We have undertaken the functional dissection of NMD pathways in plants. Using an approach that allows rapid identification of plant NMD trans factors, we demonstrated that two plant NMD pathways coexist, one eliminates mRNAs with long 3'UTRs, whereas a distinct pathway degrades mRNAs harbouring 3'UTR-located introns. We showed that UPF1, UPF2 and SMG-7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron-based NMD. The molecular mechanism of long 3'UTR-based plant NMD resembled yeast NMD, whereas the intron-based NMD was similar to mammalian NMD, suggesting that both pathways are evolutionarily conserved. Interestingly, the SMG-7 NMD component is targeted by NMD, suggesting that plant NMD is autoregulated. We propose that a complex, autoregulated NMD mechanism operated in stem eukaryotes, and that despite aspect of the mechanism being simplified in different lineages, feedback regulation was retained in all kingdoms.

Molecular cloning and characterization of the chicken cationic amino acid transporter-2 gene

The system y + cationic amino acid transporters (CATs) play an important role in the regulation of lysine and arginine utilization. We have characterized the genomic organization and tissue transcription of the chicken CAT-2 (cCAT-2) isoforms from pectoralis muscle. The primary cCAT-2 transcript is alternatively spliced within the same position of the mRNA to produce cCAT-2A and cCAT-2B isoforms that share high nucleotide homology with their mammalian counterparts. We also identified a novel third CAT-2 isoform, cCAT-2C. This isoform contains a premature termination codon and studies in the chicken LMH cell line show that cCAT-2C mRNA is degraded by the nonsense-mediated mRNA decay pathway. All three cCAT-2 isoforms are transcribed in liver, pectoralis, gastrocnemius and heart. Chicken cCAT-2A was the predominant cCAT-2 isoform in liver, pectoralis and gastrocnemius. Analysis of the 5′-untranslated region of cCAT-2 identified multiple cCAT-2 promoters. Chicken CAT-2 promoter usage was tissue dependent and was not responsible for cCAT-2 isoform production. Analysis of genomic sequence upstream cCAT-2 promoter regions revealed binding sites for transcription factors involved in amino acid sensing, hormone signaling and immune function. These results provide insight into the role of the cCAT-2 gene and its regulation of lysine and arginine utilization in aves.

Posttranscriptional Gene Regulation by Spatial Rearrangement of the 3′ Untranslated Region

Translation termination at premature termination codons (PTCs) triggers degradation of the aberrant mRNA, but the mechanism by which a termination event is defined as premature is still unclear. Here we show that the physical distance between the termination codon and the poly(A)-binding protein PABPC1 is a crucial determinant for PTC recognition in human cells. “Normal” termination codons can trigger nonsense-mediated mRNA decay (NMD) when this distance is extended; and vice versa, NMD can be suppressed by folding the poly(A) tail into proximity of a PTC or by tethering of PABPC1 nearby a PTC, indicating an evolutionarily conserved function of PABPC1 in promoting correct translation termination and antagonizing activation of NMD. Most importantly, our results demonstrate that spatial rearrangements of the 3′ untranslated region can modulate the NMD pathway and thereby provide a novel mechanism for posttranscriptional gene regulation.

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.

Molecular mechanisms of mRNA degradation

Eukaryotes have evolved quality‐control mechanisms that allow cells to remove defective messenger RNAs (mRNAs). Aberrant mRNA with premature stop codons (PTCs) are recognized and degraded by a process known as nonsense‐mediated mRNA decay (NMD). The NMD pathway is conserved from lower to higher eukaryotes. In humans, the recognition of a PTC‐containing mRNA depends on both splicing and translation.In the nucleus, a complex of proteins known as the exon junction complex (EJC) is deposited on mRNA upon splicing and stays stably bound until the nucleic acid is exported to the cytoplasm. Here, the crosstalk between a ribosome stalled at a PTC and a downstream EJC elicits a cascade of interactions that ultimately lead to the recruitment of ribonucleases and mRNA degradation. The talk will focus on our current understanding of the molecular mechanisms of NMD as elucidated by structural biology.