The nonsense-mediated mRNA decay (NMD) pathway selectively eliminates aberrant transcripts containing premature translation termination codons and regulates the levels of a number of physiological mRNAs. NMD modulates the clinical outcome of a variety of human diseases, including cancer and many genetic disorders, and may represent a target for therapeutic intervention. Here, we have developed a new multicolored bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells. Using this reporter system, we conducted a robust high-throughput small-molecule screen in human cells and, unpredictably, identified a group of cardiac glycosides, including ouabain and digoxin, as potent inhibitors of NMD. Cardiac glycoside-mediated effects on NMD are dependent on binding and inhibiting the sodium-potassium ATPase on the plasma membrane and subsequent elevation of intracellular calcium levels. Induction of calcium release from the endoplasmic reticulum also leads to inhibition of NMD. Thus, this study reveals intracellular calcium as a key regulator of NMD and has implications for exploiting NMD in the treatment of disease.

Nonsense suppression therapy is an approach to treat genetic diseases caused by nonsense mutations. This therapeutic strategy pharmacologically suppresses translation termination at Premature Termination Codons (PTCs) in order to restore expression of functional protein. However, the process of Nonsense-Mediated mRNA Decay (NMD), which reduces the abundance of mRNAs containing PTCs, frequently limits this approach. Here, we used a mouse model of the lysosomal storage disease mucopolysaccharidosis I-Hurler (MPS I-H) that carries a PTC in the Idua locus to test whether NMD attenuation can enhance PTC suppression in vivo. Idua encodes alpha-L-iduronidase, an enzyme required for degradation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. We found that the NMD attenuator NMDI-1 increased the abundance of the PTC-containing Idua transcript. Furthermore, co-administration of NMDI-1 with the PTC suppression drug gentamicin enhanced alpha-L-iduronidase activity compared to gentamicin alone, leading to a greater reduction of GAG storage in mouse tissues, including the brain. These results demonstrate that NMD attenuation significantly enhances suppression therapy in vivo.

Nonsense-mediated mRNA decay (NMD) is a RNA quality surveillance system for eukaryotes. It prevents cells from generating deleterious truncated proteins by degrading abnormal mRNAs that harbor premature termination codon (PTC). However, little is known about the molecular regulation mechanism underlying the inhibition of NMD by microRNAs.

Nonsense-mediated mRNA decay (NMD) is a cellular mRNA degradation mechanism that inhibits the expression of aberrant mRNAs harboring premature termination codons (PTCs). Recent progress in transcriptome-wide sequencing techniques has revealed that NMD also degrades approximately 5-30% of non-mutated cellular mRNAs in a way that can be regulated in response to various cellular signals. In mammals, NMD is governed by the central NMD factor UPF1, which is activated by phosphorylation after translation terminates at a nonsense codon that triggers NMD. We have found that immunoprecipitation using an antibody that is specific for phosphorylated UPF1 is a useful tool to define not only cellular NMD targets but also the nature of NMD decay intermediates and, thus, the process of NMD. To this end, we describe here a detailed protocol for what we call "NMD degradome sequencing" using high-throughput technology.

Nonsense-mediated mRNA decay (NMD) typifies an mRNA surveillance pathway. Because NMD necessitates a translation event to recognize a premature termination codon on mRNAs, truncated misfolded polypeptides (NMD-polypeptides) could potentially be generated from NMD substrates as byproducts. Here, we show that when the ubiquitin-proteasome system is overwhelmed, various misfolded polypeptides including NMD-polypeptides accumulate in the aggresome: a perinuclear nonmembranous compartment eventually cleared by autophagy. Hyperphosphorylation of the key NMD factor UPF1 is required for selective targeting of the misfolded polypeptide aggregates toward the aggresome via the CTIF-eEF1A1-DCTN1 complex: the aggresome-targeting cellular machinery. Visualization at a single-particle level reveals that UPF1 increases the frequency and fidelity of movement of CTIF aggregates toward the aggresome. Furthermore, the apoptosis induced by proteotoxic stresses is suppressed by UPF1 hyperphosphorylation. Altogether, our data provide evidence that UPF1 functions in the regulation of a protein surveillance as well as an mRNA quality control.

Nonsense-mediated mRNA decay (NMD) is a highly regulated quality control mechanism through which mRNAs harboring a premature termination codon are degraded. It is also a regulatory pathway for some genes. This mechanism is subject to various levels of regulation, including phosphorylation. To date only one kinase, SMG1, has been described to participate in NMD, by targeting the central NMD factor UPF1. Here, screening of a kinase inhibitor library revealed as putative NMD inhibitors several molecules targeting the protein kinase AKT1. We present evidence demonstrating that AKT1, a central player in the PI3K/AKT/mTOR signaling pathway, plays an essential role in NMD, being recruited by the UPF3X protein to phosphorylate UPF1. As AKT1 is often overactivated in cancer cells and as this should result in increased NMD efficiency, the possibility that this increase might affect cancer processes and be targeted in cancer therapy is discussed.

Nonsense-mediated mRNA decay (NMD) is a cellular quality-control mechanism that is thought to exacerbate the phenotype of certain pathogenic nonsense mutations by preventing the expression of semi-functional proteins. NMD also limits the efficacy of read-through compound (RTC)-based therapies. Here, we report a gene-specific method of NMD inhibition using antisense oligonucleotides (ASOs) and combine this approach with an RTC to effectively restore the expression of full-length protein from a nonsense-mutant allele.

Translation of mRNAs containing premature termination codons (PTCs) results in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance pathway responsible for detecting PTC containing transcripts. Although the molecular mechanisms governing mRNA degradation have been extensively studied, the fate of the nascent protein product remains largely uncharacterized. Here, we use a fluorescent reporter system in mammalian cells to reveal a selective degradation pathway specifically targeting the protein product of an NMD mRNA. We show that this process is post-translational and dependent on the ubiquitin proteasome system. To systematically uncover factors involved in NMD-linked protein quality control, we conducted genome-wide flow cytometry-based screens. Our screens recovered known NMD factors but suggested that protein degradation did not depend on the canonical ribosome-quality control (RQC) pathway. A subsequent arrayed screen demonstrated that protein and mRNA branches of NMD rely on a shared recognition event. Our results establish the existence of a targeted pathway for nascent protein degradation from PTC containing mRNAs, and provide a reference for the field to identify and characterize required factors.

The nonsense-mediated mRNA decay (NMD) pathway was originally identified as a pathway that degrades mRNAs with premature termination codons; however, NMD is now known to regulate natural mRNAs as well. Natural mRNAs are degraded by NMD due to the presence of specific NMD targeting features. An atypically long 3'-UTR is one of the features that has been shown to induce the rapid degradation of mRNAs by NMD in Saccharomyces cerevisiae and other organisms. S. cerevisiae CTR2 mRNAs have long 3'-UTRs and are sensitive to NMD, although the extent by which these long 3'-UTRs target the CTR2 mRNAs to the pathway is unknown. Here, we investigated the sequence elements that induce NMD of the CTR2 mRNAs and determined that the long CTR2 3'-UTR is sufficient to target an NMD-insensitive mRNA to the pathway. We also found that, although the CTR2 3'-UTR contributes to NMD-induced degradation, CTR2 mRNAs contain additional NMD-inducing features that function cooperatively with the atypically long 3'-UTR to trigger mRNA degradation. Lengthening the CTR2 ORF abrogates NMD and renders the mRNAs immune to the NMD pathway. Moreover, we found that transcription of CTR2 driven by the GPD promoter, which is not identical to the CTR2 promoter, affects degradation of the transcripts by NMD.

Growth cones enable axons to navigate toward their targets by responding to extracellular signaling molecules. Growth-cone responses are mediated in part by the local translation of axonal messenger RNAs (mRNAs). However, the mechanisms that regulate local translation are poorly understood. Here we show that Robo3.2, a receptor for the Slit family of guidance cues, is synthesized locally within axons of commissural neurons. Robo3.2 translation is induced by floor-plate-derived signals as axons cross the spinal cord midline. Robo3.2 is also a predicted target of the nonsense-mediated mRNA decay (NMD) pathway. We find that NMD regulates Robo3.2 synthesis by inducing the degradation of Robo3.2 transcripts in axons that encounter the floor plate. Commissural neurons deficient in NMD proteins exhibit aberrant axonal trajectories after crossing the midline, consistent with misregulation of Robo3.2 expression. These data show that local translation is regulated by mRNA stability and that NMD acts locally to influence axonal pathfinding.

The nonsense-mediated mRNA decay (NMD) pathway is a specialized pathway that triggers the rapid degradation of select mRNAs. Initially, identified as a pathway that degrades mRNAs with premature termination codons, NMD is now recognized as a pathway that also regulates some natural mRNAs. Since natural mRNAs do not typically contain premature termination codons, these mRNAs contain features that target them to NMD. In Saccharomyces cerevisiae mRNAs with atypically long 3'-UTRs are usually degraded by NMD, however in some conditions a constitutively expressed SSY5 mRNA with multiple NMD targeting signals including an atypically long 3'-UTR is an exception. We investigated the features of the SSY5 mRNAs that confer immunity to NMD. We found that the SSY5 mRNA 3'-UTRs are sufficient to target NMD insensitive mRNA to the pathway. Replacing the SSY5 3'-UTRs with the cyc1-512 3'-UTRs, known to target mRNAs to NMD or with the CYC1 3'-UTR, known not to target mRNAs to NMD, resulted in production of SSY5 mRNAs that were regulated by NMD. These observations suggest that the SSY5 mRNAs require sequences both within the 5'-UTR and/or ORF as well as the 3'-UTR to escape decay by NMD.

RNA-binding proteins play a crucial role in the post-transcriptional regulation of gene expression. Polypyrimidine tract binding protein (PTB in humans) has been extensively characterized as an important splicing factor, and has additional functions in 3' end processing and translation. ROD1 is a PTB paralog containing four RRM (RNA recognition motif) domains. Here, we discover a function of ROD1 in nonsense-mediated mRNA decay (NMD). We show that ROD1 and the core NMD factor UPF1 interact and co-regulate an extensive number of target genes. Using a reporter system, we demonstrate that ROD1, similarly to UPF1 and UPF2, is required for the destabilization of a known NMD substrate. Finally, we show through RIP-seq that ROD1 and UPF1 associate with a significant number of common transcripts.

Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.

Nonsense-mediated mRNA decay (NMD) is a conserved pathway that strongly influences eukaryotic gene expression. Inactivating or inhibiting NMD affects the abundance of a substantial fraction of the transcriptome in numerous species. Transcripts whose abundance is altered in NMD-deficient cells may represent either direct substrates of NMD or indirect effects of inhibiting NMD. We present a genome-wide investigation of the direct substrates of NMD in Caenorhabditis elegans Our goals were (i) to identify mRNA substrates of NMD and (ii) to distinguish those mRNAs from others whose abundance is indirectly influenced by the absence of NMD. We previously demonstrated that Upf1p/SMG-2, the central effector of NMD in all studied eukaryotes, preferentially associates with mRNAs that contain premature translation termination codons. We used this preferential association to distinguish direct from indirect effects by coupling immunopurification of Upf1/SMG-2 with high-throughput mRNA sequencing of NMD-deficient mutants and NMD-proficient controls. We identify 680 substrates of NMD, 171 of which contain novel spliced forms that (i) include sequences of annotated introns and (ii) have not been previously documented in the C. elegans transcriptome. NMD degrades unproductively spliced mRNAs with sufficient efficiency in NMD-proficient strains that such mRNAs were not previously known. Two classes of genes are enriched among the identified NMD substrates: (i) mRNAs of expressed pseudogenes and (ii) mRNAs of gene families whose gene number has recently expanded in the C. elegans genome. Our results identify novel NMD substrates and provide a context for understanding NMD's role in normal gene expression and genome evolution.

Platelet production is induced by the cytokine thrombopoietin (TPO). It is physiologically critical that TPO expression is tightly regulated, because lack of TPO causes life-threatening thrombocytopenia while an excess of TPO results in thrombocytosis. The plasma concentration of TPO is controlled by a negative feedback loop involving receptor-mediated uptake of TPO by platelets. Furthermore, TPO biosynthesis is limited by upstream open reading frames (uORFs) that curtail the translation of the TPO mRNA. uORFs are suggested to activate RNA degradation by nonsense-mediated decay (NMD) in a number of physiological transcripts. Here, we determine whether NMD affects TPO expression. We show that reporter mRNAs bearing the seventh TPO uORF escape NMD. Importantly, endogenously expressed TPO mRNA from HuH7 cells is unaffected by abrogation of NMD by RNAi. Thus, regulation of TPO expression is independent of NMD, implying that mRNAs bearing uORFs cannot generally be considered to represent NMD targets.

Nonsense-mediated mRNA decay (NMD) is an essential, highly conserved quality control pathway that detects and degrades mRNAs containing premature termination codons. Although the essentiality of NMD is frequently ascribed to its prevention of truncated protein accumulation, the extent to which NMD actually suppresses proteins encoded by NMD-sensitive transcripts is less well-understood than NMD-mediated suppression of mRNA. Here, we describe a reporter system that permits accurate quantification of both mRNA and protein levels via stable integration of paired reporters encoding NMD-sensitive and NMD-insensitive transcripts into the AAVS1 safe harbor loci in human cells. We use this system to demonstrate that NMD suppresses proteins encoded by NMD-sensitive transcripts by up to eightfold more than the mRNA itself. Our data indicate that NMD limits the accumulation of proteins encoded by NMD substrates by mechanisms beyond mRNA degradation, such that even when NMD-sensitive mRNAs escape destruction, their encoded proteins are still effectively suppressed.

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance mechanism that degrades transcripts containing premature translation termination codons, and it also influences expression of certain wild-type transcripts. Although the biochemical mechanisms of NMD have been studied intensively, its developmental functions and importance are less clear. Here, we describe the isolation and characterization of Drosophila "photoshop" mutations, which increase expression of green fluorescent protein and other transgenes. Mapping and molecular analyses show that photoshop mutations are loss-of-function mutations in the Drosophila homologs of NMD genes Upf1, Upf2, and Smg1. We find that Upf1 and Upf2 are broadly active during development, and they are required for NMD as well as for proper expression of dozens of wild-type genes during development and for larval viability. Genetic mosaic analysis shows that Upf1 and Upf2 are required for growth and/or survival of imaginal cell clones, but this defect can be overcome if surrounding wild-type cells are eliminated. By contrast, we find that the PI3K-related kinase Smg1 potentiates but is not required for NMD or for viability, implying that the Upf1 phosphorylation cycle that is required for mammalian and Caenorhabditis elegans NMD has a more limited role during Drosophila development. Finally, we show that the SV40 3' UTR, present in many Drosophila transgenes, targets the transgenes for regulation by the NMD pathway. The results establish that the Drosophila NMD pathway is broadly active and essential for development, and one critical function of the pathway is to endow proliferating imaginal cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells.

RNA studies, in recent years, have attracted much attention. These studies have broadened the understanding of the fundamental mechanisms of gene regulation. Molecular mechanisms are frequently conserved among eukaryotes. A system, or finding, in one organism can generally be extended to understanding the corresponding system in other eukaryotic organisms. Nonsense-mediated mRNA decay (NMD) components and pathways are conserved to an extent that depends on the particular eukaryotes being compared. Reports of NMD in a number of species, including higher plants, are gradually increasing so as to contribute to the elucidation of similarities and variations in the mechanisms of NMD among different species, the origin of NMD, and the evolution of NMD. It appears that the contexts of termination codons that are recognized as being "premature" can vary among different organisms. This chapter introduces and summarizes methods for the analysis of NMD-triggered mRNA degradation in plants in the hope that it will contribute to the detailed understanding of NMD.

Nonsense-mediated mRNA decay (NMD) is an essential eukaryotic process regulating transcript quality and abundance, and is involved in diverse processes including brain development and plant defenses. Although some of the NMD machinery is conserved between kingdoms, little is known about its evolution. Phosphorylation of the core NMD component UPF1 is critical for NMD and is regulated in mammals by the SURF complex (UPF1, SMG1 kinase, SMG8, SMG9 and eukaryotic release factors). However, since SMG1 is reportedly missing from the genomes of fungi and the plant Arabidopsis thaliana, it remains unclear how UPF1 is activated outside the metazoa. We used comparative genomics to determine the conservation of the NMD pathway across eukaryotic evolution. We show that SURF components are present in all major eukaryotic lineages, including fungi, suggesting that in addition to UPF1 and SMG1, SMG8 and SMG9 also existed in the last eukaryotic common ancestor, 1.8 billion years ago. However, despite the ancient origins of the SURF complex, we also found that SURF factors have been independently lost across the Eukarya, pointing to genetic buffering within the essential NMD pathway. We infer an ancient role for SURF in regulating UPF1, and the intriguing possibility of undiscovered NMD regulatory pathways.

Nonsense-mediated messenger RNA (mRNA) decay (NMD) is a surveillance pathway used by cells to control the quality mRNAs and to fine-tune transcript abundance. NMD plays an important role in cell cycle regulation, cell viability, DNA damage response, while also serving as a barrier to virus infection. Disturbance of this control mechanism caused by genetic mutations or dys-regulation of the NMD pathway can lead to pathologies, including neurological disorders, immune diseases and cancers. The role of NMD in cancer development is complex, acting as both a promoter and a barrier to tumour progression. Cancer cells can exploit NMD for the downregulation of key tumour suppressor genes, or tumours adjust NMD activity to adapt to an aggressive immune microenvironment. The latter case might provide an avenue for therapeutic intervention as NMD inhibition has been shown to lead to the production of neoantigens that stimulate an immune system attack on tumours. For this reason, understanding the biology and co-option pathways of NMD is important for the development of novel therapeutic agents. Inhibitors, whose design can make use of the many structures available for NMD study, will play a crucial role in characterizing and providing diverse therapeutic options for this pathway in cancer and other diseases.