Function In Nonsense-mediated mRNA Decay Research Articles

Inhibition of nonsense-mediated mRNA decay reduces the tumorigenicity of human fibrosarcoma cells.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA decay pathway with roles in cellular stress responses, differentiation, and viral defense. It functions in both quality control and post-transcriptional regulation of gene expression. NMD has also emerged as a modulator of cancer progression, although available evidence supports both a tumor suppressor and a pro-tumorigenic role, depending on the model. To further investigate the role of NMD in cancer, we knocked out the NMD factor SMG7 in the HT1080 human fibrosarcoma cell line, resulting in suppression of NMD function. We then compared the oncogenic properties of the parental cell line, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. We also tested the effect of a drug inhibiting the NMD factor SMG1 to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays and a mouse xenograft tumor model, we showed that suppression of NMD function severely compromises the oncogenic phenotype. Molecular pathway analysis revealed that NMD suppression strongly reduces matrix metalloprotease 9 (MMP9) expression and that MMP9 re-expression partially rescues the oncogenic phenotype. Since MMP9 promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation may contribute to the reduced tumorigenicity of NMD-suppressed cells. Collectively, our results highlight the potential value of NMD inhibition as a therapeutic approach.

Gene Variants Involved in Nonsense-Mediated mRNA Decay Suggest a Role in Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a heterogeneous neurodevelopmental condition with unclear etiology. Many genes have been associated with ASD risk, but the underlying mechanisms are still poorly understood. An important post-transcriptional regulatory mechanism that plays an essential role during neurodevelopment, the Nonsense-Mediated mRNA Decay (NMD) pathway, may contribute to ASD risk. In this study, we gathered a list of 46 NMD factors and regulators and investigated the role of genetic variants in these genes in ASD. By conducting a comprehensive search for Single Nucleotide Variants (SNVs) in NMD genes using Whole Exome Sequencing data from 1828 ASD patients, we identified 270 SNVs predicted to be damaging in 28.7% of the population. We also analyzed Copy Number Variants (CNVs) from two cohorts of ASD patients (N = 3570) and discovered 38 CNVs in 1% of cases. Importantly, we discovered 136 genetic variants (125 SNVs and 11 CNVs) in 258 ASD patients that were located within protein domains required for NMD. These gene variants are classified as damaging using in silico prediction tools, and therefore may interfere with proper NMD function in ASD. The discovery of NMD genes as candidates for ASD in large patient genomic datasets provides evidence supporting the involvement of the NMD pathway in ASD pathophysiology.

UPF2 leads to degradation of dendritically targeted mRNAs to regulate synaptic plasticity and cognitive function.

Synaptic plasticity requires a tight control of mRNA levels in dendrites. RNA translation and degradation pathways have been recently linked to neurodevelopmental and neuropsychiatric diseases, suggesting a role for RNA regulation in synaptic plasticity and cognition. While the local translation of specific mRNAs has been implicated in synaptic plasticity, the tightly controlled mechanisms that regulate local quantity of specific mRNAs remain poorly understood. Despite being the only RNA regulatory pathway that is associated with multiple mental illnesses, the nonsense-mediated mRNA decay (NMD) pathway presents an unexplored regulatory mechanism for synaptic function and plasticity. Here, we show that neuron-specific disruption of UPF2, an NMD component, in adulthood attenuates learning, memory, spine density, synaptic plasticity (L-LTP), and potentiates perseverative/repetitive behavior in mice. We report that the NMD pathway operates within dendrites to regulate Glutamate Receptor 1 (GLUR1) surface levels. Specifically, UPF2 modulates the internalization of GLUR1 and promotes its local synthesis in dendrites. We identified neuronal Prkag3 mRNA as a mechanistic substrate for NMD that contributes to the UPF2-mediated regulation of GLUR1 by limiting total GLUR1 levels. These data establish that UPF2 regulates synaptic plasticity, cognition, and local protein synthesis in dendrites, providing fundamental insight into the neuron-specific function of NMD within the brain.

Degradation of Gadd45 mRNA by nonsense-mediated decay is essential for viability.

The nonsense-mediated mRNA decay (NMD) pathway functions to degrade both abnormal and wild-type mRNAs. NMD is essential for viability in most organisms, but the molecular basis for this requirement is unknown. Here we show that a single, conserved NMD target, the mRNA coding for the stress response factor growth arrest and DNA-damage inducible 45 (GADD45) can account for lethality in Drosophila lacking core NMD genes. Moreover, depletion of Gadd45 in mammalian cells rescues the cell survival defects associated with NMD knockdown. Our findings demonstrate that degradation of Gadd45 mRNA is the essential NMD function and, surprisingly, that the surveillance of abnormal mRNAs by this pathway is not necessarily required for viability.

Identification of Interactions in the NMD Complex Using Proximity-Dependent Biotinylation (BioID).

Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly. In particular, we expressed BirA* fused to the well characterized NMD factors UPF1, UPF2 and SMG5 and detected by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) the streptavidin-purified biotinylated proteins. While the identified already known interactors confirmed the usefulness of BioID, we also found new potentially important interactors that have escaped previous detection by co-IP, presumably because they associate only weakly and/or very transiently with the NMD machinery. Our results suggest that SMG5 only transiently contacts the UPF1-UPF2-UPF3 complex and that it provides a physical link to the decapping complex. In addition, BioID revealed among others CRKL and EIF4A2 as putative novel transient interactors with NMD factors, but whether or not they have a function in NMD remains to be elucidated.

BnaC9.SMG7b Functions as a Positive Regulator of the Number of Seeds per Silique in Brassica napus by Regulating the Formation of Functional Female Gametophytes.

Number of seeds per silique (NSS) is an important determinant of seed yield potential in Brassicaceae crops, and it is controlled by naturally occurring quantitative trait loci. We previously mapped a major quantitative trait locus, qSS.C9, on the C9 chromosome that controls NSS in Brassica napus. To gain a better understanding of how qSS.C9 controls NSS in B. napus, we isolated this locus through a map-based cloning strategy. qSS.C9 encodes a predicted small protein with 119 amino acids, designated as BnaC9.SMG7b, that shows homology with the Ever ShorterTelomere1 tertratricopeptide repeats and Ever Shorter Telomere central domains of Arabidopsis (Arabidopsis thaliana) SUPPRESSOR WITH MORPHOGENETIC EFFECTS ON GENITALIA7 (SMG7). BnaC9.SMG7b plays a role in regulating the formation of functional female gametophyte, thus determining the formation of functional megaspores and then mature ovules. Natural loss or artificial knockdown of BnaC9.SMG7b significantly reduces the number of functional ovules per silique and thus, results in decreased seed number, indicating that qSS.C9 is a positive regulator of NSS in B. napus. Sequence and function analyses show that BnaC9.SMG7b experiences a subfunctionalization process that causes loss of function in nonsense-mediated mRNA decay, such as in Arabidopsis SMG7. Haplotype analysis in 84 accessions showed that the favorable BnaC9.SMG7b alleles are prevalent in modern B. napus germplasms, suggesting that this locus has been a major selection target of B. napus improvement. Our results represent the first step toward unraveling the molecular mechanism that controls the natural variation of NSS in B. napus.

Characterization of phosphorylation- and RNA-dependent UPF1 interactors by quantitative proteomics.

Human up-frameshift 1 (UPF1) is an ATP-dependent RNA helicase and phosphoprotein implicated in several biological processes but is best known for its key function in nonsense-mediated mRNA decay (NMD). Here we employed a combination of stable isotope labeling of amino acids in cell culture experiments to determine by quantitative proteomics UPF1 interactors. We used this approach to distinguish between RNA-mediated and protein-mediated UPF1 interactors and to determine proteins that preferentially bind the hypo- or the hyper-phosphorylated form of UPF1. Confirming and expanding previous studies, we identified the eukaryotic initiation factor 3 (eIF3) as a prominent protein-mediated interactor of UPF1. However, unlike previously reported, eIF3 binds to UPF1 independently of UPF1's phosphorylation state. Furthermore, our data revealed many nucleus-associated RNA-binding proteins that preferentially associate with hyper-phosphorylated UPF1 in an RNase-sensitive manner, suggesting that UPF1 gets recruited to mRNA and becomes phosphorylated before being exported to the cytoplasm as part of the mRNP.

Nonsense-mediated mRNA decay and development: shoot the messenger to survive?

NMD (nonsense-mediated mRNA decay) is a surveillance mechanism that degrades transcripts containing nonsense mutations, preventing the translation of potentially harmful truncated proteins. Although the mechanistic details of NMD are gradually being understood, the physiological role of this RNA surveillance pathway still remains largely unknown. The core NMD genes Upf1 (up-frameshift suppressor 1) and Upf2 are essential for animal viability in the fruitfly, mouse and zebrafish. These findings may reflect an important role for NMD during animal development. Alternatively, the lethal phenotypes of upf1 and upf2 mutants might be due to their function in NMD-independent processes. In the present paper, we describe the phenotypes observed when the NMD factors are mutated in various organisms, and discuss findings that might shed light on the function of NMD in cellular growth and development of an organism.

AAA+ Proteins RUVBL1 and RUVBL2 Coordinate PIKK Activity and Function in Nonsense-Mediated mRNA Decay

Phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins play essential roles in DNA-based and RNA-based processes, such as the response to DNA damage, messenger RNA (mRNA) quality control, transcription, and translation, where they contribute to the maintenance of genome integrity and accurate gene expression. The adenosine triphosphatases associated with diverse cellular activities (AAA+) family proteins RuvB-like 1 (RUVBL1) and RUVBL2 are involved in various cellular processes, including transcription, RNA modification, DNA repair, and telomere maintenance. We show that RUVBL1 and RUVBL2 associate with each PIKK family member. We also show that RUVBL1 and RUVBL2 control PIKK abundance at least at the mRNA level. Knockdown of RUVBL1 or RUVBL2 decreased PIKK abundance and impaired PIKK-mediated signaling. Analysis of SMG-1, a PIKK family member involved in nonsense-mediated mRNA decay (NMD), revealed an essential role for RUVBL1 and RUVBL2 in NMD. RUVBL1 and RUVBL2 associated with SMG-1 and the messenger ribonucleoproteins in the cytoplasm and promoted the formation of mRNA surveillance complexes during NMD. Thus, RUVBL1 and RUVBL2 regulate PIKK functions on two different levels: They control the abundance of PIKKs, and they stimulate the formation of PIKK-containing molecular complexes, such as those involved in NMD.

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.

A Novel Role for the SMG-1 Kinase in Lifespan and Oxidative Stress Resistance in Caenorhabditis elegans

The PTEN tumour suppressor encodes a phosphatase, and its daf-18 orthologue in Caenorhabditis elegans negatively regulates the insulin/IGF-1 DAF-2 receptor pathway that influences lifespan in worms and other species. In order to identify new DAF-18 regulated pathways involved in aging, we initiated a candidate RNAi feeding screen for clones that lengthen lifespan. Here, we report that smg-1 inactivation increases average lifespan in a daf-18 dependent manner. Genetic analysis is consistent with SMG-1 acting at least in part in parallel to the canonical DAF-2 receptor pathway, but converging on the transcription factor DAF-16/FOXO. SMG-1 is a serine-threonine kinase which plays a conserved role in nonsense-mediated mRNA decay (NMD) in worms and mammals. In addition, human SMG-1 has also been implicated in the p53-mediated response to genotoxic stress. The effect of smg-1 inactivation on lifespan appears to be unrelated to its NMD function, but requires the p53 tumour suppressor orthologue cep-1. Furthermore, smg-1 inactivation confers a resistance to oxidative stress in a daf-18-, daf-16- and cep-1-dependent manner. We propose that the role of SMG-1 in lifespan regulation is at least partly dependent on its function in oxidative stress resistance. Taken together, our results unveil a novel role for SMG-1 in lifespan regulation.

Upf1, an RNA Helicase Required for Nonsense-Mediated mRNA Decay, Modulates the Transcriptional Response to Oxidative Stress in Fission Yeast

In the fission yeast Schizosaccharomyces pombe, oxidative stress triggers the activation of the Spc1/Sty1 mitogen-activated protein kinase, which in turn phosphorylates the Atf1/Pcr1 heterodimeric transcription factor to effect global changes in the patterns of gene expression. This transcriptional response is also controlled by Csx1, an RNA-binding protein that directly associates with and stabilizes atf1(+) mRNA. Here we report the surprising observation that this response also requires Upf1, a component of the nonsense-mediated mRNA decay (NMD) system. Accordingly, upf1Delta and csx1Delta strains are similarly sensitive to oxidative stress, and the effects of the mutations are not additive, suggesting that Upf1 and Csx1 work in the same pathway to stabilize atf1(+) mRNA during oxidative stress. Consistent with these observations, whole-genome expression profiling studies have shown that Upf1 controls the expression of more than 100 genes that are transcriptionally induced in response to oxidative stress, the large majority of which are also controlled by Atf1 and Csx1. The unexpected connection between an NMD factor and the oxidative stress response in fission yeast may provide important new clues about the physiological function of NMD in other species.

Functions of hUpf3a and hUpf3b in nonsense-mediated mRNA decay and translation

The exon-junction complex (EJC) components hUpf3a and hUpf3b serve a dual function: They promote nonsense-mediated mRNA decay (NMD), and they also regulate translation efficiency. Whether these two functions are interdependent or independent of each other is unknown. We characterized the function of the hUpf3 proteins in a lambdaN/boxB-based tethering system. Despite the high degree of sequence similarity between hUpf3b and hUpf3a, hUpf3a is much less active than hUpf3b to induce NMD and to stimulate translation. We show that induction of NMD by hUpf3 proteins requires interaction with Y14, Magoh, BTZ, and eIF4AIII. The protein region that mediates this interaction and discriminates between hUpf3a and hUpf3b in NMD function is located in the C-terminal domain and fully contained within a small sequence that is highly conserved in Upf3b but not Upf3a proteins. Stimulation of translation is independent of this interaction and is determined by other regions of the hUpf3 protein, indicating the presence of different downstream pathways of hUpf3 proteins either in NMD or in translation.

Abnormally spliced β-globin mRNAs: a single point mutation generates transcripts sensitive and insensitive to nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) represents a phylogenetically widely conserved splicing- and translation-dependent mechanism that eliminates transcripts with premature translation stop codons and suppresses the accumulation of C-terminally truncated peptides. Elimination of frameshifted transcripts that result from faulty splicing may be an important function of NMD. To test this hypothesis directly, this study used the IVS1 + 5 G>A thalassemia mutation of the human beta-globin gene as a model system. We generated beta-globin gene constructs with this mutation and an iron-responsive element in the 5' untranslated region, which allowed specific experimental activation and inactivation of translation and, hence, NMD of this transcript. Premessenger RNAs with IVS1 + 5 G>A were spliced at normal sites and cryptic sites, enabling a direct comparison of the effect of NMD on the accumulation of normal and frameshifted messenger RNAs. In transfected HeLa cells, the predominant frameshifted transcript was degraded under conditions of active NMD, whereas accumulation to high levels occurred under conditions of specifically disabled NMD, thereby indicating an important physiologic function of NMD in the control of the splicing process. An unexpected finding was that accumulation of a second aberrant transcript remained unaffected by NMD. The IVS1 + 5 G>A mutation thus revealed the presence of an unknown cis-acting determinant that influences the NMD sensitivity of a putative NMD substrate. It can therefore serve as a useful tool for defining the mechanisms that permit specific transcripts to circumvent the NMD pathway.