Role Of Nonsense-mediated mRNA Decay Research Articles

UPF3B modulates endoplasmic reticulum stress through interaction with inositol-requiring enzyme-1α

The unfolded protein response (UPR) is a conserved and adaptive intracellular pathway that relieves the endoplasmic reticulum (ER) stress by activating ER transmembrane stress sensors. As a consequence of ER stress, the inhibition of nonsense-mediated mRNA decay (NMD) is due to an increase in the phosphorylation of eIF2α, which has the effect of inhibiting translation. However, the role of NMD in maintaining ER homeostasis remains unclear. In this study, we found that the three NMD factors, up-frameshift (UPF)1, UPF2, or UPF3B, were required to negate the UPR. Among these three NMD factors, only UPF3B interacted with inositol-requiring enzyme-1α (IRE1α). This interaction inhibited the kinase activity of IRE1α, abolished autophosphorylation, and reduced IRE1α clustering for ER stress. BiP and UPF3B jointly control the activation of IRE1α on both sides of the ER membrane. Under stress conditions, the phosphorylation of UPF3B was increased and the phosphorylated sites were identified. Both the UPF3BY160D genetic mutation and phosphorylation at Thr169 of UPF3B abolished its interaction with IRE1α and UPF2, respectively, leading to activation of ER stress and NMD dysfunction. Our study reveals a key physiological role for UPF3B in the reciprocal regulatory relationship between NMD and ER stress.

Nonsense-mediated mRNA decay affects hyperactive root formation in oculo-facio-cardio-dental syndrome via up-frameshift protein 1

ObjectivesOculo-facio-cardio-dental (OFCD) syndrome is a rare X-linked genetic disorder caused by mutations in the BCL6 co-repressor (BCOR) and is mainly characterized by radiculomegaly (elongated dental roots). All BCOR mutations reported to date have been associated with premature termination codons, indicating that nonsense-mediated mRNA decay (NMD) might play a vital role in the pathogenesis of OFCD syndrome. However, the molecular mechanisms underlying NMD remain unclear. In this study, we investigated the involvement of up-frameshift protein 1 (UPF1), which plays a central role in NMD, in the hyperactive root formation caused by BCOR mutations. MethodsPeriodontal ligament cells, isolated from a Japanese woman with a c.3668delC frameshift mutation in BCOR, and primary human periodontal ligament fibroblasts (HPdLFs) were used for an RNA immunoprecipitation assay to confirm the binding of UPF1 to mutated BCOR. Additionally, the effects of UPF1 on the BCOR transcription levels and corresponding gene expression were determined by performing relative quantitative real-time polymerase chain reactions. ResultsRNA immunoprecipitation revealed that UPF1 binds to exon 9 of mutated BCOR. Additionally, UPF1 knockdown via siRNA upregulated the transcription of BCOR, whereas overexpression of wild-type and mutated BCOR with the same frameshift mutation in HPdLFs altered bone morphogenetic protein 2 (BMP2) expression. ConclusionsOur findings indicate that BCOR mutations regulate the transcription of BCOR via UPF1, which may in turn regulate the expression of BMP2. NMD, caused by a c.3668delC mutation, potentially leads to an OFCD syndrome phenotype, including elongated dental roots.

LINC02561 promotes metastasis in HCC via HIF1-α/NDRG1/UPF1 axis

In the entire world, hepatocellular carcinoma (HCC) is one of the most frequent cancers that lead to death. Experiments on the function of long non-coding RNAs in the emergence of malignancies, including HCC, are ongoing. As a crucial RNA monitoring mechanism in eucaryotic cells, nonsense-mediated mRNA decay (NMD) can recognize and destroy mRNAs, which has an premature termination codons (PTC) in the open reading frame to prevent harmful buildup of truncated protein products in the cells. Nonsense transcript regulator 1 (Up-frameshift suppressor 1, UPF1), as a highly conserved RNA helicase and ATPase, plays a key role in NMD. Our laboratory screened out the highly expressed lncRNA LINC02561 in HCC from the TCGA database. Further research found that LINC02561 enhanced the invasion and transition abilities of liver cancer cells by regulating the protein N-Myc downstream regulated 1 (NDRG1). Hypoxia inducible factor-1 (HIF-1α) can bonded to LINC02561 promoters under hypoxic conditions, thereby promoting the upregulation of LINC02561 expression in liver cancer cells. LINC02561 competes with NDRG1 mRNA to bind UPF1, thereby preventing the degradation of NDRG1 mRNA to facilitate NDRG1 protein level. Taken together, the HIF1α-LINC02561-UPF1-NDRG1 regulatory axis could be an entirely novel target of liver cancer-related treatment.

Nonsense-mediated mRNA decay suppresses injury-induced muscle regeneration via inhibiting MyoD transcriptional activity.

Skeletal muscle regeneration is a crucial physiological process that occurs in response to injury or disease. As an important transcriptome surveillance system that regulates tissue development, the role of nonsense-mediated mRNA decay (NMD) in muscle regeneration remains unclear. Here, we found that NMD inhibits myoblast differentiation by targeting the phosphoinositide-3-kinase regulatory subunit 5 gene, which leads to the suppression of the transcriptional activity of myogenic differentiation (MyoD), a key regulator of myoblast differentiation. This disruption of MyoD transcriptional activity subsequently affects the expression levels of myogenin and myosin heavy chain, crucial markers of myoblast differentiation. Additionally, through up-frameshift protein 1 knockdown experiments, we observed that inhibiting NMD can accelerate muscle regeneration in vivo. These findings highlight the potential of NMD as a novel therapeutic target for the treatment of muscle-related injuries and diseases.

Translation-coupled mRNA quality control mechanisms.

mRNA surveillance pathways are essential for accurate gene expression and to maintain translation homeostasis, ensuring the production of fully functional proteins. Future insights into mRNA quality control pathways will enable us to understand how cellular mRNA levels are controlled, how defective or unwanted mRNAs can be eliminated, and how dysregulation of these can contribute to human disease. Here we review translation-coupled mRNA quality control mechanisms, including the non-stop and no-go mRNA decay pathways, describing their mechanisms, shared trans-acting factors, and differences. We also describe advances in our understanding of the nonsense-mediated mRNA decay (NMD) pathway, highlighting recent mechanistic findings, the discovery of novel factors, as well as the role of NMD in cellular physiology and its impact on human disease.

The biological functions of nonsense-mediated mRNA decay in plants: RNA quality control and beyond.

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved quality control pathway that inhibits the expression of transcripts containing premature termination codon. Transcriptome and phenotypic studies across a range of organisms indicate roles of NMD beyond RNA quality control and imply its involvement in regulating gene expression in a wide range of physiological processes. Studies in moss Physcomitrella patens and Arabidopsis thaliana have shown that NMD is also important in plants where it contributes to the regulation of pathogen defence, hormonal signalling, circadian clock, reproduction and gene evolution. Here, we provide up to date overview of the biological functions of NMD in plants. In addition, we discuss several biological processes where NMD factors implement their function through NMD-independent mechanisms.

AKT constitutes a signal-promoted alternative exon-junction complex that regulates nonsense-mediated mRNA decay

Despite a long appreciation for the role of nonsense-mediated mRNA decay (NMD) in destroying faulty, disease-causing mRNAs and maintaining normal, physiologic mRNA abundance, additional effectors that regulate NMD activity in mammalian cells continue to be identified. Here, we describe a haploid-cell genetic screen for NMD effectors that has unexpectedly identified 13 proteins constituting the AKT signaling pathway. We show that AKT supersedes UPF2 in exon-junction complexes (EJCs) that are devoid of RNPS1 but contain CASC3, defining an unanticipated insulin-stimulated EJC. Without altering UPF1 RNA binding or ATPase activity, AKT-mediated phosphorylation of the UPF1 CH domain at T151 augments UPF1 helicase activity, which is critical for NMD and also decreases the dependence of helicase activity on ATP. We demonstrate that upregulation of AKT signaling contributes to the hyperactivation of NMD that typifies Fragile X syndrome, as exemplified using FMR1-KO neural stem cells derived from induced pluripotent stem cells.

Structures of nonsense-mediated mRNA decay factors UPF3B and UPF3A in complex with UPF2 reveal molecular basis for competitive binding and for neurodevelopmental disorder-causing mutation.

UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2′s third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B’s disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity ∼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ∼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3′s middle-domain elucidates its essential role in NMD.

Mammalian UPF3A and UPF3B can activate nonsense‐mediated mRNA decay independently of their exon junction complex binding

Nonsense‐mediated mRNA decay (NMD) is governed by the three conserved factors—UPF1, UPF2, and UPF3. While all three are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, and its paralog UPF3A is suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here, we characterize the UPF3A/B‐dependence of NMD in human cell lines deleted of one or both UPF3 paralogs. We show that in human colorectal cancer HCT116 cells, NMD can operate in a UPF3B‐dependent and ‐independent manner. While UPF3A is almost dispensable for NMD in wild‐type cells, it strongly activates NMD in cells lacking UPF3B. Notably, NMD remains partially active in cells lacking both UPF3 paralogs. Complementation studies in these cells show that EJC‐binding domain of UPF3 paralogs is dispensable for NMD. Instead, the conserved “mid” domain of UPF3 paralogs is consequential for their NMD activity. Altogether, our results demonstrate that the mammalian UPF3 proteins play a more active role in NMD than simply bridging the EJC and the UPF complex.

A role for AKT1 in nonsense-mediated mRNA decay.

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.

The loss of SMG1 causes defects in quality control pathways in Physcomitrella patens

Nonsense-mediated mRNA decay (NMD) is important for RNA quality control and gene regulation in eukaryotes. NMD targets aberrant transcripts for decay and also directly influences the abundance of non-aberrant transcripts. In animals, the SMG1 kinase plays an essential role in NMD by phosphorylating the core NMD factor UPF1. Despite SMG1 being ubiquitous throughout the plant kingdom, little is known about its function, probably because SMG1 is atypically absent from the genome of the model plant, Arabidopsis thaliana. By combining our previously established SMG1 knockout in moss with transcriptome-wide analysis, we reveal the range of processes involving SMG1 in plants. Machine learning assisted analysis suggests that 32% of multi-isoform genes produce NMD-targeted transcripts and that splice junctions downstream of a stop codon act as the major determinant of NMD targeting. Furthermore, we suggest that SMG1 is involved in other quality control pathways, affecting DNA repair and the unfolded protein response, in addition to its role in mRNA quality control. Consistent with this, smg1 plants have increased susceptibility to DNA damage, but increased tolerance to unfolded protein inducing agents. The potential involvement of SMG1 in RNA, DNA and protein quality control has major implications for the study of these processes in plants.

Possible Role of MADS AFFECTING FLOWERING 3 and B-BOX DOMAIN PROTEIN 19 in Flowering Time Regulation of Arabidopsis Mutants with Defects in Nonsense-Mediated mRNA Decay.

Eukaryotic cells use nonsense-mediated mRNA decay (NMD) to clear aberrant mRNAs from the cell, thus preventing the accumulation of truncated proteins. In Arabidopsis, two UP-Frameshift (UPF) proteins, UPF1 and UPF3, play a critical role in NMD. Although deficiency of UPF1 and UPF3 leads to various developmental defects, little is known about the mechanism underlying the regulation of flowering time by NMD. Here, we showed that the upf1-5 and upf3-1 mutants had a late-flowering phenotype under long-day conditions and the upf1-5 upf3-1 double mutants had an additive effect in delaying flowering time. RNA sequencing of the upf mutants revealed that UPF3 exerted a stronger effect than UPF1 in the UPF-mediated regulation of flowering time. Among genes known to regulate flowering time, FLOWERING LOCUS C (FLC) mRNA levels increased (up to 8-fold) in upf mutants, as confirmed by qPCR. The upf1-5, upf3-1, and upf1-5 upf3-1 mutants responded to vernalization, suggesting a role of FLC in delayed flowering of upf mutants. Consistent with the high FLC transcript levels and delayed flowering in upf mutants, levels of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) mRNAs were reduced in the upf mutants. However, RNA-seq did not identify an aberrant FLC transcript containing a premature termination codon (PTC), suggesting that FLC is not a direct target in the regulation of flowering time by NMD. Among flowering time regulators that act in an FLC-dependent manner, we found that MAF3, NF-YA2, NF-YA5, and TAF14 showed increased transcript levels in upf mutants. We also found that BBX19 and ATC, which act in an FLC-independent manner, showed increased transcript levels in upf mutants. An aberrant transcript containing a PTC was identified from MAF3 and BBX19 and the levels of the aberrant transcripts increased in upf mutants. Taking these results together, we propose that the late-flowering phenotype of upf mutants is mediated by at least two different pathways, namely, by MAF3 in an FLC-dependent manner and by BBX19 in an FLC-independent manner.

Premature termination codon readthrough in human cells occurs in novel cytoplasmic foci and requires UPF proteins.

Nonsense-mutation-containing messenger ribonucleoprotein particles (mRNPs) transit through cytoplasmic foci called P-bodies before undergoing nonsense-mediated mRNA decay (NMD), a cytoplasmic mRNA surveillance mechanism. This study shows that the cytoskeleton modulates transport of nonsense-mutation-containing mRNPs to and from P-bodies. Impairing the integrity of cytoskeleton causes inhibition of NMD. The cytoskeleton thus plays a crucial role in NMD. Interestingly, disruption of actin filaments results in both inhibition of NMD and activation of premature termination codon (PTC) readthrough, while disruption of microtubules causes only NMD inhibition. Activation of PTC readthrough occurs concomitantly with the appearance of cytoplasmic foci containing UPF proteins and mRNAs with nonsense mutations but lacking the P-body marker DCP1a. These findings demonstrate that in human cells, PTC readthrough occurs in novel 'readthrough bodies' and requires the presence of UPF proteins.

Spermatogenesis Studies Reveal a Distinct Nonsense-Mediated mRNA Decay (NMD) Mechanism for mRNAs with Long 3'UTRs.

Spermatogenesis Studies Reveal a Distinct Nonsense-Mediated mRNA Decay (NMD) Mechanism for mRNAs with Long 3'UTRs.

Physiological and pathophysiological role of nonsense-mediated mRNA decay.

Nonsense-mediated messenger RNA (mRNA) decay (NMD) is a quality control mechanism that degrades irregular or faulty mRNAs. NMD mainly degrades mRNAs, which contain a premature termination codon (PTC) and therefore encode a truncated protein. Furthermore, NMD alters the expression of different types of cellular mRNAs, the so-called endogenous NMD substrates. In this review, we focus on the impact of NMD on cellular and molecular physiology. We specify key classes of NMD substrates and provide a detailed overview of the physiological function of gene regulation by NMD. We also describe different mechanisms of NMD substrate degradation and how the regulation of the NMD machinery affects cellular physiology. Finally, we outline the physiological functions of central NMD factors.

Nonsense-mediated mRNA decay: an intricate machinery that shapes transcriptomes

Nonsense-mediated mRNA decay (NMD) is probably the best characterized eukaryotic RNA degradation pathway. Through intricate steps, a set of NMD factors recognize and degrade mRNAs with translation termination codons that are positioned in abnormal contexts. However, NMD is not only part of a general cellular quality control system that prevents the production of aberrant proteins. Mammalian cells also depend on NMD to dynamically adjust their transcriptomes and their proteomes to varying physiological conditions. In this Review, we discuss how NMD targets mRNAs, the types of mRNAs that are targeted, and the roles of NMD in cellular stress, differentiation and maturation processes.

Mechanism, factors, and physiological role of nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a translation-dependent, multistep process that degrades irregular or faulty messenger RNAs (mRNAs). NMD mainly targets mRNAs with a truncated open reading frame (ORF) due to premature termination codons (PTCs). In addition, NMD also regulates the expression of different types of endogenous mRNA substrates. A multitude of factors are involved in the tight regulation of the NMD mechanism. In this review, we focus on the molecular mechanism of mammalian NMD. Based on the published data, we discuss the involvement of translation termination in NMD initiation. Furthermore, we provide a detailed overview of the core NMD machinery, as well as several peripheral NMD factors, and discuss their function. Finally, we present an overview of diseases associated with NMD factor mutations and summarize the current state of treatment for genetic disorders caused by nonsense mutations.

Abstract 2932: SF3B1 mutations induce aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition

Abstract Recurrent heterozygous mutations of the spliceosome protein SF3B1 have been identified in myelodysplastic syndromes, chronic lymphocytic leukemia (CLL), breast, pancreatic and skin cancers. SF3B1 is a component of the U2 snRNP complex which binds to the pre-mRNA branch point site and is involved in recognition and stabilization of the spliceosome at the 3′ splice site. To understand the impact of SF3B1 mutations, we compared RNAseq profiles from tumor samples with SF3B1 hotspot mutations (SF3B1-MUT) or wild-type SF3B1 (SF3B1-WT) in breast cancer, melanoma and CLL. This analysis revealed significant increases in the usage of novel alternative splice junctions in SF3B1-MUT samples including selection of alternative 3′ splice sites and less frequently exon skipping. These events induce expression of alternative mRNAs that are translated into novel proteins or aberrant mRNAs that are decayed by cells. A common alternative splicing profile was shared across different hotspot mutations and lineages (e.g. ZDHHC16 and COASY); however, unique alternative splicing profiles were also observed suggesting lineage specific effects. RNAseq analysis of several cell lines with endogenous SF3B1 hotspot mutations confirmed the presence of the same spliced isoforms as observed in tumor samples. To prove that SF3B1-MUT were inducing alternative splicing, transient transfection of several SF3B1 hotspot mutations in 293FT cells induced the expression of the common alternatively spliced genes suggesting functional similarity. Selective shRNA depletion of mutant SF3B1 allele in SF3B1-MUT cells resulted in downregulation of the same splice isoforms. Furthermore, isogenic B-cell lines (NALM-6) expressing the most frequent SF3B1 mutation (K700E) were generated and profiled by RNAseq. As expected, similar alternatively spliced genes were observed in NALM-6 SF3B1-K700E cells exclusively. To investigate the role of nonsense-mediated mRNA decay (NMD) in eliminating aberrant mRNAs induced by SF3B1-MUT, we treated NALM-6 SF3B1-K700E cells with cycloheximide, a translation inhibitor known to inhibit NMD. In the treated samples, expression of several aberrant mRNAs was revealed and some of these transcripts were shown to be downregulated in patient samples. Taken together, these results confirm the association between different SF3B1 hotspot mutations and the presence of novel splice isoforms. We demonstrated that E7107, a potent and selective inhibitor of wild-type SF3B1, also binds and inhibits SF3B1-MUT protein. In addition, E7107 represses the expression of several common aberrant splice mRNA products in SF3B1-MUT cells in vitro and in vivo. When tested in a NALM-6 mouse model, E7107 induced tumor regression and increased the overall survival of animals implanted with NALM-6 SF3B1-K700E cells. These data suggest splicing inhibitors as a promising therapeutic approach for cancer patients carrying SF3B1 mutations. Citation Format: Silvia Buonamici, Kian Huat Lim, Jacob Feala, Eunice Park, Laura Corson, Michelle Aicher, Daniel Aird, Betty Chan, Erik Corcoran, Rachel Darman, Peter Fekkes, Gregg Keaney, Pavan Kumar, Kaiko Kunii, Linda Lee, Xiaoling Puyang, Jose Rodrigues, Anand Selvaraj, Michael Thomas, John Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Peter Smith, Yoshiharu Mizui. SF3B1 mutations induce aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition. [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 2932. doi:10.1158/1538-7445.AM2014-2932

Structures of SMG1-UPFs Complexes: SMG1 Contributes to Regulate UPF2-Dependent Activation of UPF1 in NMD

SMG1, a PI3K-related kinase, plays a critical role in nonsense-mediated mRNA decay (NMD) in mammals. SMG1-mediated phosphorylation of the UPF1 helicase is an essential step during NMD initiation. Both SMG1 and UPF1 are presumably activated by UPF2, but this regulation is incompletely understood. Here we reveal that SMG1C (a complex containing SMG1, SMG8, and SMG9) contributes to regulate NMD by recruiting UPF1 and UPF2 to distinct sites in the vicinity of the kinase domain. UPF2 binds SMG1 in an UPF1-independent manner invivo, and the SMG1C-UPF2 structure shows UPF2 recognizes the FRB domain, a region that regulates the related mTOR kinase. The molecular architectures of several SMG1C-UPFs complexes, obtained by combining electron microscopy with invivo and invitro interaction analyses, competition experiments, and mutations, suggest that UPF2 can be transferred to UPF1 within SMG1C, inducing UPF2-dependent conformational changes required to activate UPF1 within an SMG1C-UPF1-UPF2 complex.

Phosphorylation of the N‐ and C‐terminal UPF1 domains plays a critical role in plant nonsense‐mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is an essential quality control system that degrades aberrant transcripts containing premature termination codons and regulates the expression of several normal transcripts. Targets for NMD are selected during translational termination. If termination is slow, the UPF1 NMD factor binds the eRF3 protein of the termination complex and then recruits UPF2 and UPF3. Consequently, the UPF1-2-3 NMD complex induces SMG7-mediated degradation of the target mRNA. It is unknown how formation of the NMD complex and transcript degradation are linked in plants. Previously we have shown that the N- and C-terminal domains of UPF1 act redundantly and that the N-terminal domain is phosphorylated. To clarify the role of UPF1 phosphorylation in plant NMD, we generated UPF1 mutants and analyzed their phosphorylation status and the NMD competency of the mutants. We show that although several residues in the N-terminal domain of UPF1 are phosphorylated, only three phosphorylated amino acids, S3, S13 and T29, play a role in NMD. Moreover, we found that the C-terminal domain consists of redundant S/TQ-rich segments and that S1076 is involved in NMD. All NMD-relevant phosphorylation sites were in the S/TQ context. Co-localization and fluorescence resonance energy transfer-fluorescence lifetime imaging assays suggest that N-terminal and probably also C-terminal phosphorylated S/TQ residues are the binding platform for SMG7. Our data support the hypothesis that phosphorylation of UPF1 connects NMD complex formation and the SMG7-mediated target transcript degradation steps of NMD. SMG7 binds the phosphorylated S/TQ sites of the UPF1 component of the NMD complex, and then it induces the degradation of the NMD target.