The SMG5–SMG7 heterodimer directly recruits the CCR4–NOT deadenylase complex to mRNAs containing nonsense codons via interaction with POP2

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control mechanism that detects aberrant mRNAs containing nonsense codons and induces their rapid degradation. This degradation is mediated by SMG6, an NMD-specific endonuclease, as well as the SMG5 and SMG7 proteins, which recruit general mRNA decay enzymes. However, it remains unknown which specific decay factors are recruited and whether this recruitment is direct. Here, we show that SMG7 binds directly to POP2, a catalytic subunit of the CCR4–NOT deadenylase complex, and elicits deadenylation-dependent decapping and 5′-to-3′ decay of NMD targets. Accordingly, a catalytically inactive POP2 mutant partially suppresses NMD in human cells. The SMG7–POP2 interaction is critical for NMD in cells depleted of SMG6, indicating that SMG7 and SMG6 act redundantly to promote the degradation of NMD targets. We further show that UPF1 provides multiple binding sites for decapping factors. These data unveil a missing direct physical link between NMD and the general mRNA decay machinery and indicate that NMD employs diverse and partially redundant mechanisms to ensure robust degradation of aberrant mRNAs.     

An unusual arrangement of two 14-3-3-like domains in the SMG5–SMG7 heterodimer is required for efficient nonsense-mediated mRNA decay

The nonsense-mediated mRNA decay (NMD) pathway triggers the rapid degradation of aberrant mRNAs containing premature translation termination codons (PTCs). In metazoans, NMD requires three 14-3-3-like proteins: SMG5, SMG6, and SMG7. These proteins are recruited to PTC-containing mRNAs through the interaction of their 14-3-3-like domains with phosphorylated UPF1, the central NMD effector. Recruitment of SMG5, SMG6, and SMG7 causes NMD target degradation. In this study, we report the crystal structure of the Caenorhabditis elegans SMG5–SMG7 complex. The 14-3-3-like phosphopeptide recognition domains of SMG5 and SMG7 heterodimerize in an unusual perpendicular back-to-back orientation in which the peptide-binding sites face opposite directions. Structure-based mutants and functional assays indicate that the SMG5–SMG7 interaction is conserved and is crucial for efficient NMD in human cells. Notably, we demonstrate that heterodimerization increases the affinity of the SMG5–SMG7 complex for UPF1. Furthermore, we show that the degradative activity of the SMG5–SMG7 complex resides in SMG7 and that the SMG5–SMG7 complex and SMG6 play partially redundant roles in the degradation of aberrant mRNAs. We propose that the SMG5–SMG7 complex binds to phosphorylated UPF1 with high affinity and recruits decay factors to the mRNA target through SMG7, thus promoting target degradation.     

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.     

Role of SMG‐1‐mediated Upf1 phosphorylation in mammalian nonsense‐mediated mRNA decay

SMG‐1, a member of the PIKK (phosphoinositide 3‐kinase‐related kinase) family, plays a critical role in the mRNA quality control system known as nonsense‐mediated mRNA decay (NMD). NMD protects cells from the accumulation of aberrant mRNAs with premature termination codons (PTCs) which encode nonfunctional or potentially harmful truncated proteins. SMG‐1 directly phosphorylates Upf1 helicase, another key component of NMD, upon recognition of PTC on postspliced mRNA during the initial round of translation. Phosphorylated‐Upf1 recruits the SMG‐5/SMG‐7 complex to induce ribosome dissociation and decapping‐mediated decay. Phospho‐Upf1 also recruits the SMG‐6 endonuclease which might be involved in endo‐cleavage. Upf1 ATPase/helicase activities are likely required for the activation of other mRNA decay enzymes and the mRNA‐protein complex dissociation to complete NMD. At present, a variety of tools are available that can specifically suppress NMD, and it has become possible to examine the contribution of NMD in a variety of physiological and pathological conditions.     

The pioneer translation initiation complex is functionally distinct from but structurally overlaps with the steady-state translation initiation complex

The bulk of cellular proteins derive from the translation of eukaryotic translation initiation factor (eIF)4E-bound mRNA. However, recent studies of nonsense-mediated mRNA decay (NMD) indicate that cap-binding protein (CBP)80-bound mRNA, which is a precursor to eIF4E-bound mRNA, can also be translated during a pioneer round of translation. Here, we report that the pioneer round, which can be assessed by measuring NMD, is not inhibited by 4E-BP1, which is known to inhibit steady-state translation by competing with eIF4G for binding to eIF4E. Therefore, at least in this way, the pioneer round of translation is distinct from steady-state translation. eIF4GI, poly(A)-binding protein (PABP)1, eIF3, eIF4AI, and eIF2α coimmunopurify with both CBP80 and eIF4E, which suggests that each factor functions in both modes of translation. Consistent with roles for PABP1 and eIF2α in the pioneer round of translation, PABP-interacting protein 2, which is known to destabilize PABP1 binding to poly(A) and inhibit steady-state translation, as well as inactive eIF2α, which is also known to inhibit steady-state translation, also inhibit NMD. Polysome profiles indicate that CBP80-bound mRNAs are translated less efficiently than their eIF4E-bound counterparts.     

AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation

AU-rich elements (AREs, usually containing repeated copies of AUUUA), when present in the 3′-untranslated regions (UTRs) of many mammalian mRNAs, confer instability on their host RNA molecules. The viral small nuclear RNA (snRNA) Herpesvirus saimiri U RNA 1 (HSUR 1) also contains an AUUUA-rich sequence. Here, we report that this ARE induces rapid degradation of HSUR 1 itself and of other snRNAs including HSUR 2 and cellular U1. Mutational analyses of the viral ARE establish that sequence requirements for mRNA and snRNA decay are the same, suggesting a similar mechanism. Moreover, the in vivo degradation activity of mutant AREs correlates with their in vitro binding to the HuR protein, implicated previously as a component of the mRNA degradation machinery. Our results suggest that ARE-mediated instability can be uncoupled from both ongoing translation and deadenylation of the target RNA.     

Role of RNA surveillance proteins Upf1/CpaR,Upf2 and Upf3 in the translational regulation of yeast CPA1 gene

Gene CPA1, encoding one of the subunits of carbamoylphosphate synthetase (CPSase A) is subject to a translational control by arginine of which the essential element is a 25 amino acid peptide encoded by the CPA1 messenger. The peptide is the product of an open reading frame located upstream (uORF) of the coding phase of the gene, within a 250 nucleotide leader. In the past, a series of mutations impairing the repression of gene CPA1 by arginine had been selected in vivo. Most of the mutations were located in the CPA1 uORF, but mutations unlinked to the CPA1 gene were also isolated and mapped in a gene called CPAR. In this work, we show that the CPAR gene is identical to the UPF1 gene, encoding a protein responsible for the premature termination step of RNA surveillance by nonsense-mediated mRNA decay (NMD). Deletion of UPF1, or deletion of UPF2 and UPF3, the other genes involved in the NMD pathway, enhances the synthesis of CPSase A, whether arginine is present or not in the growth medium. The regulatory effect of the NMD protein complex is only observed when the uORF is present in the CPA1 messenger, indicating that the arginine-peptide repression mechanism and the RNA surveillance pathway are complementary mechanisms. Our results indicate that the NMD destabilizes the 5' end of the CPA1 message and this decay is strongly enhanced when arginine is present in the growth medium.     

SMG-8 and SMG-9, two novel subunits of the SMG-1 complex,regulate remodeling of the mRNA surveillance complex during nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature translation termination codons (PTCs). SMG-1 and Upf1 transiently form a surveillance complex termed “SURF” that includes eRF1 and eRF3 on post-spliced mRNAs during recognition of PTC. If an exon junction complex (EJC) exists downstream from the SURF complex, SMG-1 phosphorylates Upf1, the step that is a rate-limiting for NMD. We provide evidence of an association between the SURF complex and the ribosome in association with mRNPs, and we suggest that the SURF complex functions as a translation termination complex during NMD. We identified SMG-8 and SMG-9 as novel subunits of the SMG-1 complex. SMG-8 and SMG-9 suppress SMG-1 kinase activity in the isolated SMG-1 complex and are involved in NMD in both mammals and nematodes. SMG-8 recruits SMG-1 to the mRNA surveillance complex, and inactivation of SMG-8 induces accumulation of a ribosome:Upf1:eRF1:eRF3:EJC complex on mRNP, which physically bridges the ribosome and EJC through eRF1, eRF3, and Upf1. These results not only reveal the regulatory mechanism of SMG-1 kinase but also reveal the sequential remodeling of the ribosome:SURF complex to the predicted DECID (DECay InDucing) complex, a ribosome:SURF:EJC complex, as a mechanism of in vivo PTC discrimination.     

Copper tolerance of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> nonsense-mediated mRNA decay mutants

The eukaryotic nonsense-mediated mRNA (NMD) is a specialized pathway that leads to the recognition and rapid degradation of mRNAs with premature termination codons, and importantly some natural mRNAs as well. Natural mRNAs with atypically long 3′-untranslated regions (UTRs) are degraded by NMD in Saccharomyces cerevisiae. A number of S. cerevisiae mRNAs undergo alternative 3′-end processing producing mRNA isoforms that differ in their 3′-UTR lengths. Some of these alternatively 3′-end processed mRNA isoforms have atypically long 3′-UTRs and would be likely targets for NMD-mediated degradation. Here, we investigated the role NMD plays in the regulation of expression of CTR2, which encodes a vacuolar membrane copper transporter. CTR2 pre-mRNA undergoes alternative 3′-end processing to produce two mRNA isoforms with 300-nt and 2-kb 3′-UTRs. We show that both CTR2 mRNA isoforms are differentially regulated by NMD. The regulation of CTR2 mRNA by NMD has physiological consequences, since nmd mutants are more tolerant to toxic levels of copper relative to wild-type yeast cells and the copper tolerance of nmd mutants is dependent on the presence of CTR2.