SMG7 is a 14-3-3-like adaptor in the nonsense-mediated mRNA decay pathway

In metazoa, regulation of the phosphorylation state of UPF1 is crucial for nonsense-mediated mRNA decay (NMD), a process by which aberrant mRNAs containing nonsense mutations are degraded. UPF1 is targeted for dephosphorylation by three related proteins, SMG5, SMG6, and SMG7. We report here the crystal structure of the N-terminal domain of SMG7. The structure reveals that SMG7 contains a 14-3-3-like domain. Residues that bind phosphoserine-containing peptides in 14-3-3 are conserved at the equivalent positions in SMG7. Mutation of these residues impairs UPF1 binding to SMG7 in vitro and UPF1 recruitment to cytoplasmic mRNA decay foci in vivo, suggesting that SMG7 acts as an adaptor in targeting mRNAs associated with phosphorylated UPF1 for degradation. The 14-3-3 site of SMG7 is conserved in SMG5 and SMG6. These data also imply that the homologous human Est1 might have a 14-3-3 function at telomeres, and that phosphorylation events may be important for telomerase regulation.     

Phospho-dependent and phospho-independent interactions of the helicase UPF1 with the NMD factors SMG5–SMG7 and SMG6

Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes mRNAs with premature stop codons and targets them for rapid degradation. Evidence from previous studies has converged on UPF1 as the central NMD factor. In human cells, the SMG1 kinase phosphorylates UPF1 at the N-terminal and C-terminal tails, in turn allowing the recruitment of the NMD factors SMG5, SMG6 and SMG7. To understand the molecular mechanisms, we recapitulated these steps of NMD in vitro using purified components. We find that a short C-terminal segment of phosphorylated UPF1 containing the last two Ser-Gln motifs is recognized by the heterodimer of SMG5 and SMG7 14–3–3-like proteins. In contrast, the SMG6 14–3–3-like domain is a monomer. The crystal structure indicates that the phosphoserine binding site of the SMG6 14–3–3-like domain is similar to that of SMG5 and can mediate a weak phospho-dependent interaction with UPF1. The dominant SMG6–UPF1 interaction is mediated by a low-complexity region bordering the 14–3–3-like domain of SMG6 and by the helicase domain and C-terminal tail of UPF1. This interaction is phosphorylation independent. Our study demonstrates that SMG5–SMG7 and SMG6 exhibit different and non-overlapping modes of UPF1 recognition, thus pointing at distinguished roles in integrating the complex NMD interaction network.     

A novel phosphorylation-independent interaction between SMG6 and UPF1 is essential for human NMD

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD substrates can be degraded by different routes that all require phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of the three known NMD factors thought to be recruited to nonsense mRNAs via an interaction with P-UPF1, leading to eventual mRNA degradation. By artificial tethering of SMG6 and mutants thereof to a reporter mRNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 to reduce reporter mRNA levels. Using in vivo and in vitro approaches, we further document that SMG6 and the unique stalk region of the UPF1 helicase domain, along with a contribution from the SQ domain, form a novel interaction and we also show that this region of the UPF1 helicase domain is critical for SMG6 function and NMD. Our results show that this interaction is required for NMD and for the capability of tethered SMG6 to degrade its bound RNA, suggesting that it contributes to the intricate regulation of UPF1 and SMG6 enzymatic activities.     

Processing bodies are not required for mammalian nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality-control mechanism that recognizes and degrades mRNAs with premature termination codons (PTCs). In yeast, PTC-containing mRNAs are targeted to processing bodies (P-bodies), and yeast strains expressing an ATPase defective Upf1p mutant accumulate P-bodies. Here we show that in human cells, an ATPase-deficient UPF1 mutant and a fraction of UPF2 and UPF3b accumulate in cytoplasmic foci that co-localize with P-bodies. Depletion of the P-body component Ge-1, which prevents formation of microscopically detectable P-bodies, also impairs the localization of mutant UPF1, UPF2, and UPF3b in cytoplasmic foci. However, the accumulation of the ATPase-deficient UPF1 mutant in P-bodies is independent of UPF2, UPF3b, or SMG1, and the ATPase-deficient UPF1 mutant can localize into the P-bodies independent of its phosphorylation status. Most importantly, disruption of P-bodies by depletion of Ge-1 affects neither the mRNA levels of PTC-containing reporter genes nor endogenous NMD substrates. Consistent with the recently reported decapping-independent SMG6-mediated endonucleolytic decay of human nonsense mRNAs, our results imply that microscopically detectable P-bodies are not required for mammalian NMD.     

Nonsense-mediated mRNA decay in Drosophila: at the intersection of the yeast and mammalian pathways

The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature stop codons (PTCs). In Caenorhabditis elegans, seven genes (smg1-7) playing an essential role in NMD have been identified. Only SMG2-4 (known as UPF1-3) have orthologs in Saccharomyces cerevisiae. Here we show that the Drosophila orthologs of UPF1-3, SMG1, SMG5 and SMG6 are required for the degradation of PTC-containing mRNAs, but that there is no SMG7 ortholog in this organism. In contrast, orthologs of SMG5-7 are encoded by the human genome and all three are required for NMD. In human cells, exon boundaries have been shown to play a critical role in defining PTCs. This role is mediated by components of the exon junction complex (EJC). Contrary to expectation, however, we show that the components of the EJC are dispensable for NMD in Drosophila cells. Consistently, PTC definition occurs independently of exon boundaries in DROSOPHILA: Our findings reveal that despite conservation of the NMD machinery, different mechanisms have evolved to discriminate premature from natural stop codons in metazoa.     

SMG1-UPF1-eRF1-eRF3复合体参与贾第虫无义介导的mRNA降解激活

无义介导的mRNA降解（NMD）是一种重要的真核生物mRNA质量监控途径。NMD可识别并降解含有提前终止密码子（PTC）的异常mRNA（PTC-mRNA）。但NMD途径对PTC-mRNA的识别和降解机制尚无阐明。蓝氏贾第虫（Giardia lamblia）是一种寄生性的原生动物,进化上处于真核生物基部,对其NMD途径的研究有利于了解NMD途径的机制与进化。本研究通过双分子荧光互补实验、酵母双杂交实验和体外pull-down实验,分析了贾第虫的UPF1 (GlUPF1)、SMG1 (GlSMG1)和肽链释放因子（GleRF1、GleRF3）之间的相互作用关系。结果表明,贾第虫的肽链释放因子都能够与GlUPF1发生相互作用,且GlUPF1的CH结构域与GleRF3能够形成较稳定的复合体,而GlSMG1的激酶结构域PIKK能与UPF1的C端和N端结构域相互作用。进一步研究证实,GlSMG1的PIKK结构域能使GlUPF1两种截短体GlUPF1（1~500 aa）和GlUPF1（501~1 304 aa）发生磷酸化修饰,说明GlUPF1 的N端和C端均有GlSMG1的磷酸化位点。进一步分析证实,T111是GlUPF1上的1个磷酸化位点。我们的研究结果表明,贾第虫NMD途径起始阶段,首先在mRNA的PTC处的核糖体上形成SMG1-UPF1-eRF1-eRF3(SURF)复合体,并且GlSMG1磷酸化修饰GlUPF1,由此激活NMD途径,可能招募XRN1和SKI7d等酶参与无义mRNA的降解。     

CK2-mediated TEL2 phosphorylation augments nonsense-mediated mRNA decay (NMD) by increase of SMG1 stability

Nonsense-mediated mRNA decay (NMD) is the best-characterized mRNA surveillance mechanism that degrades a premature-termination codon (PTC)-containing mRNA. During mammalian NMD, SMG1 and UPF1, key proteins in NMD, join at a PTC and form an SMG1–UPF1–eRF1–eRF3 (SURF) complex by binding UPF1 to eRF3 after PTC-recognition by the translating ribosome. Subsequently, UPF1 is phosphorylated after UPF1–SMG1 moves onto the downstream exon junction complex (EJC). However, the cellular events that induce UPF1 and SMG1 complex formation and increase NMD efficiency before PTC recognition remain unclear. Here, we show that telomere-maintenance 2 (TEL2) phosphorylation by casein-kinase 2 (CK2) increases SMG1 stability, which increases UPF1 phosphorylation and, ultimately, augments NMD. Inhibition of CK2 activity or downregulation of TEL2 impairs NMD. Intriguingly, loss of TEL2 phosphorylation reduces UPF1-bound PTC-containing mRNA and the formation of the SMG1–UPF1 complex. Thus, our results identify a new function of CK2-mediated TEL2 phosphorylation in a mammalian NMD.     

SMG1 regulates adipogenesis via targeting of staufen1-mediated mRNA decay

Suppressor of morphogenesis in genitalia 1 (SMG1), a member of the phosphatidylinositol 3-kinase-related kinase family, is involved in nonsense-mediated mRNA decay (NMD). SMG1 phosphorylates Upf1, a key NMD factor. Subsequently, hyperphosphorylated Upf1 associates with SMG5-7 or proline-rich nuclear receptor coregulatory protein (PNRC2) to elicit rapid mRNA degradation. Upf1 is also known to be involved in staufen 1 (Stau1)-mediated mRNA decay (SMD), which is closely related to NMD. However, the biological and molecular roles of SMG1 in SMD remain unknown. Here, we provide evidence that SMG1 is involved in SMD. The immunoprecipitation results show that SMG1 is complexed with Stau1, Upf1, and Dcp1a. Downregulation of SMG1 or overexpression of a kinase-inactive mutant of SMG1 inhibits SMD efficiency. In addition, downregulation of SMG1 inhibits rapid degradation elicited by artificially tethered Stau1 or Upf1 downstream of the normal termination codon. Furthermore, Stau1 and Upf1 colocalize in processing bodies in an SMG1-dependent manner. We also find that the level of SMG1 increases during adipogenesis. Accordingly, downregulation of SMG1 causes the reduction in the level of Upf1 phosphorylation and delays adipogenesis, suggesting the functional involvement of SMG1 in adipogenesis via SMD.     

Structures of the PIN domains of SMG6 and SMG5 reveal a nuclease within the mRNA surveillance complex

SMG6 and SMG5 are essential factors in nonsense-mediated mRNA decay, a conserved pathway that degrades mRNAs with premature translation termination codons. Both SMG5 and SMG6 have been predicted to contain a C-terminal PIN (PilT N-terminus) domain, present in proteins with ribonuclease activity. We have determined the structures of human SMG5 and SMG6 PIN domains. Although they share a similar overall fold related to ribonucleases of the RNase H family, they have local differences at the putative active site. SMG6 has the canonical triad of acidic residues that are crucial in RNase H for nuclease activity, while SMG5 lacks key catalytic residues. The structural differences are reflected at the functional level. Only the PIN domain of SMG6 has degradation activity on single-stranded RNA in vitro. This difference in catalytic activity is conserved in Drosophila, where an SMG6 with an inactive PIN domain inhibits NMD in a dominant-negative manner. Our findings suggest that the NMD machinery has intrinsic nuclease activity that is likely to contribute to the rapid decay of mRNAs that terminate translation prematurely.     

Role for Upf2p phosphorylation in Saccharomyces cerevisiae nonsense-mediated mRNA decay

Premature termination (nonsense) codons trigger rapid mRNA decay by the nonsense-mediated mRNA decay (NMD) pathway. Two conserved proteins essential for NMD, UPF1 and UPF2, are phosphorylated in higher eukaryotes. The phosphorylation and dephosphorylation of UPF1 appear to be crucial for NMD, as blockade of either event in Caenorhabditis elegans and mammals largely prevents NMD. The universality of this phosphorylation/dephosphorylation cycle pathway has been questioned, however, because the well-studied Saccharomyces cerevisiae NMD pathway has not been shown to be regulated by phosphorylation. Here, we used in vitro and in vivo biochemical techniques to show that both S. cerevisiae Upf1p and Upf2p are phosphoproteins. We provide evidence that the phosphorylation of the N-terminal region of Upf2p is crucial for its interaction with Hrp1p, an RNA-binding protein that we previously showed is essential for NMD. We identify specific amino acids in Upf2p's N-terminal domain, including phosphorylated serines, which dictate both its interaction with Hrp1p and its ability to elicit NMD. Our results indicate that phosphorylation of UPF1 and UPF2 is a conserved event in eukaryotes and for the first time provide evidence that Upf2p phosphorylation is crucial for NMD.     

Interactions between UPF1, eRFs, PABP and the exon junction complex suggest an integrated model for mammalian NMD pathways

Nonsense-mediated mRNA decay (NMD) represents a key mechanism to control the expression of wild-type and aberrant mRNAs. Phosphorylation of the protein UPF1 in the context of translation termination contributes to committing mRNAs to NMD. We report that translation termination is inhibited by UPF1 and stimulated by cytoplasmic poly(A)-binding protein (PABPC1). UPF1 binds to eRF1 and to the GTPase domain of eRF3 both in its GTP- and GDP-bound states. Importantly, mutation studies show that UPF1 can interact with the exon junction complex (EJC) alternatively through either UPF2 or UPF3b to become phosphorylated and to activate NMD. On this basis, we discuss an integrated model where UPF1 halts translation termination and is phosphorylated by SMG1 if the termination-promoting interaction of PABPC1 with eRF3 cannot readily occur. The EJC, with UPF2 or UPF3b as a cofactor, interferes with physiological termination through UPF1. This model integrates previously competing models of NMD and suggests a mechanistic basis for alternative NMD pathways.