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.     

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.     

Unexpected roles for UPF1 in HIV-1 RNA metabolism and translation

The HIV-1 ribonucleoprotein (RNP) contains the major structural protein, pr55(Gag), viral genomic RNA, as well as the host protein, Staufen1. In this report, we show that the nonsense-mediated decay (NMD) factor UPF1 is also a component of the HIV-1 RNP. We investigated the role of UPF1 in HIV-1-expressing cells. Depletion of UPF1 by siRNA resulted in a dramatic reduction in steady-state HIV-1 RNA and pr55(Gag). Pr55(Gag) synthesis, but not the cognate genomic RNA, was efficiently rescued by expression of an siRNA-insensitive UPF1, demonstrating that UPF1 positively influences HIV-1 RNA translatability. Conversely, overexpression of UPF1 led to a dramatic up-regulation of HIV-1 expression at the RNA and protein synthesis levels. The effects of UPF1 on HIV-1 RNA stability were observed in the nucleus and cytoplasm and required ongoing translation. We also demonstrate that the effects exerted by UPF1 on HIV-1 expression were dependent on its ATPase activity, but were separable from its role in NMD and did not require interaction with UPF2.     

A new function for nonsense-mediated mRNA-decay factors

mRNAs often contain premature-termination (nonsense) codons as a result of mutations and RNA splicing errors. These nonsense codons cause rapid decay of the mRNAs that contain them, a phenomenon called nonsense-mediated mRNA decay (NMD). This response is thought to be a quality-control mechanism that protects cells from truncated dominant-negative proteins. Surprisingly, recent evidence strongly suggests that the NMD factors UPF1, UPF2, UPF3B, RNPS1, Y14 and MAGOH also promote translation of normal mRNAs in mammalian cells. This, along with an earlier discovery that NMD factors appear to dictate efficient translation termination, suggests that NMD factors do not merely function in RNA surveillance. These findings lead to the interesting question of why NMD factors evolved; are they for RNA-quality control or to promote efficient translation initiation and termination?     

Purification and characterization of the Upf1 protein: a factor involved in translation and mRNA degradation.

mRNA degradation is an important control point in the regulation of gene expression and has been shown to be linked to the process of translation. One clear example of this linkage is the observation that nonsense mutations in a gene can accelerate the decay of the corresponding mRNA. In the yeast Saccharomyces cerevisiae, the product of the UPF1 gene, harboring zinc finger, NTP hydrolysis, and helicase motifs, was shown to be a trans-acting factor in this decay pathway. A UPF1 gene disruption results in stabilization of nonsense-containing mRNAs and leads to a nonsense suppression phenotype. As a first step toward understanding the molecular and biochemical mechanism of nonsense-mediated mRNA decay, we have purified Upf1p from a yeast extract and characterized its nucleic acid-dependent NTPase activity, helicase activity, and nucleic acid binding properties. The results presented in this paper demonstrate that Upf1p contains both RNA- and DNA-dependent ATPase activities and RNA and DNA helicase activities. In the absence of ATP, Upf1p binds to single-stranded RNA or DNA, whereas hydrolysis of ATP facilitates its release from single-stranded nucleic acid. Based on these results, the role of Upf1p's biochemical activities in mRNA decay and translation are discussed.     

The majority of yeast UPF1 co-localizes with polyribosomes in the cytoplasm.

In Saccharomyces cerevisiae the UPF1 protein is required for nonsense-mediated mRNA decay, the accelerated turnover of mRNAs containing a nonsense mutation. Several lines of evidence suggest that translation plays an important role in the mechanism of nonsense mRNA decay, including a previous report that nonsense mRNAs assemble in polyribosomes. In this study we show that UPF1 and ribosomal protein L1 co-localize in the cytoplasm and that UPF1 co-sediments with polyribosomes. To detect UPF1, three copies of the influenza hemagglutinin epitope were placed at the C-terminus. The tagged protein, UPF1-3EP, retains 86% (+/- 5%) of function. Using immunological detection, we found that UPF1-3EP is primarily cytoplasmic and was not detected either in the nucleus or in the mitochondrion. UPF1-3EP and L1 co-distributed with polyribosomes fractionated in a 7-47% sucrose gradient. The sucrose sedimentation profiles for UPF1-3EP and L1 exhibited similar changes using three different sets of conditions that altered the polyribosome profile. When polyribosomes were disaggregated, UPF1-3EP and L1 accumulated in fractions coincident with 80S ribosomal particles. These results suggest that UPF1-3EP associates with polyribosomes. L3 and S3 mRNAs, which code for ribosomal proteins of the 60S and 40S ribosomal subunits, respectively, were on average about 100-fold more abundant than UPF1 mRNA. Assuming that translation rates for L3, S3, and UPF1 mRNA are similar, this result suggests that there are far fewer UPF1 molecules than ribosomes per cell. Constraints imposed by the low UPF1 abundance on the functional relationships between UPF1, polyribosomes, and nonsense mRNA turnover are discussed.     

Crystal structure of the UPF2-interacting domain of nonsense-mediated mRNA decay factor UPF1

UPF1 is an essential eukaryotic RNA helicase that plays a key role in various mRNA degradation pathways, notably nonsense-mediated mRNA decay (NMD). In combination with UPF2 and UPF3, it forms part of the surveillance complex that detects mRNAs containing premature stop codons and triggers their degradation in all organisms studied from yeast to human. We describe the 3 A resolution crystal structure of the highly conserved cysteine-histidine-rich domain of human UPF1 and show that it is a unique combination of three zinc-binding motifs arranged into two tandem modules related to the RING-box and U-box domains of ubiquitin ligases. This UPF1 domain interacts with UPF2, and we identified by mutational analysis residues in two distinct conserved surface regions of UPF1 that mediate this interaction. UPF1 residues we identify as important for the interaction with UPF2 are not conserved in UPF1 homologs from certain unicellular parasites that also appear to lack UPF2 in their genomes.     

The Double Life of UPF1 in RNA and DNA Stability Pathways

The DNA and RNA helicase UPF1 is well known for its central role in Nonsense Mediated RNA Decay (NMD), which promotes degradation of mRNAs containing premature stop codons. However, we have recently demonstrated that human UPF1 is also essential for DNA replication and S phase progression. This function appears to be independent of NMD, which is not required for cell cycle progression. UPF1 physically interacts with the replicative DNA polymerase δ and it associates with chromatin during S phase and upon DNA damage in an ATR-dependent manner. Intriguingly, the human NMD kinase SMG1 is also involved in genome stability pathways and the human NMD-factor EST1A/SMG6 is telomerase-associated and has been implicated in telomere maintenance. Here we review the recent findings, which uncovered the direct roles of UPF1 and other NMD-factors in DNA replication and genome maintenance pathways and suggest functional connections between RNA and DNA metabolism.     

Identification and characterization of mutations in the UPF1 gene that affect nonsense suppression and the formation of the Upf protein complex but not mRNA turnover.

To understand the relationship between translation and mRNA decay, we have been studying how premature translation termination accelerates the degradation of mRNAs. In the yeast Saccharomyces cerevisiae, the Upf1 protein (Upf1p), which contains a cysteine- and histidine-rich region and nucleoside triphosphate hydrolysis and helicase motifs, was shown to be a trans-acting factor in this decay pathway. A UPF1 gene disruption results in the stabilization of nonsense-containing mRNAs and leads to a nonsense suppression phenotype. Biochemical analysis of the wild-type Upf1p demonstrated that it has RNA-dependent ATPase, RNA helicase, and RNA binding activities. In the work described in the accompanying paper (Y. Weng, K. Czaplinski, and S. W. Peltz, Mol. Cell. Biol. 16:5477-5490, 1996) mutations in the helicase region of Upf1p that inactivated its mRNA decay function but prevented suppression of leu2-2 and tyr7-1 nonsense alleles are identified. On the basis of these results, we suggested that Upf1p is a multifunctional protein involved in modulating mRNA decay and translation termination at nonsense codons. If this is true, we predict that UPF1 mutations with the converse phenotype should be identified. In this report, we describe the identification and biochemical characterization of mutations in the amino-terminal cysteine- and histidine-rich region of Upf1p that have normal nonsense-mediated mRNA decay activities but are able to suppress leu2-2 and tyr7-1 nonsense alleles. Biochemical characterization of these mutant proteins demonstrated that they have altered RNA binding properties. Furthermore, using the two-hybrid system, we characterized the Upf1p-Upf2p interactions and demonstrated that Upf2p interacts with Upf3p. Mutations in the cysteine- and histidine-rich region of Upf1p abolish Upf1p-Upf2p interaction. On the basis of these results, the role of the Upf complex in nonsense-mediated mRNA decay and nonsense suppression is discussed.     

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.     

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.     

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.