Phospho-dependent and phospho-independent interactions of the helicase UPF1 with the NMD factors SMG5-SMG7 and SMG6.
ABSTRACT: 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.
Project description: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.
Project description:In mammals, nonsense-mediated mRNA decay (NMD) functions in post-transcriptional gene regulation as well as mRNA surveillance. A key NMD factor, Upf1, becomes hyperphosphorylated by SMG1 kinase during the recognition of NMD substrates. Hyperphosphorylated Upf1 interacts with several factors including SMG5, SMG6, SMG7 and PNRC2 to trigger rapid mRNA degradation. However, the possible cross-talk among these factors and their selective use during NMD remain unknown. Here, we show that PNRC2 is preferentially complexed with SMG5, but not with SMG6 or SMG7, and that downregulation of PNRC2 abolishes the interaction between SMG5 and Dcp1a, a component of the decapping complex. In addition, tethering experiments reveal the function of Upf1, SMG5 and PNRC2 at the same step of NMD and the requirement of SMG6 for Upf1 for efficient mRNA degradation. Intriguingly, microarray results reveal the significant overlap of SMG5-dependent NMD substrates more with PNRC2-dependent NMD substrates than with SMG7-dependent NMD substrates, suggesting the functional dominance of SMG5-PNRC2, rather than SMG5-SMG7, under normal conditions. The results provide evidence that, to some extent, endogenous NMD substrates have their own binding preference for Upf1-interacting adaptors or effectors.
Project description:Nonsense-mediated mRNA decay (NMD) controls gene expression by eliminating mRNAs with premature or aberrant translation termination. Degradation of NMD substrates is initiated by the central NMD factor UPF1, which recruits the endonuclease SMG6 and the deadenylation-promoting SMG5/7 complex. The extent to which SMG5/7 and SMG6 contribute to the degradation of individual substrates and their regulation by UPF1 remains elusive. Here we map transcriptome-wide sites of SMG6-mediated endocleavage via 3' fragment capture and degradome sequencing. This reveals that endogenous transcripts can have NMD-eliciting features at various positions, including upstream open reading frames (uORFs), premature termination codons (PTCs), and long 3' UTRs. We find that NMD substrates with PTCs undergo constitutive SMG6-dependent endocleavage, rather than SMG7-dependent exonucleolytic decay. In contrast, the turnover of NMD substrates containing uORFs and long 3' UTRs involves both SMG6- and SMG7-dependent endo- and exonucleolytic decay, respectively. This suggests that the extent to which SMG6 and SMG7 degrade NMD substrates is determined by the mRNA architecture.
Project description:The term "nonsense-mediated mRNA decay" (NMD) originally described the degradation of mRNAs with premature translation-termination codons (PTCs), but its meaning has recently been extended to be a translation-dependent post-transcriptional regulator of gene expression affecting 3%-10% of all mRNAs. The degradation of NMD target mRNAs involves both exonucleolytic and endonucleolytic pathways in mammalian cells. While the latter is mediated by the endonuclease SMG6, the former pathway has been reported to require a complex of SMG5-SMG7 or SMG5-PNRC2 binding to UPF1. However, the existence, dominance, and mechanistic details of these exonucleolytic pathways are divisive. Therefore, we have investigated the possible exonucleolytic modes of mRNA decay in NMD by examining the roles of UPF1, SMG5, SMG7, and PNRC2 using a combination of functional assays and interaction mapping. Confirming previous work, we detected an interaction between SMG5 and SMG7 and also a functional need for this complex in NMD. In contrast, we found no evidence for the existence of a physical or functional interaction between SMG5 and PNRC2. Instead, we show that UPF1 interacts with PNRC2 and that it triggers 5'-3' exonucleolytic decay of reporter transcripts in tethering assays. PNRC2 interacts mainly with decapping factors and its knockdown does not affect the RNA levels of NMD reporters. We conclude that PNRC2 is probably an important mRNA decapping factor but that it does not appear to be required for NMD.
Project description:The nonsense-mediated mRNA decay (NMD) pathway promotes rapid degradation of mRNAs containing premature translation termination codons (PTCs or nonsense codons), preventing accumulation of potentially detrimental truncated proteins. In metazoa, seven genes (upf1, upf2, upf3, smg1, smg5, smg6, and smg7) have been identified as essential for NMD; here we show that the zebrafish genome encodes orthologs of upf1, upf2, smg1, and smg5 to smg7 and two upf3 paralogs. We also show that Upf1 is required for degradation of PTC-containing mRNAs in zebrafish embryos. Moreover, its depletion has a severe impact on embryonic development, early patterning, and viability. Similar phenotypes are observed in Upf2-, Smg5-, or Smg6-depleted embryos, suggesting that zebrafish embryogenesis requires an active NMD pathway. Using cultured cells, we demonstrate that the ability of a PTC to trigger NMD is strongly stimulated by downstream exon-exon boundaries. Thus, as in mammals and plants but in contrast to invertebrates and fungi, NMD is coupled to splicing in zebrafish. Our results together with previous studies show that NMD effectors are essential for vertebrate embryogenesis and suggest that the coupling of splicing and NMD has been maintained in vertebrates but lost in fungi and invertebrates.
Project description:The nonsense-mediated messenger RNA (mRNA) decay (NMD) pathway is a cellular quality control and post-transcriptional gene regulatory mechanism and is essential for viability in most multicellular organisms . A complex of proteins has been identified to be required for NMD function to occur; however, there is an incomplete understanding of the individual contributions of each of these factors to the NMD process. Central to the NMD process are three proteins, Upf1 (SMG-2), Upf2 (SMG-3), and Upf3 (SMG-4), which are found in all eukaryotes, with Upf1 and Upf2 being absolutely required for NMD in all organisms in which their functions have been examined. The other known NMD factors, Smg1, Smg5, Smg6, and Smg7, are more variable in their presence in different orders of organisms and are thought to have a more regulatory role. Here we present the first genetic analysis of the NMD factor Smg5 in Drosophila Surprisingly, we find that unlike the other analyzed Smg genes in this organism, Smg5 is essential for NMD activity. We found this is due in part to a requirement for Smg5 in both the activity of Smg6-dependent endonucleolytic cleavage, as well as an additional Smg6-independent mechanism. Redundancy between these degradation pathways explains why some Drosophila NMD genes are not required for all NMD-pathway activity. We also found that while the NMD component Smg1 has only a minimal role in Drosophila NMD during normal conditions, it becomes essential when NMD activity is compromised by partial loss of Smg5 function. Our findings suggest that not all NMD complex components are required for NMD function at all times, but instead are utilized in a context-dependent manner in vivo.
Project description:Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase upframeshift 1 (UPF1). Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for premature termination codon (PTC)-containing reporter mRNAs when compared with their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1)-binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD target marker. p-UPF1 is enriched on NMD target 3' untranslated regions (UTRs) along with suppressor with morphogenic effect on genitalia 5 (SMG5) and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with Förster resonance energy transfer (FRET) experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3' UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3' UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay.
Project description:Eukaryotic gene expression is constantly regulated and controlled by the translation-coupled nonsense-mediated mRNA decay (NMD) pathway. Aberrant translation termination leads to NMD activation and robust clearance of NMD targets via two seemingly independent and redundant mRNA degradation branches. Here, we uncover that the loss of the first SMG5-SMG7-dependent pathway also inactivates the second SMG6-dependent branch, indicating an unexpected functional hierarchy of the final NMD steps. Transcriptome-wide analyses of SMG5-SMG7-depleted cells confirm complete NMD inhibition resulting in massive transcriptomic alterations. The NMD activity conferred by SMG5-SMG7 is determined to varying degrees by their interaction with the central NMD factor UPF1, heterodimer formation and the initiation of deadenylation. Surprisingly, we find that SMG5 functionally substitutes SMG7 and vice versa. Our data support an improved model for NMD execution that features two-factor authentication involving UPF1 phosphorylation and SMG5-SMG7 recruitment to access SMG6 activity.
Project description:The Smg proteins Smg5, Smg6 and Smg7 are involved in nonsense-mediated RNA decay (NMD) in metazoans, but no orthologs have been found in the budding yeast Saccharomyces cerevisiae. Sequence alignments reveal that yeast Ebs1p is similar in structure to the human Smg5-7, with highest homology to Smg7. We demonstrate here that Ebs1p is involved in NMD and behaves similarly to human Smg proteins. Indeed, both loss and overexpression of Ebs1p results in stabilization of NMD targets. However, Ebs1-loss in yeast or Smg7-depletion in human cells only partially disrupts NMD and in the latter, Smg7-depletion is partially compensated for by Smg6. Ebs1p physically interacts with the NMD helicase Upf1p and overexpressed Ebs1p leads to recruitment of Upf1p into cytoplasmic P-bodies. Furthermore, Ebs1p localizes to P-bodies upon glucose starvation along with Upf1p. Overall our findings suggest that NMD is more conserved in evolution than previously thought, and that at least one of the Smg5-7 proteins is conserved in budding yeast.
Project description: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.