Inactivation of NMD increases viability of <Emphasis Type="Italic">sup45</Emphasis> nonsense mutants in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

The nonsense-mediated mRNA decay (NMD) pathway promotes the rapid degradation of mRNAs containing premature termination codons (PTCs). In yeast Saccharomyces cerevisiae, the activity of the NMD pathway depends on the recognition of the PTC by the translational machinery. Translation termination factors eRF1 (Sup45) and eRF3 (Sup35) participate not only in the last step of protein synthesis but also in mRNA degradation and translation initiation via interaction with such proteins as Pab1, Upf1, Upf2 and Upf3.     

Translational competence of ribosomes released from a premature termination codon is modulated by NMD factors

In addition to their well-documented roles in the promotion of nonsense-mediated mRNA decay (NMD), yeast Upf proteins (Upf1, Upf2/Nmd2, and Upf3) also manifest translational regulatory functions, at least in vitro, including roles in premature translation termination and subsequent reinitiation. Here, we find that all upfΔ strains also fail to reinitiate translation after encountering a premature termination codon (PTC) in vivo, a result that led us to seek a unifying mechanism for all of these translation phenomena. Comparisons of the in vitro translational activities of wild-type (WT) and upf1Δ extracts were utilized to test for a Upf1 role in post-termination ribosome reutilization. Relative to WT extracts, non-nucleased extracts lacking Upf1 had approximately twofold decreased activity for the translation of synthetic CAN1/LUC mRNA, a defect paralleled by fewer ribosomes per mRNA and reduced efficiency of the 60S joining step at initiation. These deficiencies could be complemented by purified FLAG-Upf1, or 60S subunits, and appeared to reflect diminished cycling of ribosomes from endogenous PTC-containing mRNAs to exogenously added synthetic mRNA in the same extracts. This hypothesis was tested, and supported, by experiments in which nucleased WT or upf1Δ extracts were first challenged with high concentrations of synthetic mRNAs that were templates for either normal or premature translation termination and then assayed for their capacity to translate a normal mRNA. Our results indicate that Upf1 plays a key role in a mechanism coupling termination and ribosome release at a PTC to subsequent ribosome reutilization for another round of translation initiation.     

Targeting of aberrant mRNAs to cytoplasmic processing bodies

In eukaryotes, a specialized pathway of mRNA degradation termed nonsense-mediated decay (NMD) functions in mRNA quality control by recognizing and degrading mRNAs with aberrant termination codons. We demonstrate that NMD in yeast targets premature termination codon (PTC)-containing mRNA to P-bodies. Upf1p is sufficient for targeting mRNAs to P-bodies, whereas Upf2p and Upf3p act, at least in part, downstream of P-body targeting to trigger decapping. The ATPase activity of Upf1p is required for NMD after the targeting of mRNAs to P-bodies. Moreover, Upf1p can target normal mRNAs to P-bodies but not promote their degradation. These observations lead us to propose a new model for NMD wherein two successive steps are used to distinguish normal and aberrant mRNAs.     

Selective destabilization of polypeptides synthesized from NMD-targeted transcripts

The translation of mRNAs that contain a premature termination codon (PTC) generates truncated proteins that may have toxic dominant negative effects. Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that degrades PTC-containing mRNAs to limit the production of truncated proteins. NMD activation requires a ribosome terminating translation at a PTC, but what happens to the polypeptides synthesized during the translation cycle needed to activate NMD is incompletely understood. Here, by establishing reporter systems that encode the same polypeptide sequence before a normal termination codon or PTC, we show that termination of protein synthesis at a PTC is sufficient to selectively destabilize polypeptides in mammalian cells. Proteasome inhibition specifically rescues the levels of nascent polypeptides produced from PTC-containing mRNAs within an hour, but also disrupts mRNA homeostasis within a few hours. PTC-terminated polypeptide destabilization is also alleviated by depleting the central NMD factor UPF1 or SMG1, the kinase that phosphorylates UPF1 to activate NMD, but not by inhibiting SMG1 kinase activity. Our results suggest that polypeptide degradation is linked to PTC recognition in mammalian cells and clarify a framework to investigate these mechanisms.     

An NMD pathway in yeast involving accelerated deadenylation and exosome-mediated 3'-->5' degradation

Eukaryotic mRNAs containing premature termination codons are subjected to accelerated turnover, known as nonsense-mediated decay (NMD). Recognition of translation termination events as premature requires a surveillance complex, which includes the RNA helicase Upf1p. In Saccharomyces cerevisiae, NMD provokes rapid decapping followed by 5'-->3' exonucleolytic decay. Here we report an alternative, decapping-independent NMD pathway involving deadenylation and subsequent 3'-->5' exonucleolytic decay. Accelerated turnover via this pathway required Upf1p and was blocked by the translation inhibitor cycloheximide. Degradation of the deadenylated mRNA required the Rrp4p and Ski7p components of the cytoplasmic exosome complex, as well as the putative RNA helicase Ski2p. We conclude that recognition of NMD substrates by the Upf surveillance complex can target mRNAs to rapid deadenylation and exosome-mediated degradation.     

The role of SMG-1 in nonsense-mediated mRNA decay

SMG-1, a member of the PIKK (phosphoinositide 3-kinase related kinases) family, plays a critical role in the mRNA quality control system termed nonsense-mediated mRNA decay (NMD). NMD protects the cells from the accumulation of aberrant mRNAs with premature termination codons (PTCs) that encode nonfunctional or potentially harmful truncated proteins. SMG-1 directly phosphorylates Upf1, another key component of NMD, and this phosphorylation occurs upon recognition of PTC on post-spliced mRNA during the initial round of translation. At present, a variety of tools are available that can specifically suppress NMD, and it is possible to examine the contribution of NMD in a variety of physiological and pathological conditions.     

CBP80 promotes interaction of Upf1 with Upf2 during nonsense-mediated mRNA decay in mammalian cells

In mammalian cells, nonsense-mediated messenger RNA decay (NMD) targets newly synthesized nonsense-containing mRNA bound by the cap-binding-protein heterodimer CBP80-CBP20 and at least one exon-junction complex (EJC). An EJC includes the NMD factors Upf3 or Upf3X and Upf2, and Upf2 recruits Upf1. Once this pioneer translation initiation complex is remodeled so that CBP80-CBP20 is replaced by eukaryotic initiation factor 4E, the mRNA is no longer detectably targeted for NMD. Here, we provide evidence that CBP80 augments the efficiency of NMD but not of Staufen1 (Stau1)-mediated mRNA decay (SMD). SMD depends on the recruitment of Upf1 by the RNA-binding protein Stau1 but does not depend on the other Upf proteins. We find that CBP80 interacts with Upf1 and promotes the interaction of Upf1 with Upf2 but not with Stau1.     

Evidence that the Upf1-related molecular motor scans the 3'-UTR to ensure mRNA integrity

Upf1 is a highly conserved RNA helicase essential for nonsense-mediated mRNA decay (NMD), an mRNA quality-control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). For the activation of NMD, UPF1 interacts first with a translation-terminating ribosome and then with a downstream exon-junction complex (EJC), which is deposited at exon-exon junctions during splicing. Although the helicase activity of Upf1 is indispensable for NMD, its roles and substrates have yet to be fully elucidated. Here we show that stable RNA secondary structures between a PTC and a downstream exon-exon junction increase the levels of potential NMD substrates. We also demonstrate that a stable secondary structure within the 3'-untranslated region (UTR) induces the binding of Upf1 to mRNA in a translation-dependent manner and that the Upf1-related molecules are accumulated at the 5'-side of such a structure. Furthermore, we present evidence that the helicase activity of Upf1 is used to bridge the spatial gap between a translation-termination codon and a downstream exon-exon junction for the activation of NMD. Based on these findings, we propose a model that the Upf1-related molecular motor scans the 3'-UTR in the 5'-to-3' direction for the mRNA-binding factors including EJCs to ensure mRNA integrity.     

A competition between stimulators and antagonists of Upf complex recruitment governs human nonsense-mediated mRNA decay

The nonsense-mediated decay (NMD) pathway subjects mRNAs with premature termination codons (PTCs) to rapid decay. The conserved Upf1–3 complex interacts with the eukaryotic translation release factors, eRF3 and eRF1, and triggers NMD when translation termination takes place at a PTC. Contrasting models postulate central roles in PTC-recognition for the exon junction complex in mammals versus the cytoplasmic poly(A)-binding protein (PABP) in other eukaryotes. Here we present evidence for a unified model for NMD, in which PTC recognition in human cells is mediated by a competition between 3′ UTR–associated factors that stimulate or antagonize recruitment of the Upf complex to the terminating ribosome. We identify cytoplasmic PABP as a human NMD antagonizing factor, which inhibits the interaction between eRF3 and Upf1 in vitro and prevents NMD in cells when positioned in proximity to the termination codon. Surprisingly, only when an extended 3′ UTR places cytoplasmic PABP distally to the termination codon does a downstream exon junction complex enhance NMD, likely through increasing the affinity of Upf proteins for the 3′ UTR. Interestingly, while an artificial 3′ UTR of >420 nucleotides triggers NMD, a large subset of human mRNAs contain longer 3′ UTRs but evade NMD. We speculate that these have evolved to concentrate NMD-inhibiting factors, such as PABP, in spatial proximity of the termination codon.     

Caenorhabditis elegans SMG-2 selectively marks mRNAs containing premature translation termination codons

Eukaryotic mRNAs containing premature translation termination codons (PTCs) are rapidly degraded by a process termed "nonsense-mediated mRNA decay" (NMD). We examined protein-protein and protein-RNA interactions among Caenorhabditis elegans proteins required for NMD. SMG-2, SMG-3, and SMG-4 are orthologs of yeast (Saccharomyces cerevisiae) and mammalian Upf1, Upf2, and Upf3, respectively. A combination of immunoprecipitation and yeast two-hybrid experiments indicated that SMG-2 interacts with SMG-3, SMG-3 interacts with SMG-4, and SMG-2 interacts indirectly with SMG-4 via shared interactions with SMG-3. Such interactions are similar to those observed in yeast and mammalian cells. SMG-2-SMG-3-SMG-4 interactions require neither SMG-2 phosphorylation, which is abolished in smg-1 mutants, nor SMG-2 dephosphorylation, which is reduced or eliminated in smg-5 mutants. SMG-2 preferentially associates with PTC-containing mRNAs. We monitored the association of SMG-2, SMG-3, and SMG-4 with mRNAs of five endogenous genes whose mRNAs are alternatively spliced to either contain or not contain PTCs. SMG-2 associates with both PTC-free and PTC-containing mRNPs, but it strongly and preferentially associates with ("marks") those containing PTCs. SMG-2 marking of PTC-mRNPs is enhanced by SMG-3 and SMG-4, but SMG-3 and SMG-4 are not detectably associated with the same mRNPs. Neither SMG-2 phosphorylation nor dephosphorylation is required for selective association of SMG-2 with PTC-containing mRNPs, indicating that SMG-2 is phosphorylated only after premature terminations have been discriminated from normal terminations. We discuss these observations with regard to the functions of SMG-2 and its phosphorylation during NMD.     

Upf1 stimulates degradation of the product derived from aberrant messenger RNA containing a specific nonsense mutation by the proteasome

Aberrant messenger RNAs containing a premature termination codon (PTC) are eliminated by the nonsense‐mediated mRNA decay (NMD) pathway. Here, we show that a crucial NMD factor, up frameshift 1 protein (Upf1), is required for rapid proteasome‐mediated degradation of an aberrant protein (PTC product) derived from a PTC‐containing mRNA. Western blot and pulse–chase analyses revealed that Upf1 stimulates the degradation of specific PTC products by the proteasome. Moreover, the Upf1‐dependent, proteasome‐mediated degradation of the PTC product was also stimulated by mRNAs harbouring a faux 3′ untranslated region (3′‐UTR). These results indicate that protein stability might be regulated by an aberrant mRNA 3′‐UTR.     

The influence of UPF genes on the severity of SUP45 mutations

Eukaryotic cells possess special mechanism of the degradation of mRNAs containing premature termination codons (PTCs)--nonsense-mediated mRNA decay (NMD) pathway. In yeast Saccharomyces cerevisiae, the activity of this pathway depends on the recognition of the PTC by the translational machinery and interaction of translation termination factors eRF1 and eRF3 with Upf1, Upf2 and Upf3 proteins. Previously we have shown that decreasing of eRF1 amount causes an impairment of NMD. Here we show that deletion of either UPF1 or UPF2 increased viability of sup45 mutants, while effect of UPF3 deletion is allele-specific. Two-hybrid data have shown that aa 1-555 of eRF1 participate in interaction with Upf1. Deletion of each UPF gene leads to allosuppresson of ade1-14 mutation without changing eRF1 amount. Depletion of Upf1 does not influence synthetic lethality of sup45 and prion [PSI+]. It is possible that the absence of Upf1 (or its activator Upf2) leads to more effective formation of the translation termination complex and, consequently, increased viability of cells containing mutant termination factors.