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.     

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.     

Testing the faux-UTR model for NMD: analysis of Upf1p and Pab1p competition for binding to eRF3/Sup35p

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that accelerates the degradation of mRNAs containing premature translation termination codons. This quality control pathway depends on the NMD-specific factors, Upf1p, Upf2p/Nmd2p, and Upf3p, as well as the two release factors, eRF1 and eRF3 (respectively designated Sup45p and Sup35p in yeast). NMD activation is also enabled by the absence of the poly(A)-binding protein, Pab1p, downstream of a termination event. Since Sup35p interacts with both Upf1p and Pab1p we considered the possibility that differential binding of the latter factors to Sup35p may be a critical determinant of NMD sensitivity for an mRNA. Here we describe three approaches to assess this hypothesis. First, we tethered fragments or mutant forms of Sup35p downstream of a premature termination codon in a mini-pgk1 nonsense-containing mRNA and showed that the inhibition of NMD by tethered Sup35p does not depend on the domain necessary for the recruitment of Pab1p. Second, we examined the Sup35p interaction properties of Upf1p and Pab1p in vitro and showed that these two proteins bind differentially to Sup35p. Finally, we examined competitive binding between the three proteins and observed that Upf1p inhibits Pab1p binding to Sup35p whereas the interaction between Upf1p and Sup35p is relatively unaffected by Pab1p. These data indicate that the binding of Upf1p and Pab1p to Sup35p may be more complex than anticipated and that NMD activation could involve more than just simple competition between these factors. We conclude that activation of NMD at a premature termination codon is not solely based on the absence of Pab1p and suggest that a specific recruitment step must commit Upf1p to the process and Upf1p-associated mRNAs to NMD.     

The Upf-dependent decay of wild-type PPR1 mRNA depends on its 5'-UTR and first 92 ORF nucleotides

mRNAs containing premature translation termination codons (nonsense mRNAs) are targeted for deadenylation-independent degradation in a mechanism that depends on Upf1p, Upf2p and Upf3p. This decay pathway is often called nonsense- mediated mRNA decay (NMD). Nonsense mRNAs are decapped by Dcp1p and then degraded 5′ to 3′ by Xrn1p. In the yeast Saccharomyces cerevisiae, a significant number of wild-type mRNAs accumulate in upf mutants. Wild-type PPR1 mRNA is one of these mRNAs. Here we show that PPR1 mRNA degradation depends on the Upf proteins, Dcp1p, Xrn1p and Hrp1p. We have mapped an Upf1p-dependent destabilizing element to a region located within the 5′-UTR and the first 92 bases of the PPR1 ORF. This element targets PPR1 mRNA for Upf-dependent decay by a novel mechanism.     

Upf proteins: highly conserved factors involved in nonsense mRNA mediated decay

Over 10% of genetic diseases are caused by mutations that introduce a premature termination codon in protein-coding mRNA. Nonsense-mediated mRNA decay (NMD) is an essential cellular pathway that degrades these mRNAs to prevent the accumulation of harmful partial protein products. NMD machinery is also increasingly appreciated to play a role in other essential cellular functions, including telomere homeostasis and the regulation of normal mRNA turnover, and is misregulated in numerous cancers. Hence, understanding and designing therapeutics targeting NMD is an important goal in biomedical science. The central regulator of NMD, the Upf1 protein, interacts with translation termination factors and contextual factors to initiate NMD specifically on mRNAs containing PTCs. The molecular details of how these contextual factors affect Upf1 function remain poorly understood. Here, we review plausible models for the NMD pathway and the evidence for the variety of roles NMD machinery may play in different cellular processes.     

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 RNA helicase Ddx5/p68 binds to hUpf3 and enhances NMD of Ddx17/p72 and Smg5 mRNA

Non-sense-mediated mRNA decay (NMD) is a mechanism of translation-dependent mRNA surveillance in eukaryotes: it degrades mRNAs with premature termination codons (PTCs) and contributes to cellular homeostasis by downregulating a number of physiologically important mRNAs. In the NMD pathway, Upf proteins, a set of conserved factors of which Upf1 is the central regulator, recruit decay enzymes to promote RNA cleavage. In mammals, the degradation of PTC-containing mRNAs is triggered by the exon–junction complex (EJC) through binding of its constituents Upf2 and Upf3 to Upf1. The complex formed eventually induces translational repression and recruitment of decay enzymes. Mechanisms by which physiological mRNAs are targeted by the NMD machinery in the absence of an EJC have been described but still are discussed controversially. Here, we report that the DEAD box proteins Ddx5/p68 and its paralog Ddx17/p72 also bind the Upf complex by physical interaction with Upf3, thereby interfering with the binding of EJC. By activating the NMD machinery, Ddx5 is shown to regulate the expression of its own, Ddx17 and Smg5 mRNAs. For NMD triggering, the adenosine triphosphate-binding activity of Ddx5 and the 3′-untranslated region of substrate mRNAs are essential.     

Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1

One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.     

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.     

Upf1 potentially serves as a RING-related E3 ubiquitin ligase via its association with Upf3 in yeast

Three Upf proteins are essential to the nonsense-mediated mRNA decay (NMD) pathway. Although these proteins assemble on polysomes for recognition of aberrant mRNAs containing premature termination codons, the significance of this assembly remains to be elucidated. The Cys- and His-rich repeated N terminus (CH domain) of Upf1 has been implicated in its binding to Upf2. Here, we show that CH domain also plays a RING-related role for Upf1 to exhibit E3 ubiquitin ligase activity in yeast. Despite the sequence divergence from typical E3-RING fingers, the CH domain of yeast Upf1 specifically and directly interacted with the yeast E2 Ubc3. Interestingly, Upf1 served as a substrate for the in vitro self-ubiquitination, and the modification required its association with Upf3 rather than Upf2. Substitution of the coordinated Cys and His residues in the CH domain impaired not only self-ubiquitination of Upf1 but also rapid decay of aberrant mRNAs. These results suggest that Upf1 may serve as an E3 ubiquitin ligase upon its association with Upf3 and play an important role in signaling to the NMD pathway.     

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.     

Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4)

Nonsense-mediated mRNA decay (NMD), also called mRNA surveillance, is an important pathway used by all organisms that have been tested to degrade mRNAs that prematurely terminate translation and, as a consequence, eliminate the production of aberrant proteins that could be potentially harmful. In mammalian cells, NMD appears to involve splicing-dependent alterations to mRNA as well as ribosome-associated components of the translational apparatus. To date, human (h) Upf1 protein (p) (hUpf1p), a group 1 RNA helicase named after its Saccharomyces cerevisiae orthologue that functions in both translation termination and NMD, has been the only factor shown to be required for NMD in mammalian cells. Here, we describe human orthologues to S. cerevisiae Upf2p and S. cerevisiae Upf3p (Caenorhabditis elegans SMG-4) based on limited amino acid similarities. The existence of these orthologues provides evidence for a higher degree of evolutionary conservation of NMD than previously appreciated. Interestingly, human orthologues to S. cerevisiae Upf3p (C. elegans SMG-4) derive from two genes, one of which is X-linked and both of which generate multiple isoforms due to alternative pre-mRNA splicing. We demonstrate using immunoprecipitations of epitope-tagged proteins transiently produced in HeLa cells that hUpf2p interacts with hUpf1p, hUpf3p-X, and hUpf3p, and we define the domains required for the interactions. Furthermore, we find by using indirect immunofluorescence that hUpf1p is detected only in the cytoplasm, hUpf2p is detected primarily in the cytoplasm, and hUpf3p-X localizes primarily to nuclei. The finding that hUpf3p-X is a shuttling protein provides additional indication that NMD has both nuclear and cytoplasmic components.     

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.