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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.