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.     

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.     

Interaction of PABPC1 with the translation initiation complex is critical to the NMD resistance of AUG-proximal nonsense mutations

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'-5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.     

Influence of <Emphasis Type="Italic">UPF</Emphasis> genes on severity of <Emphasis Type="Italic">SUP45</Emphasis> mutations

Eukaryotic cells possess a special mechanism for the degradation of mRNAs containing premature termination codons (PTCs), referred to as NMD (nonsense-mediated mRNA decay). The strength of this pathway depends on the recognition of the PTCs by translational machinery and the interaction of translation termination factors eRF1 and eRF3 with Upf1, Upf2 and Upf3 proteins in Sachromyces cerevisiae yeast. Previously, we have shown that the decrease of eRF1 protein amounts in sup45 nonsense mutants leads to the impairment of NMD. Here we show that the deletion of UPF1 or UPF2 genes leads to an increase in the viability of sup45 mutants, while the effect of UPF3 gene deletion is allele-specific. Two-hybrid data have shown that amino acid residues 1–555 of Upf1 protein interact with eRF1. Any UPF gene deletion leads to allosupression of the adel1-14 mutation without a change in eRF1 content. The Upf1 depletion does not influence the synthetic lethality of sup45 mutations and the [PSI ⁺] prion. It is possible that the absence of Upf1 (or its activator Upf2) leads to a more effective formation of the translation termination complex and consequently to the increased viability of the cells containing mutant termination factors.     

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.     

Dual function of UPF3B in early and late translation termination

Nonsense‐mediated mRNA decay (NMD) is a cellular surveillance pathway that recognizes and degrades mRNAs with premature termination codons (PTCs). The mechanisms underlying translation termination are key to the understanding of RNA surveillance mechanisms such as NMD and crucial for the development of therapeutic strategies for NMD‐related diseases. Here, we have used a fully reconstituted in vitro translation system to probe the NMD proteins for interaction with the termination apparatus. We discovered that UPF3B (i) interacts with the release factors, (ii) delays translation termination and (iii) dissociates post‐termination ribosomal complexes that are devoid of the nascent peptide. Furthermore, we identified UPF1 and ribosomes as new interaction partners of UPF3B. These previously unknown functions of UPF3B during the early and late phases of translation termination suggest that UPF3B is involved in the crosstalk between the NMD machinery and the PTC‐bound ribosome, a central mechanistic step of RNA surveillance.     

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?     

Leaky termination at premature stop codons antagonizes nonsense-mediated mRNA decay in S. cerevisiae

The Nonsense-Mediated mRNA Decay (NMD) pathway mediates the rapid degradation of mRNAs that contain premature stop mutations in eukaryotic organisms. It was recently shown that mutations in three yeast genes that encode proteins involved in the NMD process, UPF1, UPF2, and UPF3, also reduce the efficiency of translation termination. In the current study, we compared the efficiency of translation termination in a upf1Delta strain and a [PSI(+)] strain using a collection of translation termination reporter constructs. The [PSI(+)] state is caused by a prion form of the polypeptide chain release factor eRF3 that limits its availability to participate in translation termination. In contrast, the mechanism by which Upf1p influences translation termination is poorly understood. The efficiency of translation termination is primarily determined by a tetranucleotide termination signal consisting of the stop codon and the first nucleotide immediately 3' of the stop codon. We found that the upf1Delta mutation, like the [PSI(+)] state, decreases the efficiency of translation termination over a broad range of tetranucleotide termination signals in a unique, context-dependent manner. These results suggest that Upf1p may associate with the termination complex prior to polypeptide chain release. We also found that the increase in readthrough observed in a [PSI(+)]/upf1Delta strain was larger than the readthrough observed in strains carrying either defect alone, indicating that the upf1Delta mutation and the [PSI(+)] state influence the termination process in distinct ways. Finally, our analysis revealed that the mRNA destabilization associated with NMD could be separated into two distinct forms that correlated with the extent the premature stop codon was suppressed. The minor component of NMD was a 25% decrease in mRNA levels observed when readthrough was >/=0.5%, while the major component was represented by a larger decrease in mRNA abundance that was observed only when readthrough was 相似文献   

Human nonsense-mediated mRNA decay factor UPF2 interacts directly with eRF3 and the SURF complex

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that regulates gene expression and mRNA quality. A complex network of macromolecular interactions regulates NMD initiation, which is only partially understood. According to prevailing models, NMD begins by the assembly of the SURF (SMG1–UPF1–eRF1–eRF3) complex at the ribosome, followed by UPF1 activation by additional factors such as UPF2 and UPF3. Elucidating the interactions between NMD factors is essential to comprehend NMD, and here we demonstrate biochemically and structurally the interaction between human UPF2 and eukaryotic release factor 3 (eRF3). In addition, we find that UPF2 associates with SURF and ribosomes in cells, in an UPF3-independent manner. Binding assays using a collection of UPF2 truncated variants reveal that eRF3 binds to the C-terminal part of UPF2. This region of UPF2 is partially coincident with the UPF3-binding site as revealed by electron microscopy of the UPF2–eRF3 complex. Accordingly, we find that the interaction of UPF2 with UPF3b interferes with the assembly of the UPF2–eRF3 complex, and that UPF2 binds UPF3b more strongly than eRF3. Together, our results highlight the role of UPF2 as a platform for the transient interactions of several NMD factors, including several components of SURF.     

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.     

Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay

mRNA surveillance pathways selectively clear defective mRNAs from the cell. As such, these pathways serve as important modifiers of genetic disorders. Nonsense-mediated decay (NMD), the most intensively studied surveillance pathway, recognizes mRNAs with premature termination codons (PTCs). In mammalian systems the location of a PTC more than 50 nucleotides 5' to the terminal exon-exon junction is a critical determinant of NMD. However, mRNAs with nonsense codons that fulfill this requirement but are located very early in the open reading frame can effectively evade NMD. The unexpected resistance of such mRNAs with AUG-proximal PTCs to accelerated decay suggests that important determinants of NMD remain to be identified. Here, we report that an NMD-sensitive mRNA can be stabilized by artificially tethering the cytoplasmic poly(A) binding protein 1, PABPC1, at a PTC-proximal position. Remarkably, the data further suggest that NMD of an mRNA with an AUG-proximal PTC can also be repressed by PABPC1, which might be brought into proximity with the PTC during cap-dependent translation and 43S scanning. These results reveal a novel parameter of NMD in mammalian cells that can account for the stability of mRNAs with AUG-proximal PTCs. These findings serve to expand current mechanistic models of NMD and mRNA translation.     

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.