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.     

Pioneer round of translation mediated by nuclear cap-binding proteins CBP80/20 occurs during prolonged hypoxia

Nonsense-mediated mRNA decay (NMD) is one of the mRNA surveillance mechanisms, which eliminates aberrant mRNAs harboring premature termination codons. NMD targets only mRNAs bound by the nuclear cap-binding protein complex CBP80/20 which directs the pioneer round of translation. Here we demonstrate that NMD occurs efficiently during prolonged hypoxia in which steady-state translation is drastically inhibited. Accordingly, CBP80 remains in the nucleus, and processing bodies are unaffected with regard to their abundance and number under prolonged hypoxic conditions. These results indicate that mRNAs enter the pioneer round of translation during prolonged hypoxia.     

Efficiency of the pioneer round of translation affects the cellular site of nonsense-mediated mRNA decay

In mammalian cells, nonsense-mediated mRNA decay (NMD) is a consequence of nonsense codon recognition during a pioneer round of translation. This round can occur largely before or largely after the release of newly synthesized mRNA from nuclei, depending on the mRNA, and likely utilizes cytoplasmic ribosomes. We show that increasing the cellular concentration of the splicing factor SF2/ASF augments the efficiency of NMD and ultimately shifts NMD that takes place after mRNA export to the cytoplasm to NMD that occurs before mRNA release from nuclei. These changes are accompanied by an increased association of pioneer translation initiation complexes with SF2/ASF, translationally active ribosomes, and the translational activator TAP. Increased TAP binding correlates with increased SF2/ASF binding, but not increased REF/Aly or Y14 binding. Our results uncover an additional role for SF2/ASF and indicate that the efficiency of the pioneer round of translation influences the efficiency of subsequent rounds of translation.     

NMD resulting from encephalomyocarditis virus IRES-directed translation initiation seems to be restricted to CBP80/20-bound mRNA

Nonsense-mediated messenger RNA decay (NMD) generally degrades mRNAs that prematurely terminate translation as a means of quality control. NMD in mammalian cells targets newly spliced mRNA that is bound by the cap-binding protein heterodimer CBP80/20 and one or more post-splicing exon junction complexes during a pioneer round of translation. NMD targets mRNA that initiates translation using the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), therefore NMD might target not only CBP80/20-bound mRNA but also its remodelled product, eIF4E-bound mRNA. Here, we provide evidence that NMD triggered by translation initiation at the EMCV IRES, similar to NMD triggered by translation initiation at an mRNA cap, targets CBP80/20-bound mRNA but does not detectably target eIF4E-bound mRNA. We show that EMCV IRES-initiated translation undergoes a CBP80/20-associated pioneer round of translation that results in CBP80/20-dependent and Upf factor-dependent NMD when translation terminates prematurely.     

Pioneer round of translation occurs during serum starvation

The pioneer round of translation plays a role in translation initiation of newly spliced and exon junction complex (EJC)-bound mRNAs. Nuclear cap-binding protein complex CBP80/20 binds to those mRNAs at the 5'-end, recruiting translation initiation complex. As a consequence of the pioneer round of translation, the bound EJCs are dissociated from mRNAs and CBP80/20 is replaced by the cytoplasmic cap-binding protein eIF4E. Steady-state translation directed by eIF4E allows for an immediate and rapid response to changes in physiological conditions. Here, we show that nonsense-mediated mRNA decay (NMD), which restricts only to the pioneer round of translation but not to steady-state translation, efficiently occurs even during serum starvation, in which steady-state translation is drastically abolished. Accordingly, CBP80 remains in the nucleus and processing bodies are unaffected in their abundance and number in serum-starved conditions. These results suggest that mRNAs enter the pioneer round of translation during serum starvation and are targeted for NMD if they contain premature termination codons.     

Ectopic expression of eIF4E-transporter triggers the movement of eIF4E into P-bodies, inhibiting steady-state translation but not the pioneer round of translation

Nonsense-mediated mRNA decay (NMD) is the best-characterized mRNA surveillance mechanism; this process removes faulty mRNAs harboring premature termination codons (PTCs). NMD targets newly synthesized mRNAs bound by nuclear cap-binding proteins 80/20 (CBP80/20) and exon junction complex (EJC), the former of which is thought to recruit the ribosome to initiate the pioneer round of translation. After completion of the pioneer round of translation, CBP80/20 is replaced by the cytoplasmic cap-binding protein eIF4E, which mediates steady-state translation in the cytoplasm. Here, we show that overexpression of eIF4E-T preferentially inhibits cap-dependent steady-state translation, but not the pioneer round of translation. We also demonstrate that overexpression of eIF4E-T or Dcp1a triggers the movement of eIF4E into the processing bodies. These results suggest that the pioneer round of translation differs from steady-state translation in terms of ribosome recruitment.     

SMG6 is the catalytic endonuclease that cleaves mRNAs containing nonsense codons in metazoan

Messenger RNAs harboring nonsense codons (or premature translation termination codons [PTCs]) are degraded by a conserved quality-control mechanism known as nonsense-mediated mRNA decay (NMD), which prevents the accumulation of truncated and potentially harmful proteins. In Drosophila melanogaster, degradation of PTC-containing messages is initiated by endonucleolytic cleavage in the vicinity of the nonsense codon. The endonuclease responsible for this cleavage has not been identified. Here, we show that SMG6 is the long sought NMD endonuclease. First, cells expressing an SMG6 protein mutated at catalytic residues fail to degrade PTC-containing messages. Moreover, the SMG6-PIN domain can be replaced with the active PIN domain of an unrelated protein, indicating that its sole function is to provide endonuclease activity for NMD. Unexpectedly, we found that the catalytic activity of SMG6 contributes to the degradation of PTC-containing mRNAs in human cells. Thus, SMG6 is a conserved endonuclease that degrades mRNAs terminating translation prematurely in metazoa.     

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.     

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.     

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.     

Nonsense-mediated mRNA decay in Drosophila: at the intersection of the yeast and mammalian pathways

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.     

Resistance of mRNAs with AUG-proximal nonsense mutations to nonsense-mediated decay reflects variables of mRNA structure and translational activity

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature termination codons (PTCs). The level of sensitivity of a PTC-containing mRNA to NMD is multifactorial. We have previously shown that human β-globin mRNAs carrying PTCs in close proximity to the translation initiation AUG codon escape NMD. This was called the ‘AUG-proximity effect’. The present analysis of nonsense codons in the human α-globin mRNA illustrates that the determinants of the AUG-proximity effect are in fact quite complex, reflecting the ability of the ribosome to re-initiate translation 3′ to the PTC and the specific sequence and secondary structure of the translated ORF. These data support a model in which the time taken to translate the short ORF, impacted by distance, sequence, and structure, not only modulates translation re-initiation, but also impacts on the exact boundary of AUG-proximity protection from NMD.