Previous contrasting models for PTC-recognition in NMD invoke either 3′ UTR–associated factors that stimulate NMD, the EJC in human cells [2
], and DSE-binding proteins in yeast [21
], or factors that stimulate normal translation termination and antagonize NMD [1
]. Our observations, together with the observations in the paper by Eberle et al. [46
], are consistent with a unified model for human NMD, in which the balance between NMD-antagonizing (such as PABPC1) and NMD-stimulating (such as the EJC) factor(s) that are associated with the mRNA 3′ UTR, determines whether termination is considered normal or premature (A). According to this model, a translation termination event proximal to cytoplasmic PABP (), or other unknown NMD-antagonizing factors, precludes the interaction of hUpf1 with eRF3 (C) and thus prohibits NMD (A, top). By contrast, if hUpf1 associates with eRF3, NMD ensues (A, bottom). This occurs when cytoplasmic PABP, or other inhibitory factors, are spatially distant from the termination event () and is enhanced when a splicing event downstream of a termination codon results in deposition of an EJC (), which provides higher affinity for the hUpf complex (A, bottom). However, an exon-exon junction in the 3′ UTR is not sufficient for NMD (). This suggests that a proximal cytoplasmic PABP is dominant over 3′ UTR exon-exon junctions, which is consistent with the observation that the affinity of PABPC1 for eRF3 appears to be several orders of magnitude higher than that of hUpf1 ( and unpublished data). However, while introns are observed to only stimulate NMD of the substrates tested in this study, it cannot be ruled out that a subset of human mRNAs requires downstream introns for NMD. Previous experiments, in which EJC or hUpf proteins tethered to an mRNA 3′ UTR were observed to trigger NMD, may have been assisted by the extended 3′ UTRs resulting from insertion of multiple tethering sites and/or by the recruitment of multiple NMD-promoting factors [15
]. The model depicted in A may be extended to eukaryotes other than mammals and is consistent with the observation in Drosophila
S2 cells that the decay of an NMD reporter mRNA is inhibited upon cytoplasmic PABP depletion [5
]. In this case it is predicted that a large subset of normally stable endogenous mRNAs become NMD substrates, thus out-titrating the NMD pathway.
A Competition between Stimulators and Antagonists of Upf Complex Recruitment in Human NMD
How does cytoplasmic PABP antagonize NMD? While PABPC1 can out-compete the association of hUpf1 with eRF3 in vitro (B), a more complex relationship may exist between these proteins in the cell. For example, we failed to observe exogenously expressed PABPC1 out-compete the co-IP of endogenous hUpf1 with eRF3 (unpublished data). Moreover, in S. cerevisiae
, cytoplasmic PABP truncated of its C-terminal eRF3-interaction region was capable of suppressing NMD when tethered in proximity of a PTC [7
]. However, we found no loss of eRF3-association of a similarly truncated PABPC1 in co-IP assays between exogenously expressed human proteins (unpublished data), suggesting that eRF3 may form a complex with PABPC1 through additional regions. Understanding the specific mechanism by which NMD is antagonized by cytoplasmic PABP, and likely other 3′ UTR–associated factors, is an important goal for future studies and could involve both direct competition with the Upf complex as well as modulation of the translation termination event that excludes Upf complex recruitment in a more indirect manner. Another open question is how the interplay between eRF3, PABP, and the Upf complex influences events downstream of translation termination. Interestingly, it was previously observed that the interaction between eRF3 and cytoplasmic PABP stimulates mRNA deadenylation in yeast [50
], and that deadenylation can be an early step in NMD [51
]. Clearly, a great deal remains to be learned about the relationship between eRF3, the Upf complex, and cytoplasmic PABP and how it controls the fates of mRNAs after translation termination.
It is likely that 3′ UTR–associated factors (indicated by a question mark in A) other than cytoplasmic PABP can antagonize NMD. This hypothesis is consistent with the observation that in yeast cells, cytoplasmic PABP is not required for discriminating tested NMD substrates from their normal counterparts [11
]. An excellent candidate for such an activity is the yeast protein Pub1p, which has been identified as a factor that binds downstream of upstream open reading frames (uORFs) in GCN4 and YAP1 mRNAs to prevent NMD [54
]. It is possible that Pub1p and factors with similar activities are found in a subset of normal 3′ UTRs. It remains to be tested whether Pub1p acts on the terminating ribosome in a manner similar to cytoplasmic PABP. Similarly, factors other than the EJC could provide an enhanced affinity for the Upf complex and stimulate NMD. For example, the protein Hrp1p appears to serve such a role in the yeast PGK1 NMD substrate [21
]. Moreover, human Staufen1 and histone mRNA stem loop binding protein have been shown to recruit hUpf1 to the 3′ UTR of specific mRNAs to trigger NMD-like mRNA decay [55
]. Thus, our observations suggest that the NMD pathway is much more conserved between mammals and other eukaryotes than previously appreciated. Nevertheless, there is evidence that differences exist between yeast and mammalian cells as to which round of translation can stimulate NMD [28
Our observations suggest that while artificial long 3′ UTRs trigger NMD (), a subset of mRNAs containing long 3′ UTRs have evolved mechanisms to evade NMD (). Future studies should reveal the mechanism by which this is accomplished. This could conceivably be achieved by (i) induced looping of the 3′ UTR, thus placing the poly(A) tail and cytoplasmic PABP in close spatial proximity to the translation termination event (B, top), or (ii) by recruitment of factors that antagonize NMD (such as PABPC1 or Pub1p) to the 3′ UTR in proximity to the termination codon (B, bottom). The observation that cytoplasmic PABP alleviates NMD when placed in the vicinity of a PTC () [5
] and the finding in the paper by Eberle et al. that artificially induced 3′ UTR looping rescues reporter mRNAs with extended 3′ UTRs from NMD [46
], provides proof-of-principle evidence for each of these models. The mechanism by which specific mRNAs evade the NMD pathway is an important subject for future investigation and is likely to vary between individual mRNAs.
After the submission of this paper, we have become aware of two other studies reporting that cytoplasmic PABP antagonizes human NMD when placed in proximity to a PTC [60