For NMD to protect the cell from aberrant proteins, it is crucial to destroy PTC-containing mRNAs as soon as possible and before multiple truncated polypeptides have been translated. Could this destruction happen as early as when the mRNA is still in the nucleus? Although the possibility of 'nuclear translation' has recently received renewed attention [27
], cytoplasmic translation appears to be required for NMD [13
]; the first round of translation, that is, the first time that the mRNA passes through the ribosome, is considered to be particularly important [29
]. What distinguishes the initial round from subsequent rounds of translation? Conceivably, NMD could take place while the mRNA is being exported from the nucleus. During this phase, nuclear factors bound to the mRNA could be removed and potentially replaced by cytosolic proteins. Is this initial round of translation initiated by the canonical translation initiation factors or does it involve any special translation initiation factors?
In the nucleus, the 7
mGpppN cap structure of mRNAs is bound by a heterodimeric cap-binding complex (CBC, consisting of CBP20 and CBP80), which can affect splicing and 3' end processing [30
]. It appears that mRNAs are exported with their 5' ends first, exposing the CBC to the cytoplasm early in the transport process [31
]. The initiation factor eIF4E binds the cap structure in the cytoplasm, displacing the CBC, and mediates translation by recruiting eIF4G, other translation factors and ultimately the ribosome. This raises the question of whether the CBC and the cytoplasmic eIF4E exchange before, during or after the initial round of translation (Figure ). Notably, both yeast and mammalian CBCs can, like eIF4E, interact with eIF4G (via their CBP80 subunits) [32
], and the yeast CBC has been shown to be able to support cell-free translation, albeit quite inefficiently [32
Recently, Ishigaki et al.
] proposed that a "pioneer round of translation" may be initiated while the mRNA is still associated with the CBC, and that this could act to survey the mRNA for the presence of PTCs. Two versions of a human β-globin mRNA served as a reporter system for the experiments of Ishigaki et al.
: a β-globin mRNA with a PTC ('β-globin PTC') and one without ('β-globin normal') [34
]. Following immunoprecipitation with antibodies against CBP80, the level of co-immunoprecipitated β-globin PTC RNA was reduced to 20% of the level of β-globin normal RNA. This reduction quantitatively reflects NMD of the β-globin PTC RNA in the total cellular RNA fraction, and corresponds to the level of reduction in the β-globin PTC RNA (15%) that was precipitatable with antibodies to eIF4E. Thus, the authors suggest that surveillance and NMD of β-globin PTC take place while the mRNA is associated with CBP80 and before the remaining intact RNA is handed over to eIF4E [34
Ishigaki et al.
] also showed that hUpf2, hUpf3 (but not hUpf1) and the nuclear poly(A)-binding protein (PABP2) co-immunoprecipitated with anti-CBP80 antibodies in an RNA-dependent manner, whereas complexes precipitated with the anti-eIF4E antibody did not contain any of these proteins. Given that hUpf2 and hUpf3 are involved in NMD, these results can be interpreted to suggest that NMD occurs while the mRNA is still bound by CBP80, but not detectably when the RNA is bound to eIF4E [34
Do the majority of mRNAs undergo the initial round of translation without the support of eIF4E, which was thought to be a very important translation initiation factor? This idea offers a very interesting and unexpected twist to our perception of the translation and NMD pathway, and will surely stimulate many interesting future experiments. Before accepting the notion of a 'pioneer round' of mRNA translation under the guidance of CBP80 as a general aspect of mRNA export, however, some additional considerations may be warranted. At least in vitro
, the CBC is an ineffective translation-initiation factor compared with eIF4E [32
]. Moreover, a yeast strain with the CBP80
gene deleted does not display any overt translational defects nor any lack of NMD [35
]; CBP80 is thus not essential either for NMD or for translation in yeast. The function of the CBC in mammalian cells may differ from its functions in yeast, however, and/or it may act as a more efficient translation factor in vivo
than in vitro
. The finding that the amount of β-globin PTC mRNA associated with CBP80 was reduced to 20% of the level of β-globin normal mRNA [34
] certainly suggests that translation and NMD can occur before CBC has been exchanged for eIF4E. Only about 15%-30% of the CBC and eIF4E proteins and their associated RNAs from the extract was accessible to immunoprecipitation [34
], however, and we therefore do not know about the mRNAs associated with the other 70-85% of these proteins. Comparisons of mRNA levels were made between PTC-containing and wild-type RNAs each co-immunoprecipitated with either CBP80 or eIF4E, but we do not know the percentage of the β-globin PTC and β-globin normal mRNAs present in the cells that was analyzed [34
]. Thus, we do not yet know to what extent the analyzed mRNA-protein complexes (mRNPs) are representative of the majority of mRNAs being exported from the nucleus and being surveyed for NMD. Undoubtedly, future experiments will address this question as well as some of the intriguing implications of this work.
From a broader perspective, the recent progress highlights the dynamic nature of mRNP remodeling during mRNA processing, export, translation and degradation. We are on the way to a better understanding of how the different levels of gene expression are integrated and coordinated, how they affect and influence each other, and what the players and mechanisms are at the biochemical level.