Here we report a new role for 4E-T in mammalian mRNA degradation. We demonstrate that 4E-T inhibits translation, colocalizes with decapping factors in cytoplasmic P-bodies, and decreases mRNA stability. 4E-T interacts with eIF4E through a shared recognition motif (YXXXXLΦ) also found in eIF4G (Dostie et al., 2000a
). Similar to other cap-dependent translation inhibitors, 4E-T is likely to inhibit translation by competing with eIF4G for binding to eIF4E and preventing formation of the eIF4F complex (Pause et al., 1994
; Poulin et al., 1998
). Accordingly, overexpression of 4E-T wt
, but not of a mutant defective in eIF4E binding, strongly inhibited cap-dependent translation of a reporter mRNA in vivo ().
The transition of an mRNA from a translationally active mRNP to one destined for decay is believed to be a consequence of translational inhibition, especially in yeast (Tharun and Parker, 2001
; Teixeira et al., 2005
). Remarkably, 4E-T is present in P-bodies, which are cytoplasmic foci containing factors involved in mRNA decay (Ingelfinger et al., 2002
; van Dijk et al., 2002
; Sheth and Parker, 2003
; Cougot et al., 2004
; Liu et al., 2004
) (). In this study, we also show that eIF4E is localized to mammalian P-bodies (). Overexpression of 4E-T in HeLa cells caused a marked increase of eIF4E in P-bodies (). This observation suggests that translation inhibition and targeting of eIF4E to P-bodies are causally related. The accumulation of eIF4E in P-bodies requires interaction with 4E-T, because an eIF4E mutant that fails to bind to 4E-T cannot localize to P-bodies (), and depletion of 4E-T resulted in loss of eIF4E from P-bodies (). However, the localization of 4E-T to P-bodies does not require interaction with eIF4E, because a 4E-T mutant defective in binding to eIF4E also localizes to P-bodies ().
eIF4GI and eIF4A did not localize to mammalian processing bodies (), which is in agreement with studies that demonstrated that mRNA pools from P-bodies are distinct from translating pools (Schwartz and Parker, 1999
; Tharun and Parker, 2001
; Teixeira et al., 2005
). These observations favor a model whereby translation inhibition that results from disruption of the translation initiation complex occurs outside P-bodies, and precedes P-body formation.
As was shown for Dcp1a (Sheth and Parker, 2003
; Cougot et al., 2004
), cycloheximide treatment of HeLa cells reduced the amount of 4E-T associated with P-bodies (). This result indicates that ongoing mRNA translation regulates the association of 4E-T with P-bodies, and suggests that 4E-T is not a permanent P-body constituent, but rather localizes to these structures in an mRNA-dependent manner. Importantly, 4E-T RNAi treatment results in decreased decapping activity in HeLa cells as evidenced by diminished localization of Dcp1a and p54 to P-bodies. Taken together, these data support a model whereby interaction of 4E-T with eIF4E acts as a priming event that leads to mRNP remodeling and mRNA decay.
In addition to their role in mRNA decay, P-bodies were suggested to function as mRNA storage sites (Sheth and Parker, 2003
; Coller and Parker, 2004
). For example, the Drosophila
decapping factor Me31B, is concentrated in cytoplasmic granules in germline cells where bicoid
mRNAs are translationally masked (Nakamura et al., 2001
). In addition, the Xenopus
equivalent, Xp54, is a major constituent of maternal mRNA storage particles where translation is repressed (Ladomery et al., 1997
; Minshall and Standart, 2004
). Thus, it is possible that under certain conditions, the presence of eIF4E within P-bodies might permit the transition of a translationally repressed/stored mRNA to a translationally competent state.
Homology searches using Blast algorithms failed to identify 4E-T yeast homologues (Dostie et al., 2000a
), and eIF4E was shown not to localize to P-bodies in yeast (unpublished data). Therefore, 4E-T might represent a more evolutionarily complex mRNA decay/storage regulation pathway in higher eukaryotes. The Drosophila
4E-T homologue, Cup, was reported to mediate translational repression of nanos
by interacting with eIF4E and 3′ trans-acting factors (Wilhelm and Smibert, 2005
). Interestingly, the RNA-binding protein Smaug, which interacts with Cup, recently was shown to recruit the CCR4 deadenylase complex in Drosophila
embryos (Semotok et al., 2005
An important finding in this paper is that reduction in 4E-T results in an increase in mRNA stability (). All of the mRNAs tested here are ARE-containing mRNAs, which are believed to be subject to 3′-5′ degradation by the exosome, based on in vitro decay assays (Chen et al., 2001
; Mukherjee et al., 2002
). The major deadenylase of AU-containing mRNAs in mammalian cells is believed to be poly (A) ribonuclease (Gao et al., 2000
; Lai et al., 2003
). Therefore, 3′-5′degradation would require destabilization of the interaction between PABP and eIF4G to allow entry and association of poly (A) ribonuclease with the cap (Wilusz et al., 2001
). Therefore, 4E-T may instigate the dissociation of PABP from eIF4G by binding to eIF4E and displacing eIF4G. The decay machinery, which processes mRNA through the 5′-3′ exonucleolytic pathway, is present in P-bodies. Recent studies have implied that the 5′-3′ and 3′-5′ pathways converge at these sites. For instance, mRNA decay enzymes involved in 5′-3′ and 3′-5′ decay are recruited by the ARE binding proteins, tristetraprolin and butyrate response factor (Lykke-Andersen and Wagner, 2005
). Moreover, tristetraprolin, which mediates ARE degradation by recruiting the exosome (Chen et al., 2001
), also is found localized in P-bodies (Kedersha et al., 2005
). Because the disappearance of P-bodies with 4E-T RNAi was not synonymous with complete stabilization of p21 mRNA, it is possible that this process occurs outside of P-bodies or that its deadenylation/decay occurs by way of a distinct pathway from c-fos
and GM-CSF mRNAs.
In conclusion, the interaction of 4E-T with eIF4E in cells has two consequences: import of eIF4E to the nucleus (Dostie et al., 2000a
) and the targeting of eIF4E to sites of mRNA decay. The function of eIF4E in the nucleus is under investigation; a proposed nuclear function of eIF4E is to export a subset of mRNAs from the nucleus (Lai and Borden, 2000
; Rousseau et al., 1996
). The dual role of 4E-T also might serve to sequester the limiting factor in translation initiation, eIF4E, as a means of maintaining translational homeostasis. It will be important to examine how 4E-T is regulated. It is reported that 4E-T is a phosphoprotein (Pyronnet et al., 2001
); therefore, it would be interesting to know what pathways regulate its phosphorylation and activity.