The role in translational control of a class of predominantly nuclear mRNA binding proteins involved in splicing regulation has recently emerged as a common theme in the field of gene expression. However, little is known about the relationship between the subcellular localization of these trans
-acting factors and their ability to regulate translation. We have shown here that hnRNP A1, an abundant mRNA binding protein present in the nucleus of mammalian cells, is a translation trans
-acting factor that modulates the IRES activity of two IRES-containing mRNAs. Three sets of data have allowed us to demonstrate that hnRNP A1 promotes IRES activity of the HRV mRNA but that it inhibits IRES-mediated translation of the apaf-1 mRNA. First, we have shown that hnRNP A1 binds specifically and with an affinity ranging from 200 to 270 nM to both IRESs (). Additionally, we have provided evidence that expression of a cytoplasmically restricted mutant of hnRNP A1 deleted of its M9 carboxy-terminal shuttling domain either stimulates HRV IRES activity () or inhibits apaf-1 IRES activity (), as measured after transfection of bicistronic reporter mRNAs containing these IRESs. Finally, siRNA-mediated knockdown of hnRNP A1 expression results in decreased HRV IRES activity () and increased IRES-mediated apaf-1 mRNA translation after UVC irradiation (). Importantly, the IRES activities were generally evaluated after transfection of in vitro synthesized mRNAs, ensuring that our results reflect a true IRES activity and not the use of cryptic promoters or splice sites that would have been activated upon DNA transfection (see Supplemental Figure S2 for the apaf-1 IRES). Interestingly, we have recently shown that hnRNP A1 is an IRES trans
-acting factor that activates translation of the FGF-2 mRNA (Bonnal et al., 2005
) but inhibits translation of the XIAP (X-linked inhibitor of apoptosis) mRNA (Lewis et al., 2007
). The role of hnRNP A1 as an ITAF now extends to four mRNAs, and we predict that it controls IRES-mediated translation of many more genes. The ability of hnRNP A1 to be either an activator or an inhibitor of IRES function (depending on its target IRES) may be related to its ability to catalyze RNA–RNA annealing (Kumar and Wilson, 1990
; Munroe and Dong, 1992
), because structural remodeling of the 5′ UTR may either potentiate or destroy the IRES. Alternatively, hnRNP A1 may compete with other ITAFs (either positive or negative regulators) for binding to specific IRES elements and thus regulate IRES activity in this manner.
Because hnRNP A1 is a predominantly nuclear protein, but it is able to relocalize in the cytoplasm in a regulated manner (i.e., picornavirus infection [Gustin and Sarnow, 2001
] or stress stimuli such as osmotic shock or UVC irradiation in a p38 mitogen-activated protein kinase (MAP)-dependent pathway [van der Houven van Oordt et al.
, 2003]), the expectation is that this relocalization has a consequence for translational control. Two hypotheses could explain how this relocalization affects translational control. First, hnRNP A1 is preassembled with its target mRNA in the nucleus and is transported together with the mRNA into the cytoplasm, where it subsequently affects translation. In this case, translational control would be an indirect consequence of the cytoplasmic relocalization. Second, hnRNP A1 binds to its target mRNA in the cytoplasm and the cytoplasmic redistribution of hnRNP A1 constitutes a direct switch to control translation. Here, we have provided data that support this second hypothesis from our study of IRES-mediated translational control of two different mRNAs (HRV and apaf-1) that are regulated by two different stimuli (viral infection and UVC irradiation), raising the possibility that our conclusions may be generalized to other mRNAs.
Concerning the human rhinovirus, we have found that the HRV IRES is less active after RNA transfection than DNA transfection (). We reasoned that this weaker activity after RNA transfection might be due to the lack of a sufficient amount of hnRNP A1 in the cytoplasm. We have indeed demonstrated that HRV IRES activity after RNA transfection is insensitive to siRNA-mediated knockdown of hnRNP A1 expression (), but it is stimulated upon cytoplasmic relocalization of hnRNP A1 ( and 4). As a way to force the cytoplasmic relocalization of hnRNP A1, we have either produced a mutant hnRNP A1 protein deleted of its carboxy-terminal M9 shuttling domain () or used physiological conditions, such as the infection by the rhinovirus (), which lead to hnRNP A1 redistribution to the cytoplasm due to inhibition of its nuclear import (Gustin and Sarnow, 2001
). Furthermore, rhinovirus infection has been recently shown to induce phosphorylation and activation of the p38 MAP kinase (Dumitru et al., 2006
). The cytoplasmic relocalization of hnRNP A1 after rhinovirus infection may therefore also be dependent on the p38 MAP kinase pathway as it is during stress conditions (van der Houven van Oordt et al.
, 2003). Numerous reports have demonstrated that IRES elements have important functions in the viral life cycle, mostly to ensure efficient viral translation when components of the host translation machinery are limited due to virus-induced modifications or host-induced antiviral responses, such as the phosphorylation of eukaryotic initiation factor 2α (Hellen and Sarnow, 2001
; Vagner et al., 2001
). However, no evidence exists to support a direct positive influence on virus IRES-mediated translation after infection. Here, we have shown that rhinovirus infection exerts a direct positive switch to control its IRES-mediated translation through relocalization of an ITAF. Several other ITAFs have been described for the rhinovirus IRES, including unr, hnRNP I/PTB, and hnRNP E2 (Hunt et al., 1999
; Walter et al., 1999
; Boussadia et al., 2003
). However, the in vivo role of these ITAFs in HRV IRES function after rhinovirus infection, as well as their respective role in IRES function in general, may warrant further investigations. Interestingly, we have not been able to detect strong and conclusive interactions between hnRNP A1 and either unr or PTB (data not shown), excluding the possibility of the existence of a multimeric complex involved in IRES function.
Concerning the apaf-1 gene, we have forced the cytoplasmic redistribution of hnRNP A1 by either producing a mutant hnRNP A1 protein deleted of its carboxy-terminal M9 shuttling domain or by using UVC irradiation. We have found that hnRNP A1 limits the UVC-dependent translational activation of endogenous apaf-1 mRNA translation () and apaf-1 IRES activity ( and ). Therefore, hnRNP A1 is not a factor that contributes to the UVC-dependent translational activation of the apaf-1 mRNA. The characterization and/or identification of such factors warrants further investigation. Nevertheless, our study demonstrates that cytoplasmic redistribution of hnRNP A1 exerts a direct switch to control translation of the proapoptotic apaf-1 mRNA. This is consistent with the recently reported role of hnRNP A1 in the stress response, underscored by the observation that cells lacking hnRNP A1 exhibit decreased viability rates during stress (Guil et al., 2006
). This may also be consistent with the proapoptotic effect of siRNA-mediated reduction in hnRNPA1/A2 proteins observed in various cell types, although in that case, the proapopototic effect was found to be associated with a change in the distribution of the lengths of telomeric G-tails (Patry et al., 2003
). Very interestingly, ectopic expression of a nuclear-restricted hnRNP A1 mutant was shown to enhance the susceptibility to apoptosis (Iervolino et al., 2002
). The antiapoptotic function of hnRNP A1 may therefore be linked to its cytoplasmic function in translational control, in part through the inhibition of the apaf-1 IRES activity demonstrated in this study.
The results presented in this study also imply that UVC-triggered translational control can be mediated by an IRES-based mechanism and by hnRNP A1. This demonstrates that cellular IRESs behave as translational enhancers elements regulated by specific trans
-acting mRNA binding proteins in given physiological conditions. It will be of interest to investigate UVC-induced IRES-dependent and/or hnRNP A1-dependent translational control of other mRNAs, including the 17 mRNAs that are translationally induced by UVC irradiation, which were identified in human RKO colorectal carcinoma cells (Mazan-Mamczarz et al., 2005
). This may eventually define hnRNP A1-dependent IRESs as important elements in the UVC-triggered apoptotic pathway.
Last, the control of translation initiation through relocalization of an mRNA binding protein may occur more generally, because several reports demonstrate a regulated relocalization of members of the hnRNP family involved in translation. We have recently shown that hnRNP A1 is a negative regulator of XIAP mRNA translation during osmotic shock and that a mutant cytoplasmic hnRNP A1 exerts a more potent inhibitory effect on XIAP IRES translation than the wild-type hnRNP A1 protein (Lewis et al., 2007
). Serum stimulation or constitutive activation of the extracellular signal-regulated kinase kinase 1 results in phosphorylation and cytoplasmic accumulation of hnRNP K (Habelhah et al., 2001
). Phosphorylation-dependent cytoplasmic accumulation of hnRNP K is required for its ability to silence LOX mRNA translation (Habelhah et al., 2001
). However, it has not been demonstrated whether hnRNP K binds the LOX mRNA in the nucleus or in the cytoplasm. hnRNP I (PTB) is an IRES trans
-acting factor involved in translation regulation of many IRES-containing mRNAs (Valcarcel and Gebauer, 1997
; Spriggs et al., 2005
; Bushell et al., 2006
). Interestingly, it was found that direct protein kinase A phosphorylation of PTB modulates its nucleocytoplasmic distribution (Xie et al., 2003
). In all these cases, it remains to be determined whether the cytoplasmic relocalization directly controls cytoplasmic translation. A large-scale analysis of mRNA targets bound by translation trans
-acting factors able to relocalize in a regulated manner in the cytoplasm will undoubtedly identify genes regulated at the translational level through relocalization of predominantly nuclear mRNA binding proteins.