In the present study, we provide evidence for a regulatory mechanism in which hnRNP E1 and eEF1A1 coordinate transcript-specific repression of genes required for EMT. The canonical function of eEF1A1 is to facilitate binding of aminoacyl-tRNA to the ribosomal A site in the form of a ternary complex, eEF1A1-GTP-aminoacyl-tRNA (Negrutskii and El’skaya, 1998
). Codon-anticodon recognition is followed by GTP hydrolysis on eEF1A1, with subsequent release of eEF1A1-GDP from the ribosome. Our data suggests that interaction between eEF1A1 and hnRNP E1 inhibits eEF1A1 release following hydrolysis of GTP, so that eEF1A1-GDP remains on the ribosome, thereby preventing the subsequent translocation of the aminoacyl-tRNA to the peptidyl moiety of the growing peptide chain. The ribosome is thus stalled at the eEF1A1-dependent elongation stage, resulting in translational silencing. The interaction between eEF1A1 and hnRNP E1 is largely mediated by their independent BAT element binding capacity, thus allowing for a transcript-specific translational silencing activity in mRNAs which harbor a 33-nt BAT element in their 3′-UTR. The transcript-specific restriction of the BAT element is conferred by its structural fidelity rather than a conserved sequence homology, as is evident by the fact that Dab2
mRNA differ in the nucleotide sequence of their respective BAT elements (Chaudhury et al., 2010
). Furthermore, the binding affinity of both hnRNP E1 and eEF1A1 is impaired by mutations which alter the structure of the BAT element.
Insight into the structure-function relationships of eEF1A1 and hnRNP E1 may aid in providing an understanding of how hnRNP E1 prevents release of eEF1A1 from the ribosome. Similar to other GTP-binding proteins, eEF1A1 undergoes structural changes following hydrolysis. It has been shown that upon GTP hydrolysis, domains 2 and 3 of eEF1A1 rotate as a rigid body with respect to domain 1, thus resulting in a GTP-mediated conformational change (Berchtold et al., 1993
). In vitro
binding studies demonstrated that hnRNP E1 is capable of binding to eEF1A1 via its domains 1 and 3. Possibly, hnRNP E1 binding may be affecting the interface of domain 1–3 in eEF1A1, thereby preventing the post-GTP hydrolysis conformation change necessary for eEF1A1 release. It has also been proposed that isoform-specific functions of elongation factor 1-alpha play an important role in cancer (Edmonds et al., 1996
), and while the eEF1A1 isoform was used in the present study, we cannot rule out a potential role for eEF1A2 in BAT-mediated translational silencing.
While translational regulation, through 5′ and 3′-UTR regulatory elements, has most often been demonstrated to modulate the initiation stage, several reports suggest that control may occur at the post-initiation level. For example, the translational repression of nanos
mRNA in Drosophila
embryos has been shown to be mediated by nanos
3′-UTR sequences, resulting in an altered polyribosome profile, indicating a block after initiation of translation (Clark et al., 2000
). Additionally, during C. elegans
larval development, repression of lin-14
mRNA has been shown to be directed by a small lin-4
RNA which binds the 3′-UTR of lin-14 mRNA
and mediates translational repression at a post-initiation stage of translation (Olsen and Ambrose, 1999
). Alternatively, in cell-free translation assays, it has been demonstrated that phosphorylation of eukaryotic elongation factor-1B, a guanine nucleotide exchange factor for eEF1A1, by CDK1/cyclin B, results in decreased elongation rates (Monnier et al., 2001
). While these examples are suggestive of regulation at an event post- initiation, our identification of regulation being mediated at the elongation stage, precisely through modulation of eEF1A1 function, provide a mechanistic demonstration of such a translation control mechanism.
The biological implications of the BAT-mediated translational silencing mechanism are profound. Both in vitro
and in mice, knockdown of hnRNP E1 in NMuMG caused these otherwise normal, non-invasive epithelial cells to display an inherent tumorigenic and metastatic capacity. In allograftic tumor studies, SH14 cells readily formed large, slowly growing tumors In contrast, the parental cells formed tiny, regressing nodules consisting of normal epithelial cells as evidenced by strong E-cadherin expression in tumor tissue sections. Downregulation of E-cadherin expression in SH14-derived tumors, as well as upregulation of the EMT marker vimentin and strong expression of ILEI, corroborate our morphological analysis and substantiate the findings that ILEI is necessary for tumor formation (Waerner et al., 2006
). In addition, knockdown of hnRNP E1 in the human breast cancer line MCF7 caused these cells to acquire EMT markers and to form lung metastases when injected intravenously. While these results support a direct role for hnRNP E1 in EMT and metastasis, further studies are needed to address earlier steps of metastasis such as invasion and intravasation.
While the process of EMT in development and cancer progression has been shown to encompass a wide continuum of alterations in epithelial plasticity in response to numerous signaling pathways, an important finding of this study is that a single factor, hnRNP E1, is responsible for silencing a TGFβ-mediated EMT program normally functional during embryonic development. Whereas Dab2 and ILEI protein expression is directly regulated by the BAT-mediated translational silencing mechanism, our data suggests that the combined expression of Dab2 and ILEI alone is not sufficient for mediating EMT (Chaudhury et al., 2010
). Rather, we postulate that the BAT regulatory mechanism operates upstream of key cellular pathways contributing to metastatic progression and tumor development.
An understanding of the molecular mediators that control epithelial plasticity may aid in elucidating the downstream cellular pathways that are affected by activation of silenced EMT-transcripts. A key step in EMT is disintegration of adherens junctions associated with redistribution and repression of the E-cadherin. (Thiery and Sleeman, 2006
). Snail-related zinc-finger transcriptional repressors (Snail and Slug), as well as bHLH transcription factors Twist and E12/E47, are the most prominent suppressors of E-cadherin transcription (Zavadil and Bottinger, 2005
). Interestingly, a recent report has demonstrated that translational regulation mediated by Y-box binding protein-1 (YB-1) is upstream of, and regulates the expression of transcription factors implicated in repression of E-cadherin (Evdokimova et al., 2009
). Enhanced expression of YB-1 was shown to promote EMT and the metastatic potential of normal breast epithelial cells by activating cap-independent translation of Snail1. Similarly, our findings that shRNA-mediated knockdown of hnRNP E1 results in downregulation of E-cadherin and EMT, irrespective of TGFβ-treatment, may be indicative of activated downstream EMT transcriptional programs leading to downregulation of epithelial-related genes and activation of mesenchymal genes.
Numerous studies have shown that secretion of TGFβ by tumor cell-stimulated autocrine loops and loss of sensitivity to the antiproliferative effects of TGFβ are classical hallmarks during metastatic progression of tumors (Bierie and Moses, 2006
). Additionally, activation of the PI3K/Akt pathway, as well as increased expression of Akt2 have both been shown to lead to EMT and increased cell invasiveness in tumor cells (Irie et al., 2005
; Gershtein et al., 1999
). Attenuation of the BAT mRNP complex via
TGFβ/Akt2-mediated phosphorylation of hnRNP E1 may therefore represent a downstream target of cytokine-mediated activation of EMT during tumorigenesis and metastatic progression. Although our findings suggest that the proposed translational regulatory mechanism is functionally significant in the metastatic progression of tumors, further studies are needed to determine whether this pathway is deregulated in cancer cells and tissues. Given the necessity of hnRNP E1 in the BAT mRNP complex we speculate that expression levels and/or phosphorylation status of p-Akt2 and hnRNP E1, as well as the expression of Dab2
may be directly correlative with metastatic progression of tumors.