CELF1 is an EIF4E binding protein that promotes translation of epithelial-mesenchymal transition effector mRNAs

Abstract

Mounting evidence is revealing a granularity within gene regulation that occurs at the level of mRNA translation. Within mammalian cells, canonical cap-dependent mRNA translation is dependent upon the interaction between the m 7 G cap-binding protein eukaryotic initiation factor 4E (eIF4E) and the scaffolding protein eukaryotic initiation factor 4G (eIF4G), the latter of which facilitates pre-translation initiation complex assembly, mRNA circularization, and ultimately ribosomal scanning. In breast epithelial cells, we previously demonstrated that the CELF1 RNA-binding protein promotes the translation of epithelial to mesenchymal transition (EMT) effector mRNAs containing GU-rich elements (GREs) within their 3' untranslated regions (UTRs). Here we show that within this context, CELF1 directly binds to both the eIF4E capbinding protein and Poly(A) binding protein (PABP), promoting translation of GRE-containing mRNAs in mesenchymal cells. Disruption of this CELF1/eIF4E interaction inhibits both EMT induction and experimental metastasis. Our findings illustrate a novel way in which noncanonical mechanisms of translation initiation underlie transitional cellular states within the context of development or human disease.It has now been well-documented that both oncogenes and signaling pathways regulating expression of these oncogenes together converge on the regulation of mRNA translation (2,15).Indeed, within the process of cellular transformation, mechanisms at each stage of translation and the ribosomal machinery may be co-opted to fostering translation that perpetuates the oncogenic program (16)(17)(18). This motivated us to more closely examine how CELF1 regulates translation of GRE-containing EMT effector mRNAs during EMT of breast epithelial cells. Results CELF1 interacts directly with eIF4E and PABP at the m 7 G cap, independent of intact eIF4G1Our previous work suggested that CELF1's control of GU-rich element-containing EMT effector mRNA translation is mediated at the level of 5' m 7 G cap-dependent translation initiation (10). We first confirmed and extended these findings by examining an additional CELF1 regulatory target within this context. We fused the 3' UTRs of a subset of GRE-containing mRNAs (JUNB, CRLF1, SNAI1, and SSBP2) downstream of the Renilla luciferase coding sequence in the pRL-TK-CXCR4-6x (19) reporter plasmid (Figure 1a). Transfection of the GREcontaining 3' UTR reporters into MCF10A cells, either untreated or treated with TGF-β to induce EMT, revealed a significant increase in reporter activity specific to treated cells Figure 1a), independent of any differences in relative mRNA expression (Figure 1b). In parallel, we built a battery of bicistronic constructs in which the same thymidine kinase promoter was utilized to drive expression of the firefly luciferase coding sequence, followed by the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), the Renilla luciferase open reading frame and individual GRE-containing 3' UTRs (pFR-EMCV) (Figure 1c). In contrast to cap-depen...

On the limits of inferring biophysical parameters of RBP-RNA interactions from in vitro RNA Bind’n Seq data

“…This could be interesting in light of CELF1 being a translational regulator of epithelial-mesenchymal transition via the binding of both cap-binding EIF4E and the poly(A)-binding protein. 21 …”