Abstract
Protein synthesis requires large amounts of energy and is therefore tightly controlled, primarily through regulation of translation initiation1,2,3,4. Translation initiation predominately occurs when a series of eukaryotic initiation factors (eIFs) recognize and bind the 5’ methyl guanosine (5’ 7mG) cap of the mRNA and recruit the ribosome, triggering scanning for a start codon2,3. One initiation factor, eukaryotic initiation factor 4A (eIF4A), is an ATP dependent helicase that unwinds secondary structure in the 5’ UTR of the mRNA to allow for ribosome association and scanning for a start codon7,8. Another initiation factor, eukaryotic initiation factor 4B (eIF4B), stimulates the helicase activity of eIF4A9 and binds to sections of mRNA previously unwound by eIF4A, preventing reformation of secondary structures10. Under certain conditions, primarily cellular stress, translation initiation of select mRNAs will occur without interaction with the 5’ 7mG cap, through the utilization of an internal ribosomal entry site (IRES)11. IRES mediated translation initiation often requires a subset of the canonical translation initiation factors but the specific factors needed varies between mRNAs3,14,15. Two known IRES containing mRNAs that code for proteins involved in cellular stress responses are those of insulin receptor (INSR) and insulin-like growth factor 1 receptor (IGF1R)16,17,18,19. Previous work has demonstrated that the INSR and IGF1R IRESes are resistant to inhibition of eIF4A20 and eukaryotic initiation factor 4G (eIF4G)24. The present study utilized bicistronic luciferase reporter constructs featuring the 5’ UTR of the genes of interest flanked by the open reading frames (ORFs) of renilla and firefly luciferase to examine the necessity of eIF4B in IRES mediated translation of INR and IGF1R. The reporters are constructed such that the ratio of firefly to renilla signal is indicative of the ratio of IRES mediated to canonical translation initiation. Overexpression of eIF4B resulted in increased IRES mediated translation of INR but not IGF1R and knockdown of eIF4B lead to a reduction in IRES mediated translation of both INR and IGFR. This data indicates that eIF4B likely promotes or is required for IRES mediated translation of INR and IGF1R but endogenous eIF4B levels are fully saturating for IGF1R translation initiation. This result is surprising given that eIF4B is thought to primarily function as an enhancer of eIF4A activity, which is not required for IRES mediated translation of INR and IGF1R.