Structural probing of the cap‐independent vascular endothelial growth factor messenger RNA

Abstract

Internal ribosome entry sites (IRES) are cis‐regulatory elements located at the 5′ leader region of certain messenger RNA transcripts. IRESs have the capacity to recruit the translation machinery to initiate protein production in a cap‐independent mechanism. Structural studies on viral IRESs have articulated a structure based mechanism that viral IRES RNAs adopt secondary structures that are favorable for ribosome binding and place mRNAs in the decoding center to promote initiation of protein synthesis. Despite ~10% of eukaryotic mRNAs possess 5′ IRESs, structural information towards functional elucidation of cellular IRESs are still lacking. Cellular IRESs have showcased their importance particularly under stress conditions when cap‐independent protein synthesis is not impaired as cap‐dependent translation is. Therefore, at least under certain conditions, cellular IRESs provide a mechanism of translational control for a select set of proteins. An important and well‐studied example is that of vascular endothelial growth factor (VEGF), where the 5′ untranslated region of the mRNA behaves as an IRES to enhance expression of VEGF during malignant transformation. Here we used chemical probing and cryo‐electron microscopy (cryo‐EM) to investigate the structure of VEGF IRES in solution and when it is bound to the small ribosomal subunit. Integrating experimental information from selective 2′‐hydroxyl acylation analyzed by primer extension (SHAPE) analysis, we generated a secondary structure model for the VEGF IRES RNA. In addition, we present a 10Å cryo‐EM density map of the VEGF IRES in complex with 40S ribosomal subunit. The structural information enables us to identify the location that the cellular IRES occupies on the 40S ribosomal subunit to mimic an initiation‐like state.