Spatial arrangement of conserved RNA recognition elements marks targets for post-transcriptional control

Abstract

How RNA-binding proteins recognize specific sets of target mRNAs remains poorly understood because current approaches depend heavily on sequence information alone. In this work, I demonstrate that specific recognition of mRNAs by RNA-binding proteins requires the correct spatial environment for these sequences. I characterized both the cis-acting sequence elements and the spatial restraints that define the mode of RNA-binding of the zipcode-binding protein 1 (ZBP1/IMP1/IGF2BP1) and its homolog, the insulin-like growth factor II mRNA binding protein 2 (IMP2/IGF2BP2).;ZBP1 was originally identified for its role in localization of the beta-actin mRNA by recognition of a 54-nucleotide cis-acting 'zipcode' in the beta-actin mRNA 3' untranslated region. The third and fourth KH (hnRNP K-homology) domains of ZBP1 specifically recognize a bipartite RNA element within the zipcode comprised of a 5' element, CGGAC, followed by a variable 3' element, C/A-CA-C/U that must be appropriately spaced. Remarkably, the orientation of these elements is interchangeable within target transcripts bound by ZBP1. The spatial relationship of this consensus binding site identified conserved transcripts that were verified to associate with ZBP1 in vivo. One of these transcripts, spinophilin, demonstrated reduced dendritic localization in developing ZBP1 knockout neurons.;IMP2 is one of a limited number of known genetic risk factors implicated by genome-wide association studies in the etiology of type 2 diabetes. The physiologic function of IMP2 in the development of type 2 diabetes is unknown. We used stringent in vitro selection to characterize the cis-acting sequence elements recognized by IMP2. Similar to ZBP1, the KH3 and KH4 domains of IMP2 specifically recognize a bipartite RNA element comprised of a 5' element, CUCAC, followed by a variable 3' element, A/U-GG-A/U, that must be properly spaced. The spatial relationship of these consensus RNA recognition elements identified approximately 500 conserved transcripts. Functional gene ontology analysis of IMP2 target transcripts revealed marked enrichment of mRNAs implicated in progression of metabolic diseases including type 2 diabetes, suggesting defective post-transcriptional regulation of IMP2 KH34 ligands may be the mechanistic basis for its genetic link to this condition.