The multidomain RNA-binding protein IMP3:combinatorial RNA recognition and functions during mRNP biogenesis

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The human IGF2BP proteins (Insulin like Growth Factor 2 mRNA Binding-Protein; or shortly IMP) are a family of three highly conserved mRNA-binding proteins, IMP1, IMP2, and IMP3, characterized by a unique modular structure with two N-terminal RRM motifs and four C-terminal KH domains. Here we focus on IMP3, which is particularly interesting as a pancreatic cancer marker. In the first funding period we established an integrative systematic strategy, combining single-domain-resolved SELEX-seq, including motif-spacing analyses, with in vivo iCLIP and structural studies. Our initial iCLIP analyses had shown that IMP3 binds mRNAs primarily within their 3’-UTR regions, but also within certain internal exons. Interestingly, IMP3 targets are strongly enriched for mRNAs coding for membrane and secretory proteins, suggesting a new, post-transcriptional role of IMP3 in ER-proximal localization. Our integrative approach identified the RNA-binding specificity and RNP topology of IMP3, involving all six RNA-binding domains (RBDs) and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Thereby both specificity and flexibility of IMP3-RNA recognition can be explained, providing a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation (Schneider et al., 2018).In parallel we investigated the specific and stable association of IMP3 with circular RNAs (circRNAs), a new class of noncoding RNAs generated from pre-mRNAs by alternative splicing. Based on RNA-Seq analysis of IMP3-coimmunoprecipitated RNA, we identified a subgroup of IMP3-containing circRNPs, which were validated and characterized (Schneider et al., 2016). On this basis, we plan to investigate in more detail the molecular mechanisms of RNA recognition and the functional spectrum of IMP3 as an mRNP component, focussing on these three specific aims and topics: 1. Defining and validating the general IMP3 RNA-recognition code,2. Searching for functional roles of IMP3 protein in the mRNA life cycle: 3’ processing, stability, cellular localization, translation3. Uncovering potential IMP3-mediated mRNA-circRNA networks. In sum, the major aim of our project in the second funding period will be to investigate the functional spectrum of IMP3, a prototypical multidomain RNA-binding protein. These studies will rely on the combinatorial RNA-recognition code for IMP3, which we established in the last three years and which we continue to evaluate further. In parallel, we will pursue our search for IMP3-mediated functional links and regulatory networks between mRNP and circRNP biogenesis pathways.