How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We establish an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six 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. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, allows the prediction of IMP3 targets, and provides a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

How multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. Here we focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We have established an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six 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. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, providing a paradigm for the function of multivalent interactions with multidomain RNA-binding proteins in gene regulation.

d-Galacturonic acid (GalA) is the major constituent of pectin-rich biomass, an abundant and underutilized agricultural byproduct. By one reductive step catalyzed by GalA reductases, GalA is converted to the polyhydroxy acid l-galactonate (GalOA), the first intermediate of the fungal GalA catabolic pathway, which also has interesting properties for potential applications as an additive to nutrients and cosmetics. Previous attempts to establish the production of GalOA or the full GalA catabolic pathway in Saccharomyces cerevisiae proved challenging, presumably due to the inefficient supply of NADPH, the preferred cofactor of GalA reductases. Here, we tested this hypothesis by coupling the reduction of GalA to the oxidation of the sugar alcohol sorbitol that has a higher reduction state compared to glucose and thereby yields the necessary redox cofactors. By choosing a suitable sorbitol dehydrogenase, we designed yeast strains in which the sorbitol metabolism yields a “surplus” of either NADPH or NADH. By biotransformation experiments in controlled bioreactors, we demonstrate a nearly complete conversion of consumed GalA into GalOA and a highly efficient utilization of the co-substrate sorbitol in providing NADPH. Furthermore, we performed structure-guided mutagenesis of GalA reductases to change their cofactor preference from NADPH towards NADH and demonstrated their functionality by the production of GalOA in combination with the NADH-yielding sorbitol metabolism. Moreover, the engineered enzymes enabled a doubling of GalOA yields when glucose was used as a co-substrate. This significantly expands the possibilities for metabolic engineering of GalOA production and valorization of pectin-rich biomass in general.