Logo: to the web site of Uppsala University

RNA Recognition Motifs (RRMs) are essential post-transcriptional regulators of gene expression in eukaryotic cells. The Human Musashi-1 (MSI-1) is an RNA-binding protein that recognizes (G/A)U1-3AGU and UAG sequences in diverse RNAs through two RRMs and regulates the fate of target RNA.

Here, we combined structural biology and computational approaches to analyse the binding of the RRM domains of human MSI-1 with single-stranded and structured RNAs ligands. We used our recently developed computational tool RRMScorer to design a set of mutants of the MSI-1 protein to bind novel RNA sequences to alter the binding selectivity. The in-silico predictions of the designed protein-RNA interactions are assessed by NMR and SPR. These experiments also are used to study the competition of the two RRM domains of MSI-1 for the same binding site within linear and harpin RNA. Our experimental results confirm the in-silico designed interactions, thus opening the way for the development of new biomolecules for in vitro and in vivo studies and downstream applications.

The kinetics of the interaction between Musashi-1 (MSI1) and RNA have been characterized using surface plasmon resonance biosensor analysis. Truncated variants of human MSI1 encompassing the two homologous RNA recognition motifs (RRM1 and RRM2) in tandem (aa 1-200), and the two RRMs in isolation (aa 1-103 and aa 104-200, respectively) were produced. The proteins were injected over sensor surfaces with immobilized RNA, varying in sequence and length, and with one or two RRM binding motifs. The interactions of the individual RRMs with all RNA variants were well described by a 1:1 interaction model. The interaction between the MSI1 variant encompassing both RRM motifs was bivalent and rapid for all RNA variants. Due to difficulties in fitting this complex data using standard procedures, we devised a new method to quantify the interactions. It revealed that two RRMs in tandem resulted in a significantly longer residence time than a single RRM. It also showed that RNA with double UAG binding motifs and potential hairpin structures forms less stable bivalent complexes with MSI1 than the single UAG motif containing linear RNA. Substituting the UAG binding motif with a CAG sequence resulted in a reduction of the affinity of the individual RRMs, but for MSI1, this reduction was strongly enhanced, demonstrating the importance of bivalency for specificity. This study has provided new insights into the interaction between MSI1 and RNA and an understanding of how individual domains contribute to the overall interaction. It provides an explanation for why many RNA-binding proteins contain dual RRMs.

Reporter systems are widely used to study biomolecular interactions and processes in vivo, representing one of the basic tools used to characterize synthetic regulatory circuits. Here, we developed a method that enables the monitoring of RNA–protein interactions through a reporter system in bacteria with high temporal resolution. For this, we used a Real-Time Protein Expression Assay (RT-PEA) technology for real-time monitoring of a fluorescent reporter protein, while having bacteria growing on solid media. Experimental results were analyzed by fitting a three-variable Gompertz growth model. To validate the method, the interactions between a set of RNA sequences and the RNA-binding protein (RBP) Musashi-1 (MSI1) were evaluated, as well as the allosteric modulation of the interaction by a small molecule (oleic acid). This new approach proved to be suitable to quantitatively characterize RNA–RBP interactions, thereby expanding the toolbox to study molecular interactions in living bacteria, including allosteric modulation, with special relevance for systems that are not suitable to be studied in liquid media.