A Conserved Three-nucleotide Core Motif Defines Musashi RNA Binding Specificity

• Published in: The Journal of biological chemistry Vol. 289; no. 51; pp. 35530 - 35541
• Main Authors: Zearfoss, N. Ruth, Deveau, Laura M., Clingman, Carina C., Schmidt, Eric, Johnson, Emily S., Massi, Francesca, Ryder, Sean P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 19.12.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Algorithms; Animals; Base Sequence; Binding Sites - genetics; Binding Specificity; Cell Proliferation; Conserved Sequence - genetics; Drosophila Proteins - genetics; Drosophila Proteins - metabolism; Fluorescence Anisotropy; Fluorescence Polarization; Humans; Kinetics; Magnetic Resonance Spectroscopy; Mice; Molecular Sequence Data; Mutation; Nerve Tissue Proteins - genetics; Nerve Tissue Proteins - metabolism; Nucleotide Motifs - genetics; Protein Binding; RNA; RNA - genetics; RNA - metabolism; RNA Recognition; RNA-binding Protein; RNA-Binding Proteins - biosynthesis; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; RNA-Protein Interaction; RRM; Sequence Homology, Nucleic Acid; Binding Specificity; RNA Recognition; Cell Proliferation; RNA-Protein Interaction; RNA; Fluorescence Anisotropy; RNA-binding Protein; RRM
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M114.597112

Musashi (MSI) family proteins control cell proliferation and differentiation in many biological systems. They are overexpressed in tumors of several origins, and their expression level correlates with poor prognosis. MSI proteins control gene expression by binding RNA and regulating its translation. They contain two RNA recognition motif (RRM) domains, which recognize a defined sequence element. The relative contribution of each nucleotide to the binding affinity and specificity is unknown. We analyzed the binding specificity of three MSI family RRM domains using a quantitative fluorescence anisotropy assay. We found that the core element driving recognition is the sequence UAG. Nucleotides outside of this motif have a limited contribution to binding free energy. For mouse MSI1, recognition is determined by the first of the two RRM domains. The second RRM adds affinity but does not contribute to binding specificity. In contrast, the recognition element for Drosophila MSI is more extensive than the mouse homolog, suggesting functional divergence. The short nature of the binding determinant suggests that protein-RNA affinity alone is insufficient to drive target selection by MSI family proteins. Background: The Musashi family of RNA-binding proteins promotes progenitor cell proliferation. Results: The majority of Musashi sequence specificity comes from a UAG motif. Sequences outside of UAG make minor contributions to affinity. Conclusion: UAG forms the core Musashi recognition element. Significance: Delineating the sequences that contribute to binding is critical to understanding RNA target selection.