Structure of Musashi1 in a complex with target RNA: the role of aromatic stacking interactions

Abstract

Mammalian Musashi1 (Msi1) is an RNA-binding protein that regulates the translation of target mRNAs, and participates in the maintenance of cell ‘stemness’ and tumorigenesis. Msi1 reportedly binds to the 3′-untranslated region of mRNA of Numb, which encodes Notch inhibitor, and impedes initiation of its translation by competing with eIF4G for PABP binding, resulting in triggering of Notch signaling. Here, the mechanism by which Msi1 recognizes the target RNA sequence using its Ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2 has been revealed on identification of the minimal binding RNA for each RBD and determination of the three-dimensional structure of the RBD1:RNA complex. Unique interactions were found for the recognition of the target sequence by Msi1 RBD1: adenine is sandwiched by two phenylalanines and guanine is stacked on the tryptophan in the loop between β1 and α1. The minimal recognition sequences that we have defined for Msi1 RBD1 and RBD2 have actually been found in many Msi1 target mRNAs reported to date. The present study provides molecular clues for understanding the biology involving Musashi family proteins.

RNA-Binding Protein Musashi1 Is a Central Regulator of Adhesion Pathways in Glioblastoma

“…The UAG trinucleotide, which has been previously reported as the core of the MSI1 binding site (43), was generally enriched around MSI1 iCLIP sites ( Fig. 1B and C).…”

“…Nuclear magnetic resonance structures have suggested that the two RNA recognition motif (RRM) domains of MSI1 act independently to recognize RNA motifs (43). We investigated the tendency of UAG/GUAG motif pairs, in contrast to isolated UAG or GUAG sequences, to occur at iCLIP sites compared to flanking regions in different contexts.…”

“…We then investigated how often the two RRMs act together to recognize target mRNAs. Recent in vitro work has suggested GUAG and UAG as cores of the primary sequence motif recognized by each MSI1 RRM (43). Around MSI1 iCLIP sites in 3= UTRs but not in other locations in the RNA, NUAG (where N is any nucleotide other than G) and GUAG tetramers appear significantly more often in pairs than in flanking regions, establishing that a proportion of MSI1 binding locations preferentially contain binding sequences for both RRMs.…”

Utilizing NMR to study RNA- and compound-binding mechanisms of Musashi-1, a stem/progenitor cell marker in various normal and cancer cells

“…Previously, we applied NMR methods to Msi1 RBD1 and RBD2 individually, and determined the solution structures in their free forms, identified the residues involved in RNA-binding, and assessed the backbone dynamics and discussed the origin of high RNA-binding affinity [8,9]. Our subsequent NMR analysis identified the minimal recognition RNA sequences for Msi1 RBD1 and RBD2 to be r(GUAG) and r(UAG), respectively [10]. Then, we determined the NMR solution structure of Msi1 RBD1-r(GUAGU) complex, which clearly revealed the detailed interactions between Msi1 RBD1 and r(GUAG) [10].…”

“…Our subsequent NMR analysis identified the minimal recognition RNA sequences for Msi1 RBD1 and RBD2 to be r(GUAG) and r(UAG), respectively [10]. Then, we determined the NMR solution structure of Msi1 RBD1-r(GUAGU) complex, which clearly revealed the detailed interactions between Msi1 RBD1 and r(GUAG) [10]. Furthermore, a theoretical analysis based on statistical mechanics of hydration was applied to Msi1 RBD1-r(GUAG) interaction to provide a deeper understanding into the recognition mechanism [11].…”