Human oncoprotein Musashi-2 N-terminal RNA recognition motif backbone assignment and identification of RNA-binding pocket

Abstract

RNA-binding protein Musashi-2 (MSI2) is a key regulator in stem cells, it is over-expressed in a variety of cancers and its higher expression is associated with poor prognosis. Like Musashi-1, it contains two N-terminal RRMs (RNA-recognition Motifs, also called RBDs (RNA-binding Domains)), RRM1 and RRM2, which mediate the binding to their target mRNAs. Previous studies have obtained the three-dimensional structures of the RBDs of Musashi-1 and the RBD1:RNA complex. Here we show the binding of MSI2-RRM1 to a 15nt Numb RNA in Fluorescence Polarization assay and time resolved Fluorescence Resonance Energy Transfer assay. Using nuclear magnetic resonance (NMR) spectroscopy we assigned the backbone resonances of MSI2-RRM1, and characterized the direct interaction of RRM1 to Numb RNA r(GUAGU). Our NMR titration and structure modeling studies showed that MSI2-RRM1 and MSI1-RBD1 have similar RNA binding events and binding pockets. This work adds significant information to MSI2-RRM1 structure and RNA binding pocket, and contributes to the development of MSI2 specific and MSI1/MSI2 dual inhibitors.

Crystal and solution structures of human oncoprotein Musashi‐2 N‐terminal RNA recognition motif 1

“…Based on our previously published RNA titration work, in MSI2‐RRM1, F64 and F66 in RNP1 and F24 in RNP2 are likely to be responsible for the canonical aromatic base stacking interaction with RNA. Compared with other RBPs, MSI2‐RRM1 has a potentially unique feature in that a tryptophan in the β1‐ɑ1 loop (W30) and a phenylalanine in the C‐terminus (F97) may form noncanonical base‐stacking interactions with RNA . So far, we find that this feature is likely limited to the Musashi family proteins.…”

“…MSI2‐RRM1 protein used in NMR study was purified as previously described . MSI2‐RRM1 purification for crystallization was the same as the NMR except the last step of the protein was purified in the buffer of 20 mM HEPES (pH 7.5), 100 mM NaCl.…”

“…The NMR samples contained 400 μM‐1 mM of 13 C and 15 N labeled MSI2‐RRM1 in 20 mM MES (pH 6.0), 150 mM NaCl. Backbone resonance assignments of MSI2‐RRM1 were reported previously under BMRB code 27111 . Side chain assignments were made by analyzing HCCH‐COSY and HCCH‐TOCSY (10.9 ms mixing time) spectra.…”

“…MSI2-RRM1 protein used in NMR study was purified as previously described. 27 MSI2-RRM1 purification for crystallization was the same as the NMR except the last step of the protein was purified in the buffer of 20 mM HEPES (pH 7.5), 100 mM NaCl. To remove the His-tag of purified MSI2-RRM1 for crystallization, determination of the digestion efficiency of the purified proteins by TEV protease was carried out in a total volume of 50 μL containing 100 μg of protein in assay buffer (50 mM Tris-Cl, pH 8.0, 0.5 mM EDTA, and 10 mM β-mercaptoethanol), and various amount of TEV protease (1-50 μg).…”

“…Backbone resonance assignments of MSI2-RRM1 were reported previously under BMRB code 27111. 27 Side chain assignments were made by analyzing HCCH-COSY 40 and HCCH-TOCSY 41 (10.9 ms mixing time) spectra. Distance restraints were obtained using 3D 15 N edited NOESY-HSQC 42 (120 ms mixing time) and 13 C edited NOESY-HSQC 42 (120 ms mixing time) spectra.…”