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

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In-vitro assessment of Numb RNA binding to Musachi-2 RNA-binding protein

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Many cancer treatments are available today, notably surgery, radiation therapy, chemotherapy, immunotherapy, hormone therapy, and stem cell transplants. Used alone, in combination, or sequentially, the type of treatment received by patients depends on the category of cancer and its stage of progression. Research efforts continue to seek new molecular targets, new signaling pathways, and new treatment strategies, all with a common goal: to fight cancer more effectively with new therapeutic approaches.
Supported by a growing number of papers and reviews, RNA-binding proteins (RNA-BPs) are currently accepted as modulators of cancer progression and therapeutic response. They play an important role in cancer development and therefore represent attractive targets for novel therapies. The RNA-binding proteins Musashi-1 (MSI1) and Musashi-2 (MSI2) are emerging as regulators of multiple critical biological processes relevant to cancer initiation, progression, and drug resistance.
In this article, Lan Lan and her team developed an in-vitro assay to characterize the binding of RNA moieties to MSI2. By using fluorescence techniques, titration, and competition experiments, they determined the high binding specificity of Numb, an RNA probe, to its binding pocket into the MSI2 RNA-binding protein. The data was further confirmed in NMR titrations and structure modeling studies. The RNA binding parameters, with Kd values in the low nM range, suggest the possibility of identifying novel small molecule inhibitors that are either specific to MSI2 or that target both MSI1 and MSI2.
The next generation of anti-cancer drugs is right at our doorstep. Set-up your own in-vitro assay to screen RNA/RNA-binding protein inhibitors to see if your thoughts are headed in the right direction.

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.

Details

Oncotarget, 2017 Nov 20;8(63):106587-106597.

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