13 p.-4 fig.-1 graph. abst.
Understanding how the RNA-binding domains of a protein regulator are used to recognize its RNA targets is a key problem in RNA biology, but RNA-binding domains with very low affinity do not perform well in the methods currently available to characterize protein-RNA interactions. Here, we propose to use conservative mutations that enhance the affinity of RNA-binding domains to overcome this limitation. As a proof of principle, we have designed and validated an affinity-enhanced K-homology (KH) domain mutant of the fragile X syndrome protein FMRP, a key regulator of neuronal development, and used this mutant to determine the domain’s sequence preference and to explain FMRP recognition of specific RNA motifs in the cell. Our results validate our concept and our nuclear magnetic resonance (NMR)-based workflow. While effective mutant design requires an understanding of the underlying principles of RNA recognition by the relevant domain type, we expect the method will be used effectively in many RNA-binding domains.
This work was supported by the UK Medical Research Council (MC_PC_13051, U117574558, and MR/S000305/1) and the UK BBRSC research grant S014438/1. It was also supported by University College London and by the Francis Crick Institute, which receives its core funding from Cancer Research UK (CC2029), the UK Medical Research Council (CC2029), and the Wellcome Trust (CC2029). NMR spectra were recorded at the MRC Biomedical NMR Facility Francis Crick Institute UK, which is funded by Cancer Research UK (CC1078), the UK Medical Research Council (CC1078), and the Wellcome Trust (CC1078) and at UCL NMR facilities.