Abstract
IGF2 mRNA-binding protein 1 (IMP1) is a key regulator of messenger RNA (mRNA) metabolism and transport in organismal development and, in cancer, its mis-regulation is an important component of tumour metastasis. IMP1 function relies on the recognition of a diverse set of mRNA targets that is mediated by the combinatorial action of multiple RNAbinding domains. Here, we dissect the structure and RNA-binding properties of two key RNA-binding domains of IMP1, KH1 and KH2, and we build a kinetic model for the recognition of RNA targets. Our data and model explain how the two domains are organized as an intermolecular pseudo-dimer and that the important role they play in mRNA target recognition is underpinned by the high RNA-binding affinity and fast kinetics of this KH1KH2-RNA recognition unit. Importantly, the high-affinity RNA-binding by KH1KH2 is achieved by an inter-domain coupling 50- fold stronger than that existing in a second pseudodimer in the protein, KH3KH4. The presence of this strong coupling supports a role of RNA re-modelling in IMP1 recognition of known cancer targets.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Nucleic Acids Research |
| Vol/bind | 47 |
| Udgave nummer | 8 |
| Sider (fra-til) | 4334-4348 |
| Antal sider | 15 |
| ISSN | 0305-1048 |
| DOI | |
| Status | Udgivet - 2019 |
Finansiering
We thank Drs Chris Gallagher, Virginia Castilla Llorente and Evangelos Christodoulou for assistance with cloning, Dr Virginia Castilla Llorente for advice on the analysis of IMP1 target sequences and Ms Sofia Verma for the preparation of a protein sample. We gratefully acknowledge Diamond Light Source (Grant No MX13775) and beamline IO2 for access. NMR spectra were recorded at the MRC Biomedical NMR and UCL NMR facilities and we thank Alain Oregioni and Angelo Figuereido for assistance. We thank Laura Masino for assistance in the recording of CD thermal denaturation data. Authors contributions: Cloning of the KH1KH2 construct and preparation of the protein samples was performed by R.D. NMR experiments, including relaxation and RDC measurements, were recorded and analysed by R.D. and G.K. ITC experiments were recorded and analysed by R.D. BLI experiments were recorded by R.D. and F.H. and analysed by S.M., F.H. and R.D. Kinetic modelling was performed by S.M. Crystallization was performed by R.O. and R.D. X-ray data collection, data processing, model building and refinement were performed by N.B and A.P. The project was designed by A.R. and the manuscript was written by A.R., R.D., I.T., S.M. and N.B. with help from all of the other authors. This work was supported by the UK Medical Research Council (grant numbers U117574558, MC PC 13051 to A.R.). It was also supported by University College London (A.R.) and by the Francis Crick Institute (I.A.T), which receives its core funding from Cancer Research UK (FC001178) the UK Medical Research Council (FC001178) and the Wellcome trust (FC001178). The work was supported by the Francis Crick Institute also through provision of access to the MRC Biomedical NMR Centre (Francis Crick Institute core funding by Cancer Research UK (FC001029), the UK Medical Research Council (FC001029), and the Wellcome Trust (FC001029). Funding for open access charge: RCUK to UCL. Conflict of interest statement. None declared.
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