Formation of an αCP1-KH3 complex with UC-rich RNA

• Published in: European biophysics journal 2005-07, Vol.34 (5), p.423-429
• Main Authors: Sidiqi, M., Wilce, J. A., Porter, C. J., Barker, A., Leedman, P. J., Wilce, M. C. J.
• Format: Article
• Language: English
• ISSN: 0175-7571; 1432-1017
• DOI: 10.1007/s00249-005-0467-y

The alpha CP family of proteins [also known as poly(C)-bindlng or heterogeneous nuclear ribonucleoprotein E proteins] are involved in the regulation of messenger RNA (mRNA) stability and translational efficiency. They bind via their triple heterologous nuclear ribonucleoprotein K homology (KH) domain structures to C-rich mRNA, and are thought to interact with other mRNA-binding proteins as well as provide direct nuclease protection. In particular, alpha CP1 and alpha CP2 have been shown to bind to a specific region of androgen receptor (AR) mRNA, resulting in its increased stability. The roles of each of the KH motifs in the binding affinity and the specificity is not yet understood. We report the beginning of a systematic study of each of the alpha CP KH domains, with the cloning and expression of alpha CP1-KH2 and alpha CP1-KH3. We report the ability of alpha CP1-KH3, but not alpha CP1-KH2, to bind the target AR mRNA sequence using an RNA electrophoretic mobility gel shift assay. We also report the preparation of an alpha CP1-KH3/AR mRNA complex for structural studies. super(1)H- super(15)N heteronuclear single quantum correlation NMR spectra of super(15)N-labelled alpha CP1-KH3 verified the integrity and good solution behaviour of the purified domain. The titration of the 11-nucleotide RNA target sequence from AR mRNA resulted in a rearrangement of the super(1)H- super(15)N correlations, demonstrating the complete binding of the protein to form a homogeneous protein/RNA complex suitable for future structural studies.