Formation of an aCP1-KH3 complex with UC-rich RNA

The agrCP family of proteins [also known as poly(C)-binding 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, agrCP1 and agrCP2 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 agrCP KH domains, with the cloning and expression of agrCP1-KH2 and agrCP1-KH3. We report the ability of agrCP1-KH3, but not agrCP1-KH2, to bind the target AR mRNA sequence using an RNA electrophoretic mobility gel shift assay. We also report the preparation of an agrCP1-KH3/AR mRNA complex for structural studies. 1H–15N heteronuclear single quantum correlation NMR spectra of 15N-labelled agrCP1-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 1H–15N correlations, demonstrating the complete binding of the protein to form a homogeneous protein/RNA complex suitable for future structural studies.