Formation of an alphaCP1-KH3 complex with UC-rich RNA.
The alphaCP 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, alphaCP1 and alphaCP2 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 alphaCP KH domains, with the cloning and expression of alphaCP1-KH2 and alphaCP1-KH3. We report the ability of alphaCP1-KH3, but not alphaCP1-KH2, to bind the target AR mRNA sequence using an RNA electrophoretic mobility gel shift assay. We also report the preparation of an alphaCP1-KH3/AR mRNA complex for structural studies. (1)H-(15)N heteronuclear single quantum correlation NMR spectra of (15)N-labelled alphaCP1-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 (1)H-(15)N correlations, demonstrating the complete binding of the protein to form a homogeneous protein/RNA complex suitable for future structural studies.
Sidiqi
M
M
School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, Perth, WA 6009, Australia.
Wilce
J A
JA
Porter
C J
CJ
Barker
A
A
Leedman
P J
PJ
Wilce
M C J
MC
D020816:Amino Acid Motifs; D001665:Binding Sites; D001703:Biophysics / Q000379:methods; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D034641:Heterogeneous-Nuclear Ribonucleoprotein K / Q000737:chemistry* / Q000378:metabolism; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D011944:Receptors, Androgen / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D004268:DNA-Binding Proteins; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D046911:Macromolecular Substances; C094604:PCBP1 protein, human; D020033:Protein Isoforms; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011944:Receptors, Androgen; D012313:RNA; D005982:Glutathione Transferase