Interactions of heterogeneous nuclear ribonucleoprotein D-like protein JKTBP and its domains with high-affinity binding sites.
JKTBP proteins consisting of two canonical RNA binding domains (RBDs) and a glycine-rich carboxyl domain are nucleocytoplasmic shuttling proteins. We studied in vivo and in vitro interactions between JKTBP and RNA. UV cross-linking experiments on HL-60 cells indicated that following RNA synthesis inhibition by actinomycin D, JKTBP1 accumulated in the cytoplasam is bound to poly(A)(+) RNAs. Recombinant JKTBP1 protein blots could bind poly(A)(+) RNAs, but not poly(A)(-) RNAs. For examination of RNA binding specificity of JKTBP, we enriched high binding sites from pools of 20 nt random sequence-containing RNAs by a selection/amplification method. After eight rounds of a selection and amplification, >20 sequences for each of JKTBPs 1 and 2 were identified. Their consensus high-affinity site was ACUAGC. Approximate K(d)s of JKTBPs 2 and 1 were estimated to be 6-12 nM for the selected sequences by filter binding assays. JKTBP deletion analysis indicated that not individual RBDs, both RBDs and the N-terminal 15 amino acids of the carboxyl domain are required for sequence-specific and high-affinity binding. These results indicate that JKTBP is a sequence-specific RNA binding protein differing from the related heterogeneous nuclear ribonucleoproteins A1 and D.
Kamei
Daisuke
D
Graduate School of Integrated Science, Yokohama City University, 22-2 Seto, Kanazawa, Yokohama 236-0027, Japan.
Yamada
Michiyuki
M
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D018922:HL-60 Cells; D034502:Heterogeneous-Nuclear Ribonucleoprotein D / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009843:Oligoribonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D034502:Heterogeneous-Nuclear Ribonucleoprotein D; D009843:Oligoribonucleotides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins