Interactions of heterogeneous nuclear ribonucloprotein D-like protein JKTBP and its domains with high-affinity binding sites

• Published in: Gene Vol. 298; no. 1; pp. 49 - 57
• Main Authors: Kamei, Daisuke, Yamada, Michiyuki
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 18-09-2002
• Subjects: Heterogeneous nuclear ribonucleoprotein; mRNA binding protein; Nucleocytoplasmic shuttling protein; RNA binding determinant; RNA binding specificity; Nucleocytoplasmic shuttling protein; RNA binding determinant; TPA, 12- O-tetradecanoylphorbol 13-acetate; GST, glutathione S-transferase; Heterogeneous nuclear ribonucleoprotein; hnRNP, heterogeneous nuclear ribonucleoprotein; mAb, monoclonal antibody; SDS, sodium dodecyl sulfate; SELEX, systematic evolution of ligands by exponential enrichment; DTT, dithiothreitol; RNA binding specificity; PBS(−), Ca 2+- and Mg 2+-free phosphate-buffered saline; mRNA binding protein; PMSF, phenylmethylsulfonyl fluoride; nt, nucleotide(s); PAGE, polyacrylamide gel electrophoresis; cDNA, DNA complementary to RNA; RBD, RNA binding domain
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(02)00926-5

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
ds 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.