title: The Runt domain of AML1 (RUNX1) binds a sequence-conserved RNA motif that mimics a DNA element.
abstract: AML1 (RUNX1) is a key transcription factor for hematopoiesis that binds to the Runt-binding double-stranded DNA element (RDE) of target genes through its N-terminal Runt domain. Aberrations in the AML1 gene are frequently found in human leukemia. To better understand AML1 and its potential utility for diagnosis and therapy, we obtained RNA aptamers that bind specifically to the AML1 Runt domain. Enzymatic probing and NMR analyses revealed that Apt1-S, which is a truncated variant of one of the aptamers, has a CACG tetraloop and two stem regions separated by an internal loop. All the isolated aptamers were found to contain the conserved sequence motif 5'-NNCCAC-3' and 5'-GCGMGN'N'-3' (M:A or C; N and N' form Watson-Crick base pairs). The motif contains one AC mismatch and one base bulged out. Mutational analysis of Apt1-S showed that three guanines of the motif are important for Runt binding as are the three guanines of RDE, which are directly recognized by three arginine residues of the Runt domain. Mutational analyses of the Runt domain revealed that the amino acid residues used for Apt1-S binding were similar to those used for RDE binding. Furthermore, the aptamer competed with RDE for binding to the Runt domain in vitro. These results demonstrated that the Runt domain of the AML1 protein binds to the motif of the aptamer that mimics DNA. Our findings should provide new insights into RNA function and utility in both basic and applied sciences.
authors.affiliation: Research Institute for Clinical Oncology, Saitama Cancer Center, Ina, Saitama 362-0806, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Fukunaga
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nomura
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Ryo
authors.initials: R
authors.lastname: Amano
authors.forename: Taku
authors.initials: T
authors.lastname: Tanaka
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Gota
authors.initials: G
authors.lastname: Kawai
authors.forename: Taiichi
authors.initials: T
authors.lastname: Sakamoto
authors.forename: Tomoko
authors.initials: T
authors.lastname: Kozu
mesh_terms: D052157:Aptamers, Nucleotide; D001483:Base Sequence; D017124:Conserved Sequence; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D009690:Nucleic Acid Conformation; D059372:Nucleotide Motifs; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D050676:Core Binding Factor Alpha 2 Subunit; D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; D006147:Guanine
keywords: AML1/RUNX1; NMR; RNA aptamer; SELEX