Quantitative analysis of CUG-BP1 binding to RNA repeats.
CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-3-like factors (CELF) family of RNA-binding proteins, and is involved in myotonic dystrophy type 1 (DM1). Several mRNA targets of CUG-BP1 have been identified, including the insulin receptor, muscle chloride channel, and cardiac troponin T. On the other hand, CUG-BP1 has only a weak affinity for CUG repeats. We conducted quantitative-binding assays to assess CUG-BP1 affinities for several repeat RNAs by surface plasmon resonance (SPR). Although we detected interactions between CUG-BP1 and CUG repeats, other UG-rich sequences actually showed stronger interactions. Binding constants of CUG-BP1 for RNAs indicated that the affinity for UG repeats was far stronger than for CUG repeats. We also found that N-terminal deletion mutant of CUG-BP1 has UG repeat-binding activity in a yeast three-hybrid system, although C-terminal deletion mutant does not. Our data indicates that CUG-BP1 specifically recognized UG repeats, probably through cooperative binding of RNA recognition motifs at both ends of the protein. This is the first report of a binding constant for CUG-BP1 calculated in vitro.
Department of Life Sciences, Graduate School of Arts and Sciences, the University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo, Japan.
Daisuke
D
Mori
Noboru
N
Sasagawa
Yoshihiro
Y
Kino
Shoichi
S
Ishiura
D000067879:CELF1 Protein; D004252:DNA Mutational Analysis; D006801:Humans; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D020349:Surface Plasmon Resonance; D020798:Two-Hybrid System Techniques
D000067879:CELF1 Protein; C102843:CELF1 protein, human; D009711:Nucleotides; D016601:RNA-Binding Proteins; D012313:RNA