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Journal of Biochemistry Advance Access published online on November 26, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm230
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© 2007 The Japanese Biochemical Society

Quantitative analysis of CUG-BP1 binding to RNA repeats

Daisuke Mori(1),#, Noboru Sasagawa(1),(3),#, Yoshihiro Kino(1),(2) and Shoichi Ishiura(1),*

(1)Department of Life Sciences, Graduate School of Arts and Sciences, the University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan
(2)Laboratory for Structural Neuropathology, RIKEN-Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
(3)Center for Structuring Life Sciences, Graduate School of Arts and Sciences, the University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan

*Corresponding author. Shoichi Ishiura, Tel & Fax: +81-3-5454-6739 e-mail: cishiura{at}mail.ecc.u-tokyo.ac.jp

Received November 12, 2007; Accepted November 15, 2007


  Abstract

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.

Key Words: binding constant, CUG-BP1, myotonic dystrophy, surface plasmon resonance, triplet-repeat

# These authors contributed equally.


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