Journal of Biochemistry Advance Access originally published online on November 26, 2007
Journal of Biochemistry 2008 143(3):377-383; doi:10.1093/jb/mvm230
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© 2007 The Japanese Biochemical Society.
Quantitative Analysis of CUG-BP1 Binding to RNA Repeats
1Department of Life Sciencesl; 2Center for Structuring Life Sciences, Graduate School of Arts and Sciences, the University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902; and 3Laboratory for Structural Neuropathology, RIKEN-Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
To whom correspondence should be addressed. Tel/Fax: +81-3-5454-6739, E-mail: cishiura{at}mail.ecc.u-tokyo.ac.jp.
Received November 12, 2007; Accepted November 15, 2007
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Abstract |
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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
Abbreviations: 3-AT, 3-amino triazole; CELF, CUG-BP and ETR3-like factor; DM, myotonic dystrophy; DMPK, DM protein kinase; RRM, RNA recognition motif
*The first two authors contributed equally to this work.
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