Analytical and Bioanalytical Chemistry

, Volume 402, Issue 5, pp 1889–1898

Two high-throughput screening assays for aberrant RNA–protein interactions in myotonic dystrophy type 1

  • Catherine Z. Chen
  • Krzysztof Sobczak
  • Jason Hoskins
  • Noel Southall
  • Juan J. Marugan
  • Wei Zheng
  • Charles A. Thornton
  • Christopher P. Austin
Original Paper

Abstract

Myotonic dystrophy type 1 (DM1), the most prevalent form of adult muscular dystrophy, is caused by expansion of a CTG repeat in the 3′ untranslated region of the DM protein kinase (DMPK) gene. The pathogenic effects of the CTG expansion arise from the deleterious effects of the mutant transcript. RNA with expanded CUG tracts alters the activities of several RNA binding proteins, including muscleblind-like 1 (MBNL1). MBNL1 becomes sequestered in nuclear foci in complex with the expanded CUG-repeat RNA. The resulting loss of MBNL1 activity causes misregulated alternative splicing of multiple genes, leading to symptoms of DM1. The binding interaction between MBNL1 and mutant RNA could be a key step in the pathogenesis of DM1 and serves as a potential target for therapeutic intervention. We have developed two high-throughput screens suitable assays using both homogenous time-resolved fluorescence energy transfer and AlphaScreen technologies to detect the binding of a C-terminally His-tagged MBNL1 and a biotinylated (CUG)12 RNA. These assays are homogenous and successfully miniaturized to 1,536-well plate format. Both assays were validated and show robust signal-to-basal ratios and Z′ factors.

Keywords

Myotonic dystrophy type 1 DM1 Muscleblind-like 1 MBNL1 

References

  1. 1.
    Harper P (2001) Myotonic dystrophy. Saunders, LondonGoogle Scholar
  2. 2.
    Brook JD, McCurrach ME, Harley HG, Buckler AJ, Church D, Aburatani H, Hunter K, Stanton VP, Thirion JP, Hudson T et al (1992) Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member. Cell 68(4):799–808CrossRefGoogle Scholar
  3. 3.
    Botta A, Rinaldi F, Catalli C, Vergani L, Bonifazi E, Romeo V, Loro E, Viola A, Angelini C, Novelli G (2008) The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients. J Med Genet 45(10):639–646CrossRefGoogle Scholar
  4. 4.
    Fardaei M, Rogers MT, Thorpe HM, Larkin K, Hamshere MG, Harper PS, Brook JD (2002) Three proteins, MBNL, MBLL and MBXL, co-localize in vivo with nuclear foci of expanded-repeat transcripts in DM1 and DM2 cells. Hum Mol Genet 11(7):805–814CrossRefGoogle Scholar
  5. 5.
    Mankodi A, Teng-Umnuay P, Krym M, Henderson D, Swanson M, Thornton CA (2003) Ribonuclear inclusions in skeletal muscle in myotonic dystrophy types 1 and 2. Ann Neurol 54(6):760–768CrossRefGoogle Scholar
  6. 6.
    Grammatikakis I, Goo YH, Echeverria GV, Cooper TA (2011) Identification of MBNL1 and MBNL3 domains required for splicing activation and repression. Nucleic Acids Res 39:2769–2780CrossRefGoogle Scholar
  7. 7.
    Ho TH, Charlet BN, Poulos MG, Singh G, Swanson MS, Cooper TA (2004) Muscleblind proteins regulate alternative splicing. EMBO J 23(15):3103–3112CrossRefGoogle Scholar
  8. 8.
    Kino Y, Washizu C, Oma Y, Onishi H, Nezu Y, Sasagawa N, Nukina N, Ishiura S (2009) MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1. Nucleic Acids Res 37(19):6477–6490CrossRefGoogle Scholar
  9. 9.
    Lin X, Miller JW, Mankodi A, Kanadia RN, Yuan Y, Moxley RT, Swanson MS, Thornton CA (2006) Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy. Hum Mol Genet 15(13):2087–2097CrossRefGoogle Scholar
  10. 10.
    Kanadia RN, Johnstone KA, Mankodi A, Lungu C, Thornton CA, Esson D, Timmers AM, Hauswirth WW, Swanson MS (2003) A muscleblind knockout model for myotonic dystrophy. Science 302(5652):1978–1980CrossRefGoogle Scholar
  11. 11.
    Kanadia RN, Shin J, Yuan Y, Beattie SG, Wheeler TM, Thornton CA, Swanson MS (2006) Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy. Proc Natl Acad Sci USA 103(31):11748–11753CrossRefGoogle Scholar
  12. 12.
    Wheeler TM, Sobczak K, Lueck JD, Osborne RJ, Lin X, Dirksen RT, Thornton CA (2009) Reversal of RNA dominance by displacement of protein sequestered on triplet repeat RNA. Science 325(5938):336–339CrossRefGoogle Scholar
  13. 13.
    Arambula JF, Ramisetty SR, Baranger AM, Zimmerman SC (2009) A simple ligand that selectively targets CUG trinucleotide repeats and inhibits MBNL protein binding. Proc Natl Acad Sci USA 106(38):16068–16073CrossRefGoogle Scholar
  14. 14.
    Disney MD, Lee MM, Pushechnikov A, Childs-Disney JL (2010) The role of flexibility in the rational design of modularly assembled ligands targeting the RNAs that cause the myotonic dystrophies. Chembiochem 11(3):375–382CrossRefGoogle Scholar
  15. 15.
    Gareiss PC, Sobczak K, McNaughton BR, Palde PB, Thornton CA, Miller BL (2008) Dynamic combinatorial selection of molecules capable of inhibiting the (CUG) repeat RNA-MBNL1 interaction in vitro: discovery of lead compounds targeting myotonic dystrophy (DM1). J Am Chem Soc 130(48):16254–16261CrossRefGoogle Scholar
  16. 16.
    Lee MM, Childs-Disney JL, Pushechnikov A, French JM, Sobczak K, Thornton CA, Disney MD (2009) Controlling the specificity of modularly assembled small molecules for RNA via ligand module spacing: targeting the RNAs that cause myotonic muscular dystrophy. J Am Chem Soc 131(47):17464–17472CrossRefGoogle Scholar
  17. 17.
    Pushechnikov A, Lee MM, Childs-Disney JL, Sobczak K, French JM, Thornton CA, Disney MD (2009) Rational design of ligands targeting triplet repeating transcripts that cause RNA dominant disease: application to myotonic muscular dystrophy type 1 and spinocerebellar ataxia type 3. J Am Chem Soc 131(28):9767–9779CrossRefGoogle Scholar
  18. 18.
    Warf MB, Nakamori M, Matthys CM, Thornton CA, Berglund JA (2009) Pentamidine reverses the splicing defects associated with myotonic dystrophy. Proc Natl Acad Sci USA 106(44):18551–18556CrossRefGoogle Scholar
  19. 19.
    Warf MB, Berglund JA (2007) MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T. RNA 13(12):2238–2251CrossRefGoogle Scholar
  20. 20.
    Zapp ML, Stern S, Green MR (1993) Small molecules that selectively block RNA binding of HIV-1 Rev protein inhibit Rev function and viral production. Cell 74(6):969–978CrossRefGoogle Scholar
  21. 21.
    Maroto M, Fernandez Y, Ortin J, Pelaez F, Cabello MA (2008) Development of an HTS assay for the search of anti-influenza agents targeting the interaction of viral RNA with the NS1 protein. J Biomol Screen 13(7):581–590CrossRefGoogle Scholar
  22. 22.
    Kino Y, Mori D, Oma Y, Takeshita Y, Sasagawa N, Ishiura S (2004) Muscleblind protein, MBNL1/EXP, binds specifically to CHHG repeats. Hum Mol Genet 13(5):495–507CrossRefGoogle Scholar
  23. 23.
    Hook B, Bernstein D, Zhang B, Wickens M (2005) RNA-protein interactions in the yeast three-hybrid system: affinity, sensitivity, and enhanced library screening. RNA 11(2):227–233CrossRefGoogle Scholar
  24. 24.
    Mei HY, Mack DP, Galan AA, Halim NS, Heldsinger A, Loo JA, Moreland DW, Sannes-Lowery KA, Sharmeen L, Truong HN, Czarnik AW (1997) Discovery of selective, small-molecule inhibitors of RNA complexes—I. The Tat protein/TAR RNA complexes required for HIV-1 transcription. Bioorg Med Chem 5(6):1173–1184CrossRefGoogle Scholar
  25. 25.
    Mills NL, Shelat AA, Guy RK (2007) Assay optimization and screening of RNA–protein interactions by AlphaScreen. J Biomol Screen 12(7):946–955CrossRefGoogle Scholar
  26. 26.
    Sobczak K, de Mezer M, Michlewski G, Krol J, Krzyzosiak WJ (2003) RNA structure of trinucleotide repeats associated with human neurological diseases. Nucleic Acids Res 31(19):5469–5482CrossRefGoogle Scholar
  27. 27.
    Sobczak K, Michlewski G, de Mezer M, Kierzek E, Krol J, Olejniczak M, Kierzek R, Krzyzosiak WJ (2010) Structural diversity of triplet repeat RNAs. J Biol Chem 285(17):12755–12764CrossRefGoogle Scholar
  28. 28.
    Yuan Y, Compton SA, Sobczak K, Stenberg MG, Thornton CA, Griffith JD, Swanson MS (2007) Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs. Nucleic Acids Res 35(16):5474–5486CrossRefGoogle Scholar
  29. 29.
    Inglese J, Auld DS, Jadhav A, Johnson RL, Simeonov A, Yasgar A, Zheng W, Austin CP (2006) Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci USA 103(31):11473–11478CrossRefGoogle Scholar
  30. 30.
    Kane SA, Fleener CA, Zhang YS, Davis LJ, Musselman AL, Huang PS (2000) Development of a binding assay for p53/HDM2 by using homogeneous time-resolved fluorescence. Anal Biochem 278(1):29–38CrossRefGoogle Scholar
  31. 31.
    Lopez-Crapez E, Malinge JM, Gatchitch F, Casano L, Langlois T, Pugniere M, Roquet F, Mathis G, Bazin H (2008) A homogeneous resonance energy transfer-based assay to monitor MutS/DNA interactions. Anal Biochem 383(2):301–306CrossRefGoogle Scholar
  32. 32.
    Whitfield J, Harada K, Bardelle C, Staddon JM (2003) High-throughput methods to detect dimerization of Bcl-2 family proteins. Anal Biochem 322(2):170–178CrossRefGoogle Scholar

Copyright information

© Springer-Verlag (outside the USA) 2011

Authors and Affiliations

  • Catherine Z. Chen
    • 1
  • Krzysztof Sobczak
    • 2
    • 3
  • Jason Hoskins
    • 2
  • Noel Southall
    • 1
  • Juan J. Marugan
    • 1
  • Wei Zheng
    • 1
  • Charles A. Thornton
    • 2
  • Christopher P. Austin
    • 1
  1. 1.NIH Chemical Genomics Center, National Human Genome Research InstituteNational Institutes of HealthBethesdaUSA
  2. 2.Department of Neurology, School of Medicine and DentistryUniversity of RochesterRochesterUSA
  3. 3.Department of Gene Expression, Institute of Molecular Biology and BiotechnologyAdam Mickiewicz UniversityPoznanPoland

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