A Cell-Based Double Reporter Gene Splicing Assay for Therapeutic Screening in Myotonic Dystrophy

Udosen, Inyang U.; Granados, Javier T.; Brook, John David

doi:10.2478/ebtj-2023-0011

Abstract

The study has developed a model splicing construct assay system based on splicing misregulation, one of the major molecular features associated with myotonic dystrophy. The splicing construct assay has double reporters for intron 2 splicing in chloride channel (CLCN1). The CLCN1 transgene splicing construct assay was used to transfect wild type and DM fibroblast cell lines and the clones generated showed that it enabled quantification of splicing efficiency in transgene construct. Validation of the DM fibroblasts containing transgene splicing construct was performed by differentiating the DM fibroblasts into myoblasts which exhibited a switch in CLCN1 splicing construct which was consistent with that associated with myotonic dystrophy (DM) condition. The myoblast derived from fibroblasts cell-based gene-splicing assay was subsequently applied in therapeutic screening in small throughput screens of 113 compounds which identified Protein Kinase C inhibitors- hypericin and Ro-31-8220 as potential therapeutic agents. The CLCN1 gene-splicing assay is a good model system for application in therapeutic screening in myotonic dystrophy because its double reporters facilitated quantification of effect putative drug on correction of misregulated splicing.

References

Brook, J. D., McCurrach, M. E., Harley, H. G., Buckler, A. J., Church, D., H., Aburatani, Hunter, K., Stanton, V. P., Thirion, J. P., 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, 799-808.
Search in Google Scholar Back to article
Liquori, C. L., Ricker, K., Moseley, M. L., Jacobsen, J. F., Kress, W., Naylor, S. L., Day, J. W. & Ranum, L. P. (2001) Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science, 293, 864-7.
Search in Google Scholar Back to article
Ranum, L. P. & Cooper, T. A. (2006) RNA-mediated neuromuscular disorders. Annu Rev Neurosci, 29, 259-77.
Search in Google Scholar Back to article
Charlet, B. N., Savkur, R. S., Singh, G., Philips, A. V., Grice, E. A. & Cooper, T. A.(2002) Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to mis-regulated alternative splicing. Mol Cell, 10, 45-53.
Search in Google Scholar Back to article
Dansithong, W., Paul, S., Comai, L. & Reddy, S.(2005) MBNL1 is the primary determinant of focus formation and aberrant insulin receptor splicing in DM1. J Biol Chem, 280, 5773-80.
Search in Google Scholar Back to article
Ho, T. H., Charlet, B. N., Poulous, M. G., Singh, G., Swanson, M. S. & Cooper, T. A.(2004) Muscleblind proteins regulate alternative splicing. EMBO J, 23, 3103-12.
Search in Google Scholar Back to article
Lin, X., Miller, J. W., Mankodi, A., Kanadia, R. N., Yuan, Y., Moxley, R. T., Swanson, M. S. & Thornton, C. A.(2006) Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy. Hum Mol Genet, 15, 2087-97.
Search in Google Scholar Back to article
Miller, J. W., Urbinati, C. R., Teng-Umnuay, P., Stenberg, M. G., Byrne, B. J., Thornton, C. A. & Swanson, M. S.(2000) Recruitment of human muscleblind proteins to (CUG)(n) expansions associated with myotonic dystrophy. EMBO J, 19, 4439-48.
Search in Google Scholar Back to article
Fardaei, M., Larkin, K., Brook, J. D., & Hamshere, M. G.(2001) In vivo co-localisation of MBNL protein with DMPK expanded-repeat transcripts. Nucleic Acids Res, 29, 2766-71.
Search in Google Scholar Back to article
Mankodi, A., Urbinati, C. R., Yuan, Q. P., Moxley, R. T., Sansone, V., Krym, M., Henderson, D., Schalling, M., Swanson, M. S. & Thornton, C. A.(2001) Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2. Hum Mol Genet, 10, 2165-70.
Search in Google Scholar Back to article
Kuyumcu-Martinez, N. M., Wang, G. S. & Cooper, T. A.(2007) Increased steady-state levels of CUGBP1 in myo-tonic dystrophy 1 are due to PKC-mediated hyperphosphorylation. Mol Cell, 28, 68-78.
Search in Google Scholar Back to article
Savkur, R. S., Philips, A. V. & Cooper, T. A.(2001) Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy. Nat Genet, 29, 40-7.
Search in Google Scholar Back to article
Philips, A. V., Timchenko, L. T. & Cooper, T. A.(1998) Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy. Science, 280, 737-41.
Search in Google Scholar Back to article
Kalsotra, A., Xiao, X., Ward, A. J., Castle, J. C., Johnson, J. M., Burge, C. B. & Cooper, T. A.(2008) A postnatal switch of CELF and MBNL proteins reprograms alternative splicing in the developing heart. Proc Natl Acad Sci U S A, 105, 20333-8.
Search in Google Scholar Back to article
Gareiss, P. C., Sobczak, K., McNaughton, B. R., Palde, P. B., Thornton, C. A. & Miller, B. L.(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, 16254-61.
Search in Google Scholar Back to article
Warf, M. B., Nakamori, M., Matthys, C. M., Thornton, C. A. & Berglund, J. A.(2009) Pentamidine reverses the splicing defects associated with myotonic dystrophy. Proc Natl Acad Sci U S A, 106, 18551-6.
Search in Google Scholar Back to article
Wheeler, T. M., Sobczak, K., Lueck, J. D., Osborne, R. J., Lin, X., Dirksen, R. T. & Thornton, C. A.(2009) Reversal of RNA dominance by displacement of protein sequestered on triplet repeat RNA. Science, 325, 336-9.
Search in Google Scholar Back to article
Arambula, J. F., Ramisetty, S. R., Baranger, A. M. & Zimmerman, S. C.(2009) A simple ligand that selectively targets CUG trinucleotide repeats and inhibits MBNL protein binding. Proc Natl Acad Sci U S A, 106, 16068-73.
Search in Google Scholar Back to article
Lee, M. M., Childs-Disney, J. L., Pushechnikov, A., French, J. M., Sobczak, K., Thornton, C. A. & Disney, M. D.(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, 17464-72.
Search in Google Scholar Back to article
Lee, M. M., Pushechnikov, A. & Disney, M. D.(2009) Rational and modular design of potent ligands targeting the RNA that causes myotonic dystrophy 2. ACS Chem Biol, 4, 345-55.
Search in Google Scholar Back to article
Pushechnikov, A., Lee, M. M., Childs-Disney, J. L., Sobczak, K., French, J. M., Thornton, C. A. & Disney, M. D.(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, 9767-79.
Search in Google Scholar Back to article
Disney, M. D., Lee, M. M., Pushechnikov, A. & Childs-Disney, J. L. (2010) The role of flexibility in the rational design of modularly assembled ligands targeting the RNAs that cause the myotonic dystrophies. Chembiochem, 11, 375-82.
Search in Google Scholar Back to article
O’Leary, D. A., Vargas, L., Sharif, O., Garcia, M. E., Sigal, Y. J., Chow, S. K., Schmedt, C., Caldwell, J. S., Brinker, A. & Engels, I. H. (2010) HTS-Compatible Patient-Derived Cell-Based Assay to Identify Small Molecule Modulators of Aberrant Splicing in Myotonic Dystrophy Type 1. Curr Chem Genomics, 4, 9-18.
Search in Google Scholar Back to article
Ketley, A., Chen, C. Z., Li, X., ARYA, S., Robinson, T. E., Granados-Riveron, J., Udosen, I., Morris, G. E., HOLT, I., Furling, D., Chaouch, S., Haworth, B., Southall, N., Shinn, P., Zheng, W., Austin, C. P., Hayes, C. J. & Brook, D.(2013) High-content screening identifies small molecules that remove nuclear foci, affect MBNL distribution and CELF1 protein levels via a PKC-independent pathway in myotonic dystrophy cell lines. Human Molecular Genetics 1-12.
Search in Google Scholar Back to article
Mulders, S. A., Van Den Broek, W. J., Wheeler, T. M., Croes, H. J., Van Kuik-Romeijn, P., De Kimpe, S. J., Furling, D., Platenburg, G. J., Gourdon, G., Thornton, C. A., Wieringa, B. & Wansink, D. G.(2009) Triplet-repeat oligo-nucleotide-mediated reversal of RNA toxicity in myotonic dystrophy. Proc Natl Acad Sci U S A, 106, 13915-20.
Search in Google Scholar Back to article
Wheeler, T. M., Lueck, J. D., Swanson, M. S., Dirksen, R. T. & Thornton, C. A. (2007) Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy. J Clin Invest, 117, 3952-7.
Search in Google Scholar Back to article
Francois, V., Klein, A. F., Beley, C., Jollet, A., Lemercier, C., Garcia, L. & Furling, D. (2011) Selective silencing of mutated mRNAs in DM1 by using modified hU7-snRNAs. Nat Struct Mol Biol, 18, 85-7.
Search in Google Scholar Back to article
Kanadia, R. N., Shin, J., Yuan, Y., Beattie, S. G., Wheeler, T. M., Thornton, C. A., & Swanson, M. S. (2006) Reversal of RNA missplicing and myotonia after muscleblind overexpression in a mouse poly(CUG) model for myotonic dystrophy. Proc Natl Acad Sci U S A, 103, 11748-53.
Search in Google Scholar Back to article
Hino, S., Kondo, S., Sekiya, H., Saito, A., Kanemoto, S., Murakami, T., Chihara, K., Aoki, Y., Nakamori, M., Takahashi, M. P. & Imaizumi, K. (2007) Molecular mechanisms responsible for aberrant splicing of SERCA1 in myotonic dystrophy type 1. Hum Mol Genet, 16, 2834-43.
Search in Google Scholar Back to article
Orengo, J. P., Bundman, D. & Cooper, T. A.(2006) A bichromatic fluorescent reporter for cell-based screens of alternative splicing. Nucleic Acids Res, 34, e148.
Search in Google Scholar Back to article
Wojciechowska, M., Sobczak, K., Kozlowski, P., Sedehizadeh, S., Wojtkowiak-Szlachcic, A., Czubak, K., Markus, R., Lusakowska, A., Kaminska, A. & Brook, J. D.(2018) Quantitative Methods to Monitor RNA Biomarkers in Myotonic Dystrophy. Nature 8:5885, 1-13.
Search in Google Scholar Back to article
Wang, G. S., Kuyumcu-Martinez, M. N., Sarma, S., Mathur, N., Wehrens, X. H. & Cooper, T. A.(2009) PKC inhibition ameliorates the cardiac phenotype in a mouse model of myotonic dystrophy type 1. J Clin Invest, 119, 3797-806.
Search in Google Scholar Back to article
Timchenko, L.(2022) Myotonic Dystrophy: From Molecular Pathogenesis to Therapeutics. Int. J. Mol. Sci. 23(19):11954.
Search in Google Scholar Back to article

A Cell-Based Double Reporter Gene Splicing Assay for Therapeutic Screening in Myotonic Dystrophy

Abstract

Paradigm

My account