Tags

Type your tag names separated by a space and hit enter

Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy.
Biochim Biophys Acta Mol Basis Dis. 2017 01; 1863(1):15-20.BB

Abstract

The c.2101A>G synonymous change (p.G674G) in the gene for ATR, a key player in the DNA-damage response, has been the first identified genetic cause of Seckel Syndrome (SS), an orphan disease characterized by growth and mental retardation. This mutation mainly causes exon 9 skipping, through an ill-defined mechanism. Through ATR minigene expression studies, we demonstrated that the detrimental effect of this mutation (6±1% of correct transcripts only) depends on the poor exon 9 definition (47±4% in the ATRwt context), because the change was ineffective when the weak 5' or the 3' splice sites (ss) were strengthened (scores from 0.54 to 1) by mutagenesis. Interestingly, the exonic c.2101A nucleotide is conserved across species, and the SS-causing mutation is predicted to concurrently strengthen a Splicing Silencer (ESS) and weaken a Splicing Enhancer (ESE). Consistently, the artificial c.2101A>C change, predicted to weaken the ESE only, moderately impaired exon inclusion (28±7% of correct transcripts). The observation that an antisense oligonucleotide (AONATR) targeting the c.2101A position recovers exon inclusion in the mutated context supports a major role of the underlying ESS. A U1snRNA variant (U1ATR) designed to perfectly base-pair the weak 5'ss, rescued exon inclusion (63±3%) in the ATRSS-allele. Most importantly, upon lentivirus-mediated delivery, the U1ATR partially rescued ATR mRNA splicing (from ~19% to ~54%) and protein (from negligible to ~6%) in embryonic fibroblasts derived from humanized ATRSS mice. Altogether these data elucidate the molecular mechanisms of the ATR c.2101A>G mutation and identify two potential complementary RNA-based therapies for Seckel syndrome.

Authors+Show Affiliations

Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy.Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy.Center for Genomic Science, Fondazione Istituto Italiano di Tecnologia (IIT), Via Adamello 16, Milano 20139, Italy.Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy.Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Via Luigi Borsari 46, Ferrara 44121, Italy.Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy.Center for Genomic Science, Fondazione Istituto Italiano di Tecnologia (IIT), Via Adamello 16, Milano 20139, Italy.Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, 44121 Ferrara, Italy. Electronic address: pnm@unife.it.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

27639833

Citation

Scalet, Daniela, et al. "Exploring Splicing-Switching Molecules for Seckel Syndrome Therapy." Biochimica Et Biophysica Acta. Molecular Basis of Disease, vol. 1863, no. 1, 2017, pp. 15-20.
Scalet D, Balestra D, Rohban S, et al. Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy. Biochim Biophys Acta Mol Basis Dis. 2017;1863(1):15-20.
Scalet, D., Balestra, D., Rohban, S., Bovolenta, M., Perrone, D., Bernardi, F., Campaner, S., & Pinotti, M. (2017). Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy. Biochimica Et Biophysica Acta. Molecular Basis of Disease, 1863(1), 15-20. https://doi.org/10.1016/j.bbadis.2016.09.011
Scalet D, et al. Exploring Splicing-Switching Molecules for Seckel Syndrome Therapy. Biochim Biophys Acta Mol Basis Dis. 2017;1863(1):15-20. PubMed PMID: 27639833.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Exploring Splicing-Switching Molecules For Seckel Syndrome Therapy. AU - Scalet,Daniela, AU - Balestra,Dario, AU - Rohban,Sara, AU - Bovolenta,Matteo, AU - Perrone,Daniela, AU - Bernardi,Francesco, AU - Campaner,Stefano, AU - Pinotti,Mirko, Y1 - 2016/09/14/ PY - 2016/06/14/received PY - 2016/09/05/revised PY - 2016/09/13/accepted PY - 2016/9/19/pubmed PY - 2018/12/12/medline PY - 2016/9/19/entrez KW - (up to 6) Seckel syndrome-1 KW - Antisense oligonucleotide KW - Exonic splicing silencer KW - correction approaches KW - modified U1snRNA SP - 15 EP - 20 JF - Biochimica et biophysica acta. Molecular basis of disease JO - Biochim Biophys Acta Mol Basis Dis VL - 1863 IS - 1 N2 - The c.2101A>G synonymous change (p.G674G) in the gene for ATR, a key player in the DNA-damage response, has been the first identified genetic cause of Seckel Syndrome (SS), an orphan disease characterized by growth and mental retardation. This mutation mainly causes exon 9 skipping, through an ill-defined mechanism. Through ATR minigene expression studies, we demonstrated that the detrimental effect of this mutation (6±1% of correct transcripts only) depends on the poor exon 9 definition (47±4% in the ATRwt context), because the change was ineffective when the weak 5' or the 3' splice sites (ss) were strengthened (scores from 0.54 to 1) by mutagenesis. Interestingly, the exonic c.2101A nucleotide is conserved across species, and the SS-causing mutation is predicted to concurrently strengthen a Splicing Silencer (ESS) and weaken a Splicing Enhancer (ESE). Consistently, the artificial c.2101A>C change, predicted to weaken the ESE only, moderately impaired exon inclusion (28±7% of correct transcripts). The observation that an antisense oligonucleotide (AONATR) targeting the c.2101A position recovers exon inclusion in the mutated context supports a major role of the underlying ESS. A U1snRNA variant (U1ATR) designed to perfectly base-pair the weak 5'ss, rescued exon inclusion (63±3%) in the ATRSS-allele. Most importantly, upon lentivirus-mediated delivery, the U1ATR partially rescued ATR mRNA splicing (from ~19% to ~54%) and protein (from negligible to ~6%) in embryonic fibroblasts derived from humanized ATRSS mice. Altogether these data elucidate the molecular mechanisms of the ATR c.2101A>G mutation and identify two potential complementary RNA-based therapies for Seckel syndrome. SN - 0925-4439 UR - https://www.unboundmedicine.com/medline/citation/27639833/Exploring_Splicing_Switching_Molecules_For_Seckel_Syndrome_Therapy_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0925-4439(16)30229-0 DB - PRIME DP - Unbound Medicine ER -