title: Regulation of a strong F9 cryptic 5'ss by intrinsic elements and by combination of tailored U1snRNAs with antisense oligonucleotides.
abstract: Mutations affecting specific splicing regulatory elements offer suitable models to better understand their interplay and to devise therapeutic strategies. Here we characterize a meaningful splicing model in which numerous Hemophilia B-causing mutations, either missense or at the donor splice site (5'ss) of coagulation F9 exon 2, promote aberrant splicing by inducing the usage of a strong exonic cryptic 5'ss. Splicing assays with natural and artificial F9 variants indicated that the cryptic 5'ss is regulated, among a network of regulatory elements, by an exonic splicing silencer (ESS). This finding and the comparative analysis of the F9 sequence across species showing that the cryptic 5'ss is always paralleled by the conserved ESS support a compensatory mechanism aimed at minimizing unproductive splicing. To recover splicing we tested antisense oligoribonucleotides masking the cryptic 5'ss, which were effective on exonic changes but promoted exon 2 skipping in the presence of mutations at the authentic 5'ss. On the other hand, we observed a very poor correction effect by small nuclear RNA U1 (U1snRNA) variants with increased or perfect complementarity to the defective 5'ss, a strategy previously exploited to rescue splicing. Noticeably, the combination of the mutant-specific U1snRNAs with antisense oligonucleotides produced appreciable amounts of correctly spliced transcripts (from 0 to 20-40%) from several mutants of the exon 2 5'ss. Based on the evidence of an altered interplay among ESS, cryptic and the authentic 5'ss as a disease-causing mechanism, we provide novel experimental insights into the combinatorial correction activity of antisense molecules and compensatory U1snRNAs.
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara and LTTA, Ferrara, Italy and blsdra@unife.it.
authors.forename: Dario
authors.initials: D
authors.lastname: Balestra
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara and LTTA, Ferrara, Italy and.
authors.forename: Elena
authors.initials: E
authors.lastname: Barbon
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara and LTTA, Ferrara, Italy and.
authors.forename: Daniela
authors.initials: D
authors.lastname: Scalet
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara and LTTA, Ferrara, Italy and.
authors.forename: Nicola
authors.initials: N
authors.lastname: Cavallari
authors.affiliation: Department of Chemical and Pharmaceutical Sciences, University of Ferrara, Ferrara, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Perrone
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara and LTTA, Ferrara, Italy and.
authors.forename: Silvia
authors.initials: S
authors.lastname: Zanibellato
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara and LTTA, Ferrara, Italy and.
authors.forename: Francesco
authors.initials: F
authors.lastname: Bernardi
authors.affiliation: Department of Life Sciences and Biotechnology, University of Ferrara and LTTA, Ferrara, Italy and.
authors.forename: Mirko
authors.initials: M
authors.lastname: Pinotti
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005091:Exons; D005164:Factor IX / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D007438:Introns; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D022821:RNA Splice Sites; D012342:RNA, Small Nuclear / Q000235:genetics*; D037941:Silencer Elements, Transcriptional
chemical_list: D016376:Oligonucleotides, Antisense; D022821:RNA Splice Sites; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA; D005164:Factor IX