Regulation of a strong F9 cryptic 5'ss by intrinsic elements and by combination of tailored U1snRNAs with antisense oligonucleotides

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.