Regulation of a strongF9cryptic 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.

A Compensatory U1snRNA Partially Rescues FAH Splicing and Protein Expression in a Splicing-Defective Mouse Model of Tyrosinemia Type I

“…Among them, the use of the small nuclear ribonucleoprotein U1 (U1snRNP) that, in the earliest splicing step, plays a key role in the exon definition by mediating the recognition of the 5 ss through base pair complementarity with its RNA component (U1snRNA) [3]. Variants of the U1snRNA with increased complementarity with the 5 ss of the defective exon (compensatory U1snRNA), or targeting the downstream intronic sequences (Exon specific U1snRNA, ExSpeU1), have shown the ability to rescue mRNA splicing in the presence of disease-causing mutations at 5 ss, 3 ss or within exons [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. While the correction effect has been clearly shown in several cellular models of human disease the evaluation of their therapeutic potential requires investigations in animal models harboring the disease-causing splicing mutations, which are very rare.…”

An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

“…Differently, our and other groups extensively exploited the physiological role of the U1snRNA to promote exon inclusion in the presence of exon-skipping mutations, 5,6,7,8,9,10,11,12,13,14,15,16,17 a relevant cause of severe forms of human genetic disease. 18,19,20 The first generation of engineered U1snRNA had a modified 5′ tail with increased complementarity to defective 5′ss, and were shown to rescue exon inclusion in several cellular 5,6,7,8,9,10,11,12,13,14,15,16,21 and also in vivo 17 disease models.…”

“…18,19,20 The first generation of engineered U1snRNA had a modified 5′ tail with increased complementarity to defective 5′ss, and were shown to rescue exon inclusion in several cellular 5,6,7,8,9,10,11,12,13,14,15,16,21 and also in vivo 17 disease models. However, these U1snRNAs are often tailored on the disease-causing mutation and have the intrinsic risk of off-target effects by recognizing the partially conserved donor splice site 22 in other splicing units.…”

Disease-causing variants of the conserved +2T of 5′ splice sites can be rescued by engineered U1snRNAs

“…On the basis of the frequency and relevance of these nucleotide changes and on their mechanism, we and others have devised a correction approach based on variants of the U1snRNA designed to restore complementarity with the defective 5′ss (compensatory U1snRNAs; Pinotti et al., ) or to target downstream intronic regions (exon‐specific U1snRNAs; ExSpeU1; Alanis et al., ). For different human genetic disorders, in both cellular (Balestra et al., ; Dal Mas et al., ; Glaus, Schmid, Da Costa, Berger, & Neidhardt, ; Scalet et al., ; Schmid et al., ; Tajnik et al., ; van der Woerd et al., ) and animal (Balestra et al., ; Balestra et al., ; Dal Mas, Rogalska, Bussani, & Pagani, ; Donadon et al., ; Rogalska et al., ) models, the engineered U1snRNAs were shown to be effective on variants at 5′ss but also within the exon or at the 3′ss. However, these approaches failed to rescue changes at the highly conserved nucleotides +1G and +2T of the 5′ss (Alanis et al., ; Cavallari et al., ), which are the most represented (Buratti et al., ; Krawczak et al., ) and severe ones, and commonly considered to be virtually null.…”