OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies

Claudia Sacchetto,Francisco Baralle,Laura Peretto,Iva Maestri,Dario Balestra,Francesca Tassi,Franco Pagani,Stan F J Van De Graaf,Mirko Pinotti,Francesco Bernardi

doi:10.1186/s10020-021-00418-9

Abstract

BackgroundAberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA.MethodsCreation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors.ResultsComparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects.Moreover, the interplay between the authentic and the adjacent cryptic 5′ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5′ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5′ss, was rescuable by engineered U1snRNA.ConclusionsSubtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.

Highlights

Aberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context
Subtle intronic variations explain species-specific Ornithine TransCarbamylase (OTC) splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1 small nuclear RNA (U1snRNA), which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans
Nucleotide variations at +10‐11 positions dictate species‐specific splicing patterns To infer the mechanisms underlying the remarkably different splicing patterns triggered by the OTC c.386G>A mutation in humans and mouse (Fig. 1A) (Rivera-Barahona et al 2015) we performed a sequence alignment of OTC exon 4 and the surrounding introns across species (Fig. 1B)

Summary

Introduction

Aberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. The evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era This aspect has to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. While the evolution of nucleotide variations can be evaluated by comparative genomic and RNAseq analyses, the study of their functional relevance across species, represents a major issue This aspect, hardly predictable and so far poorly investigated, has to be taken into account when exploring RNA therapeutics in animal models in the attempt to translate pre-clinical data into humans. U1snRNA variants with increased complementarity with the 5′ss of the defective exon (named compensatory U1snRNA), or targeting the downstream intronic sequences (named Exon specific U1snRNA, ExSpeU1), have demonstrated their ability to rescue exon skipping caused by different types of mutations in cellular and animal models of several human diseases (Donadon et al 2018, 2019; Scalet et al 2018, 2019; Yamazaki et al 2018; Balestra et al 2019a, 2020a; Lee et al 2019; Donegà et al 2020; Martín et al 2021)

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