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
Authors: Francesco Bernardi
Laura Peretto
Mirko Pinotti
Iva Maestri
Claudia Sacchetto
Francisco Baralle
Franco Pagani
Dario Balestra
Francesca Tassi
Stan F J van de Graaf
Journal: Molecular Medicine (2021)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Nucleotide variations; OTC deficiency; Pathogenic mRNA splicing; Spfash mouse model; U1snRNA.
Affiliations: Department of Life Sciences and Biotechnology, University of Ferrara, Via Fossato di Mortara 74, 44121, Ferrara, Italy., Liver Research Center, Q AREA Science Park, Trieste, Italy., Department of Translational Medicine and for Romagna, Pathology Unit of Pathologic Anatomy, Histology and Cytology, University of Ferrara, Ferrara, Italy., Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology and Metabolism, Academic Medical Center, Amsterdam, The Netherlands., Human Molecular Genetics, ICGEB - International Center for Genetic Engineering and Biotechnology, Trieste, Italy., Department of Life Sciences and Biotechnology, University of Ferrara, Via Fossato di Mortara 74, 44121, Ferrara, Italy. .

Abstract:  Background: 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. 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&gt;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. Methods: Creation 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. Results: Comparative 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&gt;A change, abrogating the cryptic 5'ss, was rescuable by engineered U1snRNA. Conclusions: Subtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G&gt;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. 

Retracted: No

PubPeer comments:
 Is there a splice in Figure 3A SAM68? (I didn t add the vertical black line below). Do the authors have the original scans? If there is a splice, there doesn t seem to be one in the control blot. Could the authors comment on what the black line means? ![file] ,