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Premature Termination Codon in 5′ Region of Desmoplakin and Plakoglobin Genes May Escape Nonsense-Mediated Decay through the Reinitiation of Translation
Authors:Marta Vallverdú  -Prats,Ramon Brugada,Mireia Alcalde
Affiliation:1.Cardiovascular Genetics Center, IdIBGi, University of Girona, 17190 Girona, Spain;2.Centro Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), 28029 Madrid, Spain;3.Medical Science Department, School of Medicine, University of Girona, 17071 Girona, Spain;4.Cardiology Service Hospital, University of Girona, 17007 Girona, Spain
Abstract:Arrhythmogenic cardiomyopathy is a heritable heart disease associated with desmosomal mutations, especially premature termination codon (PTC) variants. It is known that PTC triggers the nonsense-mediated decay (NMD) mechanism. It is also accepted that PTC in the last exon escapes NMD; however, the mechanisms involving NMD escaping in 5′-PTC, such as reinitiation of translation, are less known. The main objective of the present study is to evaluate the likelihood that desmosomal genes carrying 5′-PTC will trigger reinitiation. HL1 cell lines were edited by CRISPR/Cas9 to generate isogenic clones carrying 5′-PTC for each of the five desmosomal genes. The genomic context of the ATG in-frame in the 5′ region of desmosomal genes was evaluated by in silico predictions. The expression levels of the edited genes were assessed by Western blot and real-time PCR. Our results indicate that the 5′-PTC in PKP2, DSG2 and DSC2 acts as a null allele with no expression, whereas in the DSP and JUP gene, N-truncated protein is expressed. In concordance with this, the genomic context of the 5′-region of DSP and JUP presents an ATG in-frame with an optimal context for the reinitiation of translation. Thus, 5′-PTC triggers NMD in the PKP2, DSG2* and DSC2 genes, whereas it may escape NMD through the reinitiation of the translation in DSP and JUP genes, with no major effects on ACM-related gene expression.
Keywords:arrhythmogenic cardiomyopathy (ACM)   CRISPR   genetics   desmosomal genes   HL1   premature termination codon (PTC)   nonsense mediated decay (NMD)   alternative translation initiation (ATLI)
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