Premature Termination Codon in 5′ Region of Desmoplakin and Plakoglobin Genes May Escape Nonsense-Mediated Decay through the Reinitiation of Translation

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.

Alterations in Calcium Handling Are a Common Feature in an Arrhythmogenic Cardiomyopathy Cell Model Triggered by Desmosome Genes Loss

“…mRNA levels of N-DSP and DSP-KO clones were analyzed by RT-PCR for CACNA1C and PLN, which were not included in our previous study [22]. This suggested PLN downregulation in DSP-KO (Figure 6A), in contrast to the higher PLN levels observed in PKP2-KO, DSG2-KO, and DSC2-KO clones (Figure 2A).…”

“…The DSP-KO edited clones, in contrast to the PKP2-KO, DSC2-KO, and DSG2-KO clones, triggered the re-initiation of translation and were able to synthesize N-DSP [22]. Molecular and functional characterization of N-DSP and DSP-KO in this group was performed qualitatively as statistical analyses were not possible because one DSP-KO clone did not trigger re-initiation of translation.…”

“…Molecular and functional characterization of N-DSP and DSP-KO in this group was performed qualitatively as statistical analyses were not possible because one DSP-KO clone did not trigger re-initiation of translation. In contrast, the remaining clones synthesized N-DSP [22].…”

Escaping from CRISPR–Cas-mediated knockout: the facts, mechanisms, and applications

“…Examination of the cells derived from the patients revealed the presence of functional truncated protein generated by translation reinitiation from the AUG downstream R37X, which partially rescued the severe null phenotype in the patients [ 49 ]. Regarding CRISPR–Cas-mediated knockouts, translation reinitiation downstream of PTCs may be a major reason for the production of the residual proteins [ 12 , 13 , 22 , 23 , 27 , 30 , 31 , 34 , 35 ] (Fig. 2 A).…”

Characterization of the mIF4G Domains in the RNA Surveillance Protein Upf2p

“…It acts as an adapter protein when Upf1 binds to its Cterminal end and Upf3 to its N-terminal end [21][22][23][24]. Various models have been suggested to explain how these factors elicit NMD, which in turn results in the degradation of PTCcontaining RNA [25][26][27][28][29][30][31][32][33][34]. One possibility that has been studied is that Upf1 ATPase and helicase activities are regulated by Upf1 interaction with Upf2.…”