Nonsense-mediated mRNA decay (NMD) blockage promotes nonsense mRNA stabilization in protein 4.1R deficient cells carrying the 4.1R Coimbra variant of hereditary elliptocytosis

We describe a new approach to stabilize nonsense mRNA, based on the inhibition of the NMD mechanism, by combining cycloheximide-mediated inhibition of translation, and caffeine-mediated inhibition of UPF1 phosphorylation. This approach aimed to identify the impact of a 4.1R splicing mutation. This mutation is involved in a partial deficiency of 4.1R in the homozygous state in a patient with hereditary elliptocytosis and a moderated hemolytic anemia. We show that, in addition to two known minor shortened and stable spliceoforms, the mutation activates an intronic cryptic splice site, which results in a nonsense mRNA major isoform, targeted to degradation in intact cells by NMD. This accounts for the main cause of 4.1R partial deficiency. In a general perspective, blocking the NMD mechanism would help to identify a missing isoform, and pave the path for a molecular targeting strategy to circumvent a deleterious splicing pathway in favor of a therapeutic splicing pathway.