Targeted pseudouridylation: An approach for suppressing nonsense mutations in disease genes.

Nonsense mutations create premature termination codons (PTCs), activating the nonsense-mediated mRNA decay (NMD) pathway to degrade most PTC-containing mRNAs. The undegraded mRNA is translated, but translation terminates at the PTC, leading to no production of the full-length protein. This work presents targeted PTC pseudouridylation, an approach for nonsense suppression in human cells. Specifically, an artificial box H/ACA guide RNA designed to target the mRNA PTC can suppress both NMD and premature translation termination in various sequence contexts. Targeted pseudouridylation exhibits a level of suppression comparable with that of aminoglycoside antibiotic treatments. When targeted pseudouridylation is combined with antibiotic treatment, a much higher level of suppression is observed. Transfection of a disease model cell line (carrying a chromosomal PTC) with a designer guide RNA gene targeting the PTC also leads to nonsense suppression. Thus, targeted pseudouridylation is an RNA-directed gene-specific approach that suppresses NMD and concurrently promotes PTC readthrough.