Design and evaluation of antisense sequence length for modified mouse U7 small nuclear RNA to induce efficient pre-messenger RNA splicing modulation in vitro

Abstract

Pre-messenger RNA (pre-mRNA) splicing modulation is an attractive approach for investigating the mechanisms of genetic disorders caused by mis-splicing. Previous reports have indicated that a modified U7 small nuclear RNA (U7 snRNA) is a prospective tool for modulating splicing both in vitro and in vivo. To date, very few studies have investigated the role of antisense sequence length in modified U7 snRNA. In this study, we designed a series of antisense sequences with various lengths and evaluated their efficiency in inducing splicing modulation. To express modified U7 snRNAs, we constructed a series of plasmid DNA sequences which codes cytomegalovirus (CMV) enhancer, human U1 promoter, and modified mouse U7 snRNAs with antisense sequences of different lengths. We evaluated in vitro splicing modulation efficiency using a luciferase reporter system for simple and precise evaluation as well as reverse transcription-polymerase chain reaction to monitor splicing patterns. Our in vitro assay findings suggest that antisense sequences of modified mouse U7 snRNAs have an optimal length for efficient splicing modulation, which depends on the target exon. In addition, antisense sequences that were either too long or too short decreased splicing modulation efficiency. To confirm reproducibility, we performed an in vitro assay using two target genes, mouse Fas and mouse Dmd. Together, our data suggests that the antisense sequence length should be optimized for modified mouse U7 snRNAs to induce efficient splicing modulation.