A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

Examining the capacity of human U1 snRNA variants to facilitate pre-mRNA splicing

“…To test the capacity of the U1 snRNAs to catalyze pre-mRNA splicing, we applied the U1 genetic complementation assay that uses the 3-exon/2-intron Dup51p reporter ( Fig. 4 A; Sharma et al 2014 ; Martelly et al 2021 ; Wong et al 2021 ; de Vries et al 2022 ). Dup51p minigene carries mutations in the 5′-ss sequence of the second intron resulting in the skipping of exon 2 in the mature transcript in HeLa cells as measured by primer extension ( Fig.…”

“…Plasmids for the Dup51p reporter and U1 snRNA expression (pNS6U1) have been described previously ( Sharma et al 2014 ; Wong et al 2021 ). The pNS6U1 constructs expressing vU1 snRNAs were generated by in-fusion cloning (TaKaRa) using a linearized pNS6 plasmid and duplex fragments for vU1 snRNAs (Ultramer duplexes from Integrated DNA Technologies).…”

“…Primer extension to monitor splicing of the Dup51p reporter was performed using 32 P-Dup3r primer (5′-AACAGCATCAGGAGTGGACAGATCCC-3′), as described previously ( Sharma et al 2014 ; Wong et al 2021 ). The primer extension products were separated using a 10% urea–PAGE gel and visualized by the Typhoon FLA 9000 scanner.…”

“…Although vU1 snRNAs have been shown to be expressed and incorporated into snRNPs and spliceosomes, their ability to facilitate pre-mRNA splicing catalysis has not been tested so far. In this study, we applied a previously established HeLa cell-based minigene reporter assay to examine the capacity of vU1 snRNAs to support splicing ( Sharma et al 2014 ; Wong et al 2021 ). The results revealed that even though many of the human vU1s could be exogenously expressed at levels comparable to the canonical U1 snRNA and were enriched in the nucleus, the ability to support pre-mRNA splicing of only a few was appreciable.…”