Fig. 1: Development of tethered function assays for detecting direct induction of exon inclusion.
From: Large-scale evaluation of the ability of RNA-binding proteins to activate exon inclusion
a, Schematic of luciferase reporters used in the assays and resulting isoforms after cellular mRNA processing. b, Analysis workflow for calculating percent-spliced-in from luminescence measurements. c, Splicing gels of lucMAPT-30D splicing in response to co-transfection with MCP-fused positive and negative controls. Bands are generated by agarose gel electrophoresis of RT-generated cDNA amplified by minigene specific primers (shown in a) that amplify skipping and inclusion isoforms. d, Bar graph of lucMAPT-30D reporter readout as calculated from the workflow in b with the same conditions as c (mean ± s.d., n = 3 replicate transfections). e, Experimental workflow of tethering assays. The effects of recruiting 718 MCP-fused RBPs are tested in both reporter contexts. P value was calculated by independent two-sample one-tailed t-test, comparing the co-transfection of the reporter and candidates to co-transfection of the reporter and FLAG NC performed concurrently. The displayed n refers to biological replicates of candidate transfections. For FLAG NC transfections, n = 3 biological replicates for the reporter experiments and n = 6 for the splicing gel experiment. Venn diagram of final hits after all rounds of screening and verification. bp, base pairs.
