A Cell-Based High-Throughput Method for Identifying Modulators of Alternative Splicing.

Alternative splicing, a key regulatory process of gene expression, is controlled by trans-acting factors that recognize cis-elements in premature RNA transcripts to affect spliceosome assembly and splice site choices. Extracellular stimuli and signaling cascades can converge on RNA binding splicing regulators to affect alternative splicing. Defects in splicing regulation have been associated with various human diseases, and modification of disease-causing splicing events presents great therapeutic promise. Determining splicing regulators and/or upstream modulators has been difficult with low throughput, low sensitivity, and low specificity. IRAS (identifying regulators of alternative splicing) is a novel cell-based high-throughput screening strategy designed specifically to address these challenges and has achieved high throughput, high sensitivity, and high specificity. Here, we describe the IRAS method in detail with a pair of dual-fluorescence minigene reporters that produces GFP and RFP fluorescent signals to assay the two spliced isoforms exclusively. These two complementary mini-gene reporters alter GFP/RFP output ratios in the opposite direction in response to only a true splicing change. False positives from a signal screen do not stimulate opposite changes in GFP/RFP ratios. The reporter pair in conjunction with robotic liquid handlers and arrayed libraries allows IRAS to screen for both positive and negative splicing regulators with high sensitivity and specificity in a high-throughput manner.