High-Throughput Screening Method to Identify Alternative Splicing Regulators

Abstract

Misregulation of alternative pre-mRNA splicing contributes to various diseases. Understanding how alternative splicing is regulated paves the way to modulating or correcting molecular pathogenesis of the diseases. Alternative splicing is typically regulated by trans RNA binding proteins and their upstream modulators. Identification of these splicing regulators has been difficult and traditionally done piecemeal. High-throughput screening strategies to find multiple regulators of exon splicing have great potential to accelerate the discovery process, but typically confront low sensitivity and specificity of splicing assays. Here we describe a high-throughput screening method using dual-fluorescence minigene reporters to allow for sensitive detection of exon splicing changes. To enhance specificity we introduce two complementary dual-fluorescence minigenes that each express both GFP and RFP in response to exon inclusion and exclusion but oppositely. The method significantly eliminates false positives and allows for sensitive identification of true regulators of splicing. The method described here is designed to screen cDNA libraries, but can be applied to isolate splicing regulators from shRNA libraries or chemical libraries.

Methods and applications of RNA contact prediction*

“…[43] The CLIP-seq, RIP-seq, and footprinting can identify the RNA-protein contacts. [44][45][46][47] However, the available experiments work well on identifying RNA binding domains or motifs. It is still difficult to detect the exact nucleotide-nucleotide contacts accurately.…”