RNAi Screening to Identify Factors That Control Circular RNA Localization

Abstract

Thousands of eukaryotic protein-coding genes are noncanonically spliced to generate circular RNAs that have covalently linked ends. These transcripts are resistant to degradation by exonucleases, which enables some to accumulate to higher levels than the associated linear mRNA. In general, exonic circular RNAs accumulate in the cytoplasm, but functions for most of these transcripts remain unknown. It has been proposed that some may modulate the activity of microRNAs or RNA-binding proteins, be translated to yield protein products, or regulate innate immune responses. Recent work has revealed that circular RNAs are exported from the nucleus in a length-dependent manner and that the subcellular localization of these transcripts can be controlled by the DExH/D-box helicase Hel25E in Drosophila. Here, we describe how RNAi screening combined with subcellular fractionation and quantitative reverse transcription PCR (RT-qPCR) can be used to identify regulators of circular RNA localization in Drosophila cells. Long double-stranded RNAs (dsRNAs) that activate the RNA interference (RNAi) pathway are used to deplete factors of interest followed by biochemical fractionation to separate nuclear and cytoplasmic RNAs. RT-qPCR primers that amplify across the backsplicing junction of specific circular RNAs are then used to quantify the relative amounts of these transcripts in the nuclear and cytoplasmic compartments. In total, this approach can be broadly used to characterize circular RNA nuclear export and localization mechanisms, including to identify novel regulatory factors and their breadth of circular RNA targets.

Keywords: Backsplicing; Drosophila; Hel25E; Nuclear export; RNA export; RNA localization; RT-qPCR; Subcellular fractionation; circRNA; dsRNA.