RNA structural switches are key regulators of gene expression in bacteria, yet their characterization in Metazoa remains limited. Here we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3’UTR of the RORC transcript. In vivo DMS-MaPseq, coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated mRNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally-driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.
Overall design
To measure the effect of candidate RNA switches on gene expression, we used the Massively Parallel Reporter Assay. We cloned a library of candidate RNA switch sequences into a dual eGFP-mCherry fluorescent reporter vector, directly downstream of the eGFP ORF. We used eGFP fluorescence to measure the effect of candidate RNA switches on gene expression, and we used mCherry fluorescence as an endogenous control. We transduced HEK293 cells with this synthetic library, used flow-cytometry to sort cells by eGFP/mCherry expression ratio, and sequenced the genomic DNA and RNA from the resulting eight pools of cells
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