Matvei Khoroshkin, Daniel Asarnow, Shaopu Zhou, Albertas Navickas, Aidan Winters et al.
RNA structural switches are key regulators of gene expression in bacteria, but 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 3ʹ untranslated region of the RORC (RAR-related orphan receptor C) transcript. In vivo dimethyl sulfate (DMS) mutational profiling with sequencing (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 messenger RNA 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.
Matvei Khoroshkin, Daniel Asarnow, Shaopu Zhou, Albertas Navickas, Aidan Winters, Jackson Goudreau, Simon K. Zhou, Johnny Yu, Christina Palka, Lisa Fish, Ashir Borah, Kian Yousefi, Christopher Carpenter, K. Mark Ansel, Yifan Cheng, Luke A. Gilbert, Hani Goodarzi. A systematic search for RNA structural switches across the human transcriptome. 2024-07-16. https://doi.org/10.1038/s41592-024-02335-1
A lentiviral dual eGFP–mCherry reporter library tested 3,750 candidate human 3′UTR RNA-switch fragments and matched scrambled controls in HEK293 cells. Cells were sorted into eight equal fluorescence bins in two biological replicates, and construct abundance was measured from genomic DNA and reporter RNA.
A second lentiviral MPRA library tested four designed mutations per candidate RNA switch, with two perturbations favoring each of two predicted mutually exclusive RNA conformations. The same two-replicate, eight-bin HEK293 eGFP:mCherry sorting and genomic-DNA/RNA sequencing design quantified conformation-dependent reporter activity.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 4 files (ZIP)41592_2024_2335_MOESM4_ESM.xlsxGSE266058_family.soft.gzGSE266058_functional_screen.tsv.gzGSE266058_mutagenesis_screen.tsv.gz