Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, USA.
Department of Urology, University of California, San Francisco, San Francisco, CA, USA.
Nat Methods. 2024 Sep;21(9):1634-1645. doi: 10.1038/s41592-024-02335-1. Epub 2024 Jul 16.
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 3' 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.
RNA 结构开关是细菌中基因表达的关键调节剂，但在后生动物中其特征仍然有限。在这里，我们提出了 SwitchSeeker，这是一种用于系统鉴定功能 RNA 结构开关的全面计算和实验方法。我们将 SwitchSeeker 应用于人类转录组，鉴定了 245 个潜在的 RNA 开关。为了验证我们的方法，我们在 RORC（RAR 相关孤儿受体 C）转录物的 3'非翻译区中表征了一个先前未知的 RNA 开关。与冷冻电子显微镜相结合的体内二甲磺酸（DMS）突变测序（DMS-MaPseq）证实了它存在两种替代结构构象。此外，我们使用全基因组 CRISPR 筛选来鉴定通过这种 RNA 结构开关调节基因表达的转录因子。我们发现无意义介导的 mRNA 衰变以构象特异性的方式作用于该元件。SwitchSeeker 提供了一种无偏、实验驱动的方法，用于发现塑造真核基因表达景观的 RNA 结构开关。
