Stem Cell and Regenerative Biology, Genome Institute of Singapore, A*STAR, Singapore, Singapore.
Division of Biomedical Sciences, Warwick Medical School, University of Warwick, Coventry, UK.
Nat Biotechnol. 2021 Mar;39(3):336-346. doi: 10.1038/s41587-020-0712-z. Epub 2020 Oct 26.
Current methods for determining RNA structure with short-read sequencing cannot capture most differences between distinct transcript isoforms. Here we present RNA structure analysis using nanopore sequencing (PORE-cupine), which combines structure probing using chemical modifications with direct long-read RNA sequencing and machine learning to detect secondary structures in cellular RNAs. PORE-cupine also captures global structural features, such as RNA-binding-protein binding sites and reactivity differences at single-nucleotide variants. We show that shared sequences in different transcript isoforms of the same gene can fold into different structures, highlighting the importance of long-read sequencing for obtaining phase information. We also demonstrate that structural differences between transcript isoforms of the same gene lead to differences in translation efficiency. By revealing isoform-specific RNA structure, PORE-cupine will deepen understanding of the role of structures in controlling gene regulation.
目前，利用短读测序来确定 RNA 结构的方法无法捕捉到不同转录本之间的大多数差异。在这里，我们介绍了一种使用纳米孔测序（PORE-cupine）进行 RNA 结构分析的方法，它将化学修饰的结构探测与直接的长读 RNA 测序以及机器学习相结合，以检测细胞 RNA 中的二级结构。PORE-cupine 还可以捕获全局结构特征，如 RNA 结合蛋白结合位点和单核苷酸变异的反应性差异。我们表明，同一基因的不同转录本之间的共有序列可以折叠成不同的结构，这突出了获得相位信息的长读测序的重要性。我们还证明了同一基因的转录本之间的结构差异会导致翻译效率的差异。通过揭示同种基因的转录本之间的特异性 RNA 结构，PORE-cupine 将加深对结构在控制基因调控中的作用的理解。
