School of Molecular Sciences, The University of Western Australia, Crawley, Western Australia 6009, Australia.
Laboratoire d'Optique et Biosciences, École Polytechnique, CNRS, INSERM, Institut Polytechnique de Paris, 91120 Palaiseau, France.
Genome Res. 2024 Mar 20;34(2):217-230. doi: 10.1101/gr.278236.123.
Secondary structure is a principal determinant of lncRNA function, predominantly regarding scaffold formation and interfaces with target molecules. Noncanonical secondary structures that form in nucleic acids have known roles in regulating gene expression and include G-quadruplexes (G4s), intercalated motifs (iMs), and R-loops (RLs). In this paper, we used the computational tools G4-iM Grinder and QmRLFS-finder to predict the formation of each of these structures throughout the lncRNA transcriptome in comparison to protein-coding transcripts. The importance of the predicted structures in lncRNAs in biological contexts was assessed by combining our results with publicly available lncRNA tissue expression data followed by pathway analysis. The formation of predicted G4 (pG4) and iM (piM) structures in select lncRNA sequences was confirmed in vitro using biophysical experiments under near-physiological conditions. We find that the majority of the tested pG4s form highly stable G4 structures, and identify many previously unreported G4s in biologically important lncRNAs. In contrast, none of the piM sequences are able to form iM structures, consistent with the idea that RNA is unable to form stable iMs. Unexpectedly, these C-rich sequences instead form Z-RNA structures, which have not been previously observed in regions containing cytosine repeats and represent an interesting and underexplored target for protein-RNA interactions. Our results highlight the prevalence and potential structure-associated functions of noncanonical secondary structures in lncRNAs, and show G4 and Z-RNA structure formation in many lncRNA sequences for the first time, furthering the understanding of the structure-function relationship in lncRNAs.
二级结构是长链非编码 RNA（lncRNA）功能的主要决定因素，主要涉及支架形成和与靶分子的相互作用。在核酸中形成的非规范二级结构在调节基因表达方面具有已知作用，包括 G-四联体（G4s）、插入基序（iMs）和 R 环（RLs）。在本文中，我们使用计算工具 G4-iM Grinder 和 QmRLFS-finder 来预测这些结构在整个 lncRNA 转录本中的形成情况，与蛋白质编码转录本进行比较。通过将我们的结果与公开的 lncRNA 组织表达数据相结合，并进行途径分析，评估了预测结构在生物背景下 lncRNA 中的重要性。选择 lncRNA 序列中预测 G4（pG4）和 iM（piM）结构的形成在近生理条件下使用生物物理实验在体外得到了证实。我们发现，大多数经过测试的 pG4 形成高度稳定的 G4 结构，并在生物重要的 lncRNA 中鉴定出许多以前未报道过的 G4。相比之下，没有一个 piM 序列能够形成 iM 结构，这与 RNA 不能形成稳定 iM 的观点一致。出乎意料的是，这些富含 C 的序列反而形成了 Z-RNA 结构，这在以前的含有胞嘧啶重复序列的区域中从未观察到过，代表了一个有趣且尚未得到充分探索的蛋白质-RNA 相互作用的目标。我们的研究结果强调了非规范二级结构在 lncRNA 中的普遍性和潜在结构相关功能，并首次在许多 lncRNA 序列中观察到 G4 和 Z-RNA 结构的形成，进一步加深了对 lncRNA 结构-功能关系的理解。
