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.

中文翻译：

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与长非编码 RNA 中的插入基序相比，Z-RNA 优先形成

二级结构是 lncRNA 功能的主要决定因素，主要涉及支架形成和与靶分子的界面。核酸中形成的非常规二级结构在调节基因表达中具有已知的作用，包括 G 四链体 (G4)、插入基序 (iM) 和 R 环 (RL)。在本文中，我们使用计算工具 G4-iM Grinder 和 QmRLFS-finder 来预测整个 lncRNA 转录组中每种结构的形成，并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，然后进行通路分析，评估了 lncRNA 中预测结构在生物学背景中的重要性。使用接近生理条件下的生物物理实验在体外证实了选定lncRNA序列中预测的G4 (pG4)和iM (piM)结构的形成。我们发现大多数测试的 pG4 形成高度稳定的 G4 结构，并在生物学上重要的 lncRNA 中鉴定出许多以前未报道的 G4。相比之下，没有一个 piM 序列能够形成 iM 结构，这与 RNA 无法形成稳定的 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，这种结构以前在含有胞嘧啶重复的区域中没有观察到，并且代表了蛋白质-RNA 相互作用的一个有趣且尚未充分探索的目标。我们的结果强调了lncRNA中非典型二级结构的普遍性和潜在的结构相关功能，并首次显示了许多lncRNA序列中G4和Z-RNA结构的形成，进一步加深了对lncRNA结构与功能关系的理解。