Oral Presentation 50 Years Shine-Dalgarno Symposium 2023

Preferential Formation Of Z-RNA Over i-Motifs In Long Non-Coding RNA (#8)

Uditi Bhatt 1 , Anne Cucchiarini 2 , Yu Luo 2 , Cameron W Evans 1 , Jean-Louis Mergny 2 , Iyer K Swaminathan 1 , Nicole M Smith 1
  1. School of Molecular Sciences, The University of Western Australia, Perth, WA, Australia
  2. Laboratoire d’Optique et Biosciences, École Polytechnique, Paris, France

Secondary structure is a principal determinant of lncRNA function, predominantly regarding scaffold formation and interfaces with target molecules. Non-canonical nucleic acid secondary structures including G-quadruplexes (G4s), i-Motifs (iMs), and R-loops (RLs) have known roles in regulating gene expression but are significantly less well characterized in lncRNA compared to their DNA and mRNA counterparts (1–3). Left-handed double helix Z-RNA structures are known to influence RNA editing and human diseases and have mostly been studied in GC-rich sequences, however their formation in alternative RNA types and sequences has been significantly less researched (4).

We utilised G4-iM Grinder and QmRLFS_Finder computational tools to predict the formation of G4s, iMs, and RLs throughout the lncRNA transcriptome in comparison to protein-coding transcripts. The importance of the predicted lncRNA structures in biological contexts was assessed by combining our results with publicly available lncRNA tissue expression data followed by pathway analysis. Formation of predicted G4 (pG4) and iM (piM) structures in select lncRNA sequences was determined in vitro using biophysical experiments including fluorescence assays, fluorescence resonance energy transfer (FRET) melting competition, isothermal difference spectra (IDS), thermal difference spectra (TDS), UV-melt, and circular dichroism (CD).

The majority of the tested pG4 sequences formed a stable G4 structure. In contrast, none of the piM sequences were able to form iM structures even in favourable pH conditions, supporting existing literature reporting the inability of iMs to form in RNA due to poor stability (5, 6). Surprisingly, the piM lncRNA sequences instead formed left-handed double helix Z-RNA structures which have the potential to facilitate novel protein interactions and affect disease. Our results highlight the prevalence and potential structure-associated functions of non-canonical secondary structures in lncRNA. We observe G4 and Z-RNA structure formation in many lncRNA sequences for the first time, furthering understanding of the structure-function relationship in lncRNAs.

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