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Visualizing the secondary and tertiary architectural domains of lncRNA RepA

Nature Chemical Biology volume 13, pages 282–289 (2017)Cite this article

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Abstract

Long noncoding RNAs (lncRNAs) are important for gene expression, but little is known about their structures. RepA is a 1.6-kb mouse lncRNA comprising the same sequence as the 5′ region of Xist, including A and F repeats. It has been proposed to facilitate the initiation and spread of X-chromosome inactivation, although its exact role is poorly understood. To gain insight into the molecular mechanism of RepA and Xist, we determined a complete phylogenetically validated secondary-structural map of RepA through SHAPE and DMS chemical probing of a homogeneously folded RNA in vitro. We combined UV-cross-linking experiments with RNA modeling methods to produce a three-dimensional model of RepA functional domains demonstrating that tertiary architecture exists within lncRNA molecules and occurs within specific functional modules. This work provides a foundation for understanding of the evolution and functional properties of RepA and Xist and offers a framework for exploring architectural features of other lncRNAs.

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Figure 1: RepA folds into a compact species after addition of Mg2+.
Figure 2: Secondary structure of RepA derived from SHAPE and DMS probing.
Figure 3: Fragment analysis revealing independently folded domains in RepA.
Figure 4: D1 of RepA contains the functionally important A-repeat motif.
Figure 5: Characterization of the UV-cross-linking sites within RepA.

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Acknowledgements

We acknowledge M. Simon (Yale University) for sharing the in vivo DMS probing data of Xist and E. Jagdmann for the synthesis of 1M7. We thank T. Dickey, C. Zhao, O. Fedorova, and all other members of the Pyle laboratory for constructive discussion and critical reading of the manuscript. This project was supported by the National Institutes of Health (R01GM50313). A.M.P. is supported as an Investigator, and F.L. is supported as a Postdoctoral Fellow, of the Howard Hughes Medical Institute.

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  1. Srinivas Somarowthu

    Present address: Present address: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.,

Authors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA

    Fei Liu, Srinivas Somarowthu & Anna Marie Pyle

  2. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Fei Liu & Anna Marie Pyle

  3. Department of Chemistry, Yale University, New Haven, Connecticut, USA

    Anna Marie Pyle

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  1. Fei Liu
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Contributions

F.L. and A.M.P. designed the project. F.L. performed the SV-AUC, SEC, SHAPE and DMS probing, and UV-cross-linking experiments and analyzed the data obtained from the aforementioned experiments; F.L. performed the phylogenetic studies; S.S. conducted jackknife resampling, Shannon entropy, and bootstrapping analyses, and performed 3D modeling experiments. F.L., S.S., and A.M.P. wrote the manuscript.

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Correspondence to Anna Marie Pyle.

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Supplementary Results, Supplementary Figures 1–11 and Supplementary Table 1 (PDF 20667 kb)

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Liu, F., Somarowthu, S. & Pyle, A. Visualizing the secondary and tertiary architectural domains of lncRNA RepA. Nat Chem Biol 13, 282–289 (2017). https://doi.org/10.1038/nchembio.2272

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