Letter | Published:

Structural imprints in vivo decode RNA regulatory mechanisms

Nature volume 519, pages 486490 (26 March 2015) | Download Citation

  • An Erratum to this article was published on 30 September 2015

Abstract

Visualizing the physical basis for molecular behaviour inside living cells is a great challenge for biology. RNAs are central to biological regulation, and the ability of RNA to adopt specific structures intimately controls every step of the gene expression program1. However, our understanding of physiological RNA structures is limited; current in vivo RNA structure profiles include only two of the four nucleotides that make up RNA2,3. Here we present a novel biochemical approach, in vivo click selective 2′-hydroxyl acylation and profiling experiment (icSHAPE), which enables the first global view, to our knowledge, of RNA secondary structures in living cells for all four bases. icSHAPE of the mouse embryonic stem cell transcriptome versus purified RNA folded in vitro shows that the structural dynamics of RNA in the cellular environment distinguish different classes of RNAs and regulatory elements. Structural signatures at translational start sites and ribosome pause sites are conserved from in vitro conditions, suggesting that these RNA elements are programmed by sequence. In contrast, focal structural rearrangements in vivo reveal precise interfaces of RNA with RNA-binding proteins or RNA-modification sites that are consistent with atomic-resolution structural data. Such dynamic structural footprints enable accurate prediction of RNA–protein interactions and N6-methyladenosine (m6A) modification genome wide. These results open the door for structural genomics of RNA in living cells and reveal key physiological structures controlling gene expression.

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Gene Expression Omnibus

Data deposits

All genomic data sets have been deposited in the Gene Expression Omnibus under accession number GSE64169.

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Acknowledgements

We thank members of the Chang laboratory, J. Weissman and J. Doudna for comments. This work was supported by National Institutes of Health (NIH) R01HG004361 and P50HG007735, and the California Institute for Regenerative Medicine (H.Y.C.), NIH R01068122 (E.T.K.), the A.P. Giannini Foundation (R.C.S.), a Stanford Dean’s Fellowship (Q.C.Z.), NIH T32AR007422 (P.J.B.), and the Stanford Medical Scientist Training Program and NIH F30CA189514 (R.A.F.). H.Y.C. is an Early Career Scientist of the Howard Hughes Medical Institute.

Author information

Author notes

    • Robert C. Spitale
    • , Ryan A. Flynn
    •  & Qiangfeng Cliff Zhang

    These authors contributed equally to this work.

Affiliations

  1. Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Robert C. Spitale
    • , Ryan A. Flynn
    • , Qiangfeng Cliff Zhang
    • , Byron Lee
    • , Pedro J. Batista
    • , Eduardo A. Torre
    •  & Howard Y. Chang
  2. Department of Chemistry, Stanford University, Stanford, California 94305, USA

    • Pete Crisalli
    • , Jong-Wha Jung
    • , Hannes Y. Kuchelmeister
    •  & Eric T. Kool

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Contributions

R.C.S., E.T.K. and H.Y.C. conceived the study. R.C.S., P.C., J.-W.J., H.Y.K. and E.T.K. performed chemical design and synthesis. R.C.S., R.A.F., B.L., E.A.T. and P.J.B. performed biological experiments. Q.C.Z., R.C.S., R.A.F. and H.Y.C. performed data analysis. R.C.S. and H.Y.C. wrote the paper with input from all authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Eric T. Kool or Howard Y. Chang.

Extended data

Supplementary information

Excel files

  1. 1.

    Supplementary Table 1 - VTD Analysis on hexamer sequences corresponding to RNA motifs identified by RNA Compete.

    VTD information on the RBP binding motifs identified in an RNAcompete assay. Each row corresponds to aRNAcompete motif. Rows of human and mouse motifs are highlighted in green. Motifs are indexed by the Protein_name. The species and kingdom of the source organism are indicated, as well as the type(s) of RNA-binding domains contained in the protein. RNAcompete_logo is a sequence logo representing the motif obtained from RNAcompete, IUPAC_hexamerMotif are the manually curated IUPAC representation of the consensus sequences of the closest hexamers. VTD is the reactivity score difference in vivo and in vitro, as defined in the manuscript. pVaule_stable, pValue_gain and pValue_loss are p values of three permutation tests that ask: 1), whether the absolute value of the motif VTD is significantly less than a random hexamer, i.e., represents a stable region; 2), whether the motif VTD is significantly smaller than a random hexamer, i.e., represents a region that is more structured in vivo; and 3), whether the motif VTD is significantly bigger than a random hexamer, i.e., represents a region that is more structured in vitro.

  2. 2.

    Supplementary Table 2 - VTD information on hexamers that match to hexamer-seed of a mouse miRNA.

    The file contains VTD information on hexamers that match to hexamer-seed of a mouse miRNA. Each row corresponds to a mouse miRNA seed complementary hexamer. Motifs are indexed by the miRNA name; miRNAs with the same hexamer seeds are grouped together. All other values are defined the same as in the first spreadsheet.

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DOI

https://doi.org/10.1038/nature14263

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