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Revealing long noncoding RNA architecture and functions using domain-specific chromatin isolation by RNA purification

Nature Biotechnology volume 32, pages 933–940 (2014)Cite this article

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Abstract

Little is known about the functional domain architecture of long noncoding RNAs (lncRNAs) because of a relative paucity of suitable methods to analyze RNA function at a domain level. Here we describe domain-specific chromatin isolation by RNA purification (dChIRP), a scalable technique to dissect pairwise RNA-RNA, RNA-protein and RNA-chromatin interactions at the level of individual RNA domains in living cells. dChIRP of roX1, a lncRNA essential for Drosophila melanogaster X-chromosome dosage compensation, reveals a 'three-fingered hand' ribonucleoprotein topology. Each RNA finger binds chromatin and the male-specific lethal (MSL) protein complex and can individually rescue male lethality in roX-null flies, thus defining a minimal RNA domain for chromosome-wide dosage compensation. dChIRP improves the RNA genomic localization signal by >20-fold relative to previous techniques, and these binding sites are correlated with chromosome conformation data, indicating that most roX-bound loci cluster in a nuclear territory. These results suggest dChIRP can reveal lncRNA architecture and function with high precision and sensitivity.

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Figure 1: dChIRP uses antisense oligonucleotides to purify specific RNA domains and associated RNAs, proteins and chromatin.
Figure 2: dChIRP RNA co-recovery reveals roX1's topological architecture.
Figure 3: roX1 D domains interact with the MSL complex and chromatin on the X chromosome.
Figure 4: dChIRP boosts genomic occupancy signal relative to traditional ChIRP-seq.
Figure 5: CESs cluster together in a dosage-compensation territory of the nucleus.
Figure 6: roX1's D domains are independent, functional RNA subunits.

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Acknowledgements

We thank members of the Chang and Akhtar labs, P. Sharp and X. Wu (Massachusetts Institute of Technology) for meaningful discussion and E. Larschan (Brown University) for CLAMP antibody. Supported by US National Institutes of Health R01-CA118750 and R01-ES023168 (H.Y.C.), Max Planck Society (A.A.) and Bio-X Fellowship (J.J.Q.). This work was supported by DFG-funded SFB992 and SFB746 and EU-funded EpiGeneSys awarded to A.A. H.Y.C. is an Early Career Scientist of the Howard Hughes Medical Institute; A.A. is part of the BIOSS excellence initiative.

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Author notes

  1. Ibrahim A Ilik, Kun Qu and Plamen Georgiev: These authors contributed equally to this work.

Authors and Affiliations

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

    Jeffrey J Quinn, Kun Qu, Ci Chu & Howard Y Chang

  2. Department of Bioengineering, Stanford University School of Medicine and School of Engineering, Stanford, California, USA

    Jeffrey J Quinn

  3. Max Planck Institute of Immunobiology and Epigenetics, Stübeweg 51, Freiburg im Breisgau, Germany

    Ibrahim A Ilik, Plamen Georgiev & Asifa Akhtar

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Contributions

J.J.Q., I.A.I., P.G., C.C., H.Y.C. and A.A. designed the research. J.J.Q., I.A.I. and P.G. performed the research. K.Q. and J.J.Q. performed bioinformatic analyses. J.J.Q. and H.Y.C. wrote the manuscript. All authors discussed the results and reviewed the manuscript.

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Correspondence to Howard Y Chang.

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Quinn, J., Ilik, I., Qu, K. et al. Revealing long noncoding RNA architecture and functions using domain-specific chromatin isolation by RNA purification. Nat Biotechnol 32, 933–940 (2014). https://doi.org/10.1038/nbt.2943

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