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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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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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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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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DOI: https://doi.org/10.1038/nbt.2943
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