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Nat Biotechnol. 2014 Sep;32(9):933-940. doi: 10.1038/nbt.2943. Epub 2014 Jul 6.

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

Author information

1
Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford CA 94305, USA.
2
Department of Bioengineering, Stanford University Schools of Medicine and Engineering, Stanford CA 94305, USA.
3
Max Planck Institute of Immunobiology and Epigenetics, Stübeweg 51, 79108 Freiburg im Breisgau, Germany.
#
Contributed equally

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.

Comment in

PMID:
24997788
PMCID:
PMC4175979
DOI:
10.1038/nbt.2943
[Indexed for MEDLINE]
Free PMC Article

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