Non-coding RNA – Zooming in on lncRNA functions

Long non-coding RNAs (lncRNAs) are emerging as important regulators of gene expression; however, in contrast to transcription factors, their functional domain architecture remains poorly understood. Now, a new method has been developed to simultaneously map RNA–RNA, RNA–DNA and RNA–protein interactions at the level of individual RNA domains with increased sensitivity.

Quinn et al. developed a method called domain-specific chromatin isolation by RNA purification (dChIRP), in which several antisense oligonucleotide pools are used to target specific domains of lncRNAs. In this method, cells are subjected to fixation, crosslinking and sonication, and the resultant sheared chromatin is hybridized to the biotinylated oligonucleotide pools to recover chromatin fragments containing specific lncRNA domains of interest. The RNA, DNA and protein components associated with the lncRNA domains can then be analysed separately.

The authors successfully applied dChIRP to study putative functional domains in the roX1 lncRNA of Drosophila melanogaster. roX1 is essential for dosage compensation, in which expression from the single X chromosome in male flies is upregulated by twofold to match expression from the pairs of autosomal chromosomes. They also showed that the three D domains of roX1 bind directly to male-specific lethal (MSL) proteins to mediate X chromosome upregulation and that the three intervening U domains exhibit minimal binding to MSL proteins but instead associate with each other.

In addition, quantitative PCR and high-throughout DNA sequencing were used to identify genome-wide roX1-binding sites. Importantly, by focusing only on the domains that strongly associate with DNA, dChIRP improved the signal-to-noise ratio by >20-fold over traditional ChIRP (which interrogated interactions across the whole of roX1).

The wealth of information provided by dChIRP led the authors to propose a ‘three-fingered hand’ architecture and an integrated interaction model for roX1. The U domains are topologically associated to form the ‘palm’ from which the three physically distinct D domain ‘fingers’ extend, and each of these fingers independently binds to MSL proteins and DNA at dosage-compensated loci on the X chromosome.

The authors then generated transgenic constructs for each domain and showed that the D3 domain of roX1 could rescue male lethality as efficiently as full-length roX1. Notably, this represents the smallest RNA unit that is sufficient for dosage compensation.

This method enables lncRNAs to be characterized at the domain level, thus providing insights into how the modularity of specific RNA domains could contribute to the diverse functions of lncRNAs in gene expression regulation.

• Lau E. (2014) Non-coding RNA: Zooming in on lncRNA functions. Nat Rev Genet 15(9):574-5. [abstract]