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. 2017 Mar;13(3):282-289.
doi: 10.1038/nchembio.2272. Epub 2017 Jan 9.

Visualizing the Secondary and Tertiary Architectural Domains of lncRNA RepA

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Free PMC article

Visualizing the Secondary and Tertiary Architectural Domains of lncRNA RepA

Fei Liu et al. Nat Chem Biol. .
Free PMC article

Abstract

Long noncoding RNAs (lncRNAs) are important for gene expression, but little is known about their structures. RepA is a 1.6-kb mouse lncRNA comprising the same sequence as the 5' region of Xist, including A and F repeats. It has been proposed to facilitate the initiation and spread of X-chromosome inactivation, although its exact role is poorly understood. To gain insight into the molecular mechanism of RepA and Xist, we determined a complete phylogenetically validated secondary-structural map of RepA through SHAPE and DMS chemical probing of a homogeneously folded RNA in vitro. We combined UV-cross-linking experiments with RNA modeling methods to produce a three-dimensional model of RepA functional domains demonstrating that tertiary architecture exists within lncRNA molecules and occurs within specific functional modules. This work provides a foundation for understanding of the evolution and functional properties of RepA and Xist and offers a framework for exploring architectural features of other lncRNAs.

Conflict of interest statement

The authors declare no conflicts of interest.

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1:
Figure 1:. RepA folds into a compact species upon addition of Mg2+.
(a) RepA spans a region that extends from nucleotide 336 to 1965 in Xist, and includes A-repeat and F-repeat. The transcription start sites in RepA and Xist are denoted with black arrows. All the repeat regions in RepA and Xist are highlighted and labeled. (b) SV-AUC profiles of RepA obtained under native conditions in the presence of increasing concentrations of Mg2+. The graph was obtained using SedFit,. (c) Hill plot of the hydrodynamic radii (Rh, in angstroms) derived from the SV-AUC experiment described in panel a. The Rh values are derived from one independent measurement. K1/2Mg is 4.8 ± 0.2 mM and Hill coefficient (nH) is 1.4 ± 0.1. Hill plots drawn for each set of measurements are shown in Supplementary Fig. 1.
Figure 2:
Figure 2:. Secondary structure of RepA derived from SHAPE and DMS probing.
SHAPE reactivities are depicted by colored nucleotides. DMS reactivities are represented by colored dots over the nucleotides. SHAPE and DMS reactivities are denoted using the same color codes, as illustrated in the legend. Highly reactive nucleotides are indicated with red and orange, and nucleotides with low reactivities are displayed in black or blue according to their reactivity values. Covariant base-pairs in 56 mammalian sequences are highlighted in green, consistent half-flips pairs are highlighted in blue, and conserved base-pairs are highlighted in pink. UV-crosslinked nucleotide positions are indicated by arrows with dashed lines. Secondary structure was drawn using VARNA (http://varna-gui.software.informer.com/).
Figure 3:
Figure 3:. Fragment analysis reveals independently-folded domains in RepA.
(a) Schematic representation of RepA fragments with respect to their position along the sequence of full-length RepA. (b) Scatterplots comparing SHAPE reactivity of each fragment with the corresponding region in full-length RepA. Pearson correlation values (rp) between SHAPE reactivities of each fragment and the corresponding regions in full-length RepA are indicated; the standard error in correlation coefficients was estimated by bootstrapping analysis.
Figure 4:
Figure 4:. D1 of RepA contains the functionally important A-repeat motif.
(a) Secondary structural map of D1. Repeat-A units are highlighted in green and labeled accordingly. Linker regions are indicated by L1-L7. Helices in D1 are indicated by H1-H11. (b) Sequence conservation of the linker regions L3 (nucleotides 143–167), L6 (nucleotides 304–331) and L7 (nucleotides 357–373) across 27 mammalian species. The alignment of the sequences is presented and color-coded by nucleotide type, and the consensus sequence is shown on the bottom.
Figure 5:
Figure 5:. Characterization of the UV crosslinking sites within RepA.
(a) UV crosslinking patterns of RepA. Unirradiated control RNA (C) is shown in the leftmost lane. UV-treated RNA samples in the presence of 0, 5 and 10 mM Mg are shown in lanes 0, 5 and 10, respectively. The crosslinked RNA bands are labeled 1 to 8. The un-crosslinked parent RNA band is denoted by P. (b-d) Primer extension mapping of the crosslinked nucleotides. Lanes U, G and A show dideoxy sequencing ladders. Lanes C, P, 4, 5 or 7 show reverse transcription (RT) products of corresponding RNA species. The nucleotide positions of identified RT stops are indicated to the right of the gels. Original gel images are shown in Supplementary Fig. 8. (e) Primary sequence conservation of 20 mammalian species corresponding to the regions containing crosslink CL4. The alignment of the sequences is presented and color-coded by nucleotide type, and the consensus sequence is shown on the bottom. (f) Three-dimensional model of the subdomains in D2 (nucleotides 494–508 and 632–776, colored in purple) and D3 (nucleotides 1272–1304 and 1349–1406, colored in yellow). Nucleotides participating in crosslink CL4 and CL7 are colored in purple and red, respectively.

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