HOTAIR forms an intricate and modular secondary structure

Abstract

Long noncoding RNAs (lncRNAs) have recently emerged as key players in fundamental cellular processes and diseases, but their functions are poorly understood. HOTAIR is a 2,148-nt-long lncRNA molecule involved in physiological epidermal development and in pathogenic cancer progression, where it has been demonstrated to repress tumor and metastasis suppressor genes. To gain insights into the molecular mechanisms of HOTAIR, we purified it in a stable and homogenous form in vitro, and we determined its functional secondary structure through chemical probing and phylogenetic analysis. The HOTAIR structure reveals a degree of structural organization comparable to well-folded RNAs, like the group II intron, rRNA, or lncRNA steroid receptor activator. It is composed of four independently folding modules, two of which correspond to predicted protein-binding domains. Secondary structure elements that surround protein-binding motifs are evolutionarily conserved. Our work serves as a guide for "navigating" through the lncRNA HOTAIR and ultimately for understanding its function.

Figure 1. Purification and folding of HOTAIR

(A) Homogeneity of HOTAIR RNA evaluated by SEC. HOTAIR prepared via native purification (red) produces a homogeneous and monodisperse RNA sample. “Snapcool” (green) and “slowcool” (blue) denaturing protocols produce heterogeneous samples characterized by a broad distribution of elution volumes and by accumulation of aggregated material in the void volume. (B) Homogeneity of HOTAIR RNA determined by SV-AUC. HOTAIR RNA obtained through native method (red) sediments as a single homogenous species with a sedimentation coefficient of approximately 20S, whereas samples prepared by denaturation and refolding (green and blue) display a highly inhomogeneous distribution of particles. (C) SV-AUC profiles of HOTAIR obtained under native conditions in the presence of increasing concentrations of magnesium. The graph was obtained using SedFit (Brown and Schuck, 2006). (D) Hill plot of the hydrodynamic radii (R_h, in angstroms) derived from the SV-AUC experiment described in panel B (see also Figure S1).

Figure 2

Secondary structure of HOTAIR derived from SHAPE, DMS, and terbium chemical probing. SHAPE reactivities are depicted by colored nucleotides. DMS reactivities are represented by colored dots over the nucleotides. Terbium reactivities are represented by squares on the nucleotides. Highly reactive nucleotides are displayed in red and orange and low reactive nucleotides are displayed in black or blue according to the values reported in the legend. Watson-Crick and non-canonical base pairs are depicted by black and purple lines, respectively (also see Figures S2–S3, Table S1 and Table S3).

Figure 3. Shotgun fragment analysis reveals modularity in HOTAIR

(A) Schematic representation of HOTAIR fragments in respect to their position along the sequence of full-length HOTAIR. (B) Normalized SHAPE reactivity of full-length HOTAIR. (C) Scatter plots comparing shape reactivity of each fragment with corresponding region in full-length HOTAIR. Pearson correlation values (r_p) between the reactivity of each fragment and of full-length HOAIR are indicated (also see Table S2).

Figure 4. Sequence covariation in HOTAIR

(A) Secondary structure map of HOTAIR color-coded by evolutionary covariation of each base-pair in 33 mammalian sequences. Covariant base-pairs are highlighted in green, consistent half-flips pairs are highlighted in blue, and conserved base-pairs are highlighted in red. (B) One of the most highly conserved helices in the predicted PRC2-binding region (D1) of HOTAIR. The secondary structure map of H7 (nucleotides 187–216), base pairs covarying or conserved in Human and Mouse (numbered according to Genebank ID gi: 383286748) are highlighted. The alignment of the sequences of human and mouse HOTAIR is presented, color-coded by residue type. (C) Helix 10 (327–394) is not part of the predicted PRC2-binding region but it is also highly conserved between human and mouse, suggesting that helices that are not of part binding region may also play a role in HOTAIR function (also see Figure S4).