Challenges and perspectives for structural biology of lncRNAs-the example of the Xist lncRNA A-repeats

Abstract

Following the discovery of numerous long non-coding RNA (lncRNA) transcripts in the human genome, their important roles in biology and human disease are emerging. Recent progress in experimental methods has enabled the identification of structural features of lncRNAs. However, determining high-resolution structures is challenging as lncRNAs are expected to be dynamic and adopt multiple conformations, which may be modulated by interaction with protein binding partners. The X-inactive specific transcript (Xist) is necessary for X inactivation during dosage compensation in female placental mammals and one of the best-studied lncRNAs. Recent progress has provided new insights into the domain organization, molecular features, and RNA binding proteins that interact with distinct regions of Xist. The A-repeats located at the 5' end of the transcript are of particular interest as they are essential for mediating silencing of the inactive X chromosome. Here, we discuss recent progress with elucidating structural features of the Xist lncRNA, focusing on the A-repeats. We discuss the experimental and computational approaches employed that have led to distinct structural models, likely reflecting the intrinsic dynamics of this RNA. The presence of multiple dynamic conformations may also play an important role in the formation of the associated RNPs, thus influencing the molecular mechanism underlying the biological function of the Xist A-repeats. We propose that integrative approaches that combine biochemical experiments and high-resolution structural biology in vitro with chemical probing and functional studies in vivo are required to unravel the molecular mechanisms of lncRNAs.

Keywords: Xist; chemical probing; computational structure prediction; enzymatic footprinting; lncRNA; structural biology.

Figure 1

Structural arrangements of the Xist A-repeats. (A) The Xist A-repeats are located on the 5′ end of the Xist transcript. Each repeat (7.5 in mouse and 8.5 in human) is separated by a U/A-rich linker. Wutz et al. (2002) first predicted that each A-repeat formed two stable hairpins using free energy minimization; however, Duszczyk et al. (2011) showed that only the AUCG hairpin is stable while the latter drives duplex formation. (B–G) Distinct structural models for the A-repeats. Modular arrangements (where the A-repeats assemble in a modular fashion by inter-repeat duplex formation) (B–D) and non-modular arrangements (where the A-repeats are base paired in a variety of ways) (E–G) of the A-repeats. (B) Model based on NMR analysis of single and tandem repeats in vitro (see Figure 2). (C) Mouse (left) and human (right) models based on in vitro experiments: enzymatic cleavage (V1, T1, T2), chemical probing (DMS, CMCT), FRET, and comparative sequence analysis. (D) Mouse in vivo: icSHAPE (NAI-N3), PARIS (AMT), and comparative sequence analysis. (E) Mouse in vivo: Targeted Structure-Seq (DMS) and comparative sequence analysis. (F) Mouse in vitro: chemical probing using DMS and SHAPE (1 M7) and comparative sequence analysis. (G) Mouse in vivo: SHAPE-MaP (1M6, 1M7, NMIA).

Figure 2

(A) A single 26-nucleotide A-repeat region comprising two predicted hairpins. (B and C) Similarity of one-dimensional imino spectra of the dimeric single and tandem A-repeats suggests the formation of inter-repeat dimers involving the theoretical ‘hairpin 2’. (D and E) NMR structure of the stable AUCG hairpin 1 suggests this a basic folding unit of the complete A-repeat (Duszczyk et al., 2008, 2011).

Figure 3

RNA binding proteins that have been reported to bind to the Xist A-repeats by CLIP and binding shift assays.

Figure 4

Integrated approach for tertiary structure determination of lncRNAs and their RNPs. (A) SHAPE chemical probing and NMR define RNA secondary structure. (B) SAXS/SANS provide global and subdomain shapes. (C) NMR PREs yield long-range distance restraints. Crystallography can be performed in parallel. (D) Structural analysis of holo lncRNA and RNPs can be performed using cryo-EM, and dynamics and spatial arrangements can be obtained from FRET.