SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells

Abstract

The 18-kb Xist long noncoding RNA (lncRNA) is essential for X-chromosome inactivation during female eutherian mammalian development. Global structural architecture, cell-induced conformational changes, and protein-RNA interactions within Xist are poorly understood. We used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to examine these features of Xist at single-nucleotide resolution both in living cells and ex vivo. The Xist RNA forms complex welldefined secondary structure domains and the cellular environment strongly modulates the RNA structure, via motifs spanning onehalf of all Xist nucleotides. The Xist RNA structure modulates protein interactions in cells via multiple mechanisms. For example, repeatcontaining elements adopt accessible and dynamic structures that function as landing pads for protein cofactors. Structured RNA motifs create interaction domains for specific proteins and also sequester other motifs, such that only a subset of potential binding sites forms stable interactions. This work creates a broad quantitative framework for understanding structure-function interrelationships for Xist and other lncRNAs in cells.ong noncoding RNAs (lncRNAs) play central roles in the regulation of gene expression through interactions with numerous protein partners (1) and are necessary for normal health and development (2, 3). The 18-kb Xist lncRNA is essential for X-chromosome inactivation during female eutherian mammalian development and is an archetype of gene-silencing lncRNAs. During the early stages of X inactivation, Xist accumulates in cis around the future inactive X chromosome and recruits protein complexes that apply repressive chromatin modifications, leading to stable gene silencing (3,4).Genetic deletion studies have demarcated several broad regions of function within Xist. Several tandem repeat regions (labeled A-F in the mouse) show moderate conservation (5-7), and at least two of these, repeat A and the rodent-specific repeat C, are implicated in silencing and localization to the inactive X. Deletion of the final 7.5-kb exon of Xist causes a defect in its localization (8), and the 1.5-kb region encompassing repeats F and B is required for accumulation of heterochromatic marks over the inactive X (4); however, beyond these initial characterizations, the mechanisms by which gene silencing, heterochromatinization, and localization of Xist on the X chromosome occur are not well understood. In particular, the role of RNA structure in orchestrating these distinct functions remains unclear.Several previous studies have suggested the importance of RNA structures in specific regions of Xist (9-12), but overall, the locations and structures of functional domains within Xist are poorly defined. Detailed structural maps of other functional RNAs, such as ribosomal RNAs (13) and the HIV RNA genome (14-16), have been fundamental to understanding the mechanisms by which individual domains within large RNAs execute discrete cellular functions. A detailed an...

Quantifying RNA binding sites transcriptome-wide using DO-RIP-seq

“…S3). Additionally, we successfully performed DO-RIP-seq experiments for HuR in mouse cells and identified many of the same sites in mouse as we observed in human (Smola et al 2016). While producing more HuR binding sites than reported in any other study, the locations and nucleic acid sequence characteristics were similar to previous observations (Levine et al 1993;Gao et al 1994;Ma et al 1996;Lebedeva et al 2011;Mukherjee et al 2011).…”

“…It is possible that some negative IP's may introduce biases such as the binding of aptameric RNAs to antibodies. However, we have found that our antibody-based approach to identify and quantify binding was complemented by nonantibody-based Delta-SHAPE experiments determining RBP binding sites in XIST lincRNA (Smola et al 2016). This suggests that antibody-based biases have a negligible effect on DO-RIP-seq binding sites.…”

Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure

“…Our observation that these regions have higher sequence conservation suggests that interactions with the cellular environment may exert selective pressure on the RNA sequence. However, these differences do not occur with higher frequency in regions with low or high SHAPE reactivity (Supplemental Table 3), suggesting that interactions with the cellular environment are not limited to regions folding to single, well-defined conformations that have low median SHAPE (Smola et al 2016). …”

“…6B) conditions. This approach to visualizing the data enables us to identify regions of relatively high median SHAPE (less structure, more complex conformational ensemble) and relatively low median SHAPE (more structure, more likely a single conformation) (Smola et al 2016). We treated the protein:mRNA complex with 1M7 (bound), extracted RNA from cells, removing all proteins (unbound), or we transcribed RNA with T7 RNA polymerase and then treated with 1M7 (in vitro) (Supplemental Figs.…”

“…Although not statistically significant, this correlation suggests that the most highly conserved regions appear to undergo the most significant conformational rearrangements. How cellular conditions impact RNA secondary structure is an active area of research (Ding et al 2014;Rouskin et al 2014;Spitale et al 2015;Smola et al 2016;Watters et al 2016a), and conservation may be key to understanding just how the environment affects RNA structures.…”