RNA targets of multitargeted RNA-binding proteins (RBPs) can be studied by various methods including mobility shift assays, iterative in vitro selection techniques and computational approaches. These techniques, however, cannot be used to identify the cellular context within which mRNAs associate, nor can they be used to elucidate the dynamic composition of RNAs in ribonucleoprotein (RNP) complexes in response to physiological stimuli. But by combining biochemical and genomics procedures to isolate and identify RNAs associated with RNA-binding proteins, information regarding RNA-protein and RNA-RNA interactions can be examined more directly within a cellular context. Several protocols--including the yeast three-hybrid system and immunoprecipitations that use physical or chemical cross-linking--have been developed to address this issue. Cross-linking procedures in general, however, are limited by inefficiency and sequence biases. The approach outlined here, termed RNP immunoprecipitation-microarray (RIP-Chip), allows the identification of discrete subsets of RNAs associated with multi-targeted RNA-binding proteins and provides information regarding changes in the intracellular composition of mRNPs in response to physical, chemical or developmental inducements of living systems. Thus, RIP-Chip can be used to identify subsets of RNAs that have related functions and are potentially co-regulated, as well as proteins that are associated with them in RNP complexes. Using RIP-Chip, the identification and/or quantification of RNAs in RNP complexes can be accomplished within a few hours or days depending on the RNA detection method used.

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...

BackgroundSequence specific RNA binding proteins are important regulators of gene expression. Several related crosslinking-based, high-throughput sequencing methods, including PAR-CLIP, have recently been developed to determine direct binding sites of global protein-RNA interactions. However, no studies have quantitatively addressed the contribution of background binding to datasets produced by these methods.ResultsWe measured non-specific RNA background in PAR-CLIP data, demonstrating that covalently crosslinked background binding is common, reproducible and apparently universal among laboratories. We show that quantitative determination of background is essential for identifying targets of most RNA-binding proteins and can substantially improve motif analysis. We also demonstrate that by applying background correction to an RNA binding protein of unknown binding specificity, Caprin1, we can identify a previously unrecognized RNA recognition element not otherwise apparent in a PAR-CLIP study.ConclusionsEmpirical background measurements of global RNA-protein crosslinking are a necessary addendum to other experimental controls, such as performing replicates, because covalently crosslinked background signals are reproducible and otherwise unavoidable. Recognizing and quantifying the contribution of background extends the utility of PAR-CLIP and can improve mechanistic understanding of protein-RNA specificity, protein-RNA affinity and protein-RNA association dynamics.