Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo

Abstract

RNA plays a dual role as an informational molecule and a direct effector of biological tasks. The latter function is enabled by RNA’s ability to adopt complex secondary and tertiary folds and thus has motivated extensive computational1–2 and experimental3–8 efforts for determining RNA structures. Existing approaches for evaluating RNA structure have been largely limited to in vitro systems, yet the thermodynamic forces which drive RNA folding in vitro may not be sufficient to predict stable RNA structures in vivo5. Indeed, the presence of RNA binding proteins and ATP-dependent helicases can influence which structures are present inside cells. Here we present an approach for globally monitoring RNA structure in native conditions in vivo with single nucleotide precision. This method is based on in vivo modification with dimethyl sulfate (DMS), which reacts with unpaired adenine and cytosine residues9, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known mRNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome10. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast reveals that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding, thermodynamics play an incomplete role in determining mRNA structure in vivo.

Temperature-responsive in vitro RNA structurome of Yersinia pseudotuberculosis

“…In the crowded intracellular environment, where an RNA molecule encounters other RNAs, proteins, ions, and ligands, its structure might adopt a conformation different from the one mapped in vitro. The membranepermeable, adenine-and cytosine-modifying probe dimethyl sulfate was used to read out in vivo RNA structures in yeast (23) and A. thaliana (19). A cell-permeable SHAPE reagent that modifies accessible 2′-hydroxy groups of all four nucleotides allowed in vivo probing of the mouse transcriptome (22).…”

“…Global structure probing maps the structures of the entire pool of expressed RNA molecules and provides a snapshot of the RNA structurome (13). Probing at different temperatures profiles dynamic RNA structures and revealed a large number of RNAT candidates in yeast (14).Global RNA structure-probing approaches have been used with yeast cells (13, 15), Drosophila melanogaster and Caenorhabditis elegans (16), Arabidopsis thaliana (17-19), mouse (20-22), human cells (23,24), and, most recently, with Escherichia coli (25). We applied the parallel analysis of RNA structure (PARS) and experimentally determined single-and double-stranded regions in the Y. pseudotuberculosis YPIII transcriptome at three different temperatures.…”

“…Global RNA structure-probing approaches have been used with yeast cells (13,15), Drosophila melanogaster and Caenorhabditis elegans (16), Arabidopsis thaliana (17)(18)(19), mouse (20)(21)(22), human cells (23,24), and, most recently, with Escherichia coli (25). We applied the parallel analysis of RNA structure (PARS) and experimentally determined single-and double-stranded regions in the Y. pseudotuberculosis YPIII transcriptome at three different temperatures.…”

RNA structure through multidimensional chemical mapping

“…For example, viral RNA genomes interconvert between compact structures in packaged forms, less-structured cellular states that can recruit and organize host proteins, and states available for translation or replication [see, e.g. (Bothe et al 2011;Filbin & Kieft, 2009;Schneemann, 2006) Rouskin et al 2014;Spitale et al 2015)]. On the other hand, whether these conformational changes are functional or simply 'structural noise' is unknown for most regions, and the uncertainty is exacerbated by the difficulty of deconvolving the component structures from data that average over the entire ensemble of structures (Eddy, 2014;Washietl et al 2012).…”

The Elaborate Architecture of Rna

“…One month later, a group at the University of California, San Francisco, reported a comparable study of yeast and human cells 2 . The number of RNA structures they managed to resolve was "unprecedented", says Alain Laederach, an RNA biologist at the University of North Carolina at Chapel Hill (UNC).…”