Assaying RNA Structure Inside Living Cells with SHAPE

2017 ◽  
pp. 247-256 ◽  
Author(s):  
Chao Feng ◽  
Dalen Chan ◽  
Robert C. Spitale
Keyword(s):  
Rna Structure ◽  
Living Cells

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

2016 ◽  
Vol 113 (37) ◽  
pp. 10322-10327 ◽  
Author(s):  
Matthew J. Smola ◽  
Thomas W. Christy ◽  
Kaoru Inoue ◽  
Cindo O. Nicholson ◽  
Matthew Friedersdorf ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Rna Structure ◽  
Noncoding Rna ◽  
Ex Vivo ◽  
Living Cells ◽  
Mammalian Development ◽  
Cellular Environment ◽  
Nucleotide Resolution ◽  
In Cells

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 well-defined secondary structure domains and the cellular environment strongly modulates the RNA structure, via motifs spanning one-half of all Xist nucleotides. The Xist RNA structure modulates protein interactions in cells via multiple mechanisms. For example, repeat-containing 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.

scholarly journals Classification and clustering of RNA crosslink-ligation data reveal complex structures and homodimers

2021 ◽  
Author(s):  
Minjie Zhang ◽  
Irena T Fischer-Hwang ◽  
Kongpan Li ◽  
Jianhui Bai ◽  
Jian-Fu Chen ◽  
...  
Keyword(s):  
Rna Structure ◽  
De Novo ◽  
Rna Virus ◽  
Living Cells ◽  
Published Data ◽  
Rna Structures ◽  
Computational Tools ◽  
Systematic Analysis ◽  
Structure Information ◽  
Classification And Clustering

The recent development and application of methods based on the general principle of "crosslinking and proximity ligation" (crosslink-ligation) are revolutionizing RNA structure studies in living cells. However, extracting structure information from such data presents unique challenges. Here we introduce a set of computational tools for the systematic analysis of data from a wide variety of crosslink-ligation methods, specifically focusing on read mapping, alignment classification and clustering. We design a new strategy to map short reads with irregular gaps at high sensitivity and specificity. Analysis of previously published data reveals distinct properties and bias caused by the crosslinking reactions. We perform rigorous and exhaustive classification of alignments and discover 8 types of arrangements that provide distinct information on RNA structures and interactions. To deconvolve the dense and intertwined gapped alignments, we develop a net-work/graph-based tool CRSSANT (Crosslinked RNA Secondary Structure Analysis using Network Techniques), which enables clustering of gapped alignments and discovery of new alternative and dynamic conformations. We discover that multiple crosslinking and ligation events can occur on the same RNA, generating multi-segment alignments to report complex high level RNA structures and multi-RNA interactions. We find that alignments with overlapped segments are produced from potential homodimers and develop a new method for their de novo identification. Analysis of overlapping alignments revealed potential new homodimers in cellular noncoding RNAs and RNA virus genomes in the Picornaviridae family. Together, this suite of computational tools enables rapid and efficient analysis of RNA structure and interaction data in living cells.

scholarly journals RNA interactomics: recent advances and remaining challenges

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 1824 ◽  
Author(s):  
Brigitte Schönberger ◽  
Christoph Schaal ◽  
Richard Schäfer ◽  
Björn Voß
Keyword(s):  
High Throughput ◽  
Rna Structure ◽  
Living Cells ◽  
Structure And Function ◽  
Cellular Processes ◽  
Recent Advances ◽  
Regulatory Systems ◽  
Rna Duplexes ◽  
And Function

Tight regulation of cellular processes is key to the development of complex organisms but also vital for simpler ones. During evolution, different regulatory systems have emerged, among them RNA-based regulation that is carried out mainly by intramolecular and intermolecular RNA–RNA interactions. However, methods for the transcriptome-wide detection of these interactions were long unavailable. Recently, three publications described high-throughput methods to directly detect RNA duplexes in living cells. This promises to enable in-depth studies of RNA-based regulation and will narrow the gaps in our understanding of RNA structure and function. In this review, we highlight the benefits of these methods and their commonalities and differences and, in particular, point to methodological shortcomings that hamper their wider application. We conclude by presenting ideas for how to overcome these problems and commenting on the prospects we see in this area of research.

scholarly journals RNA structure probing to characterize RNA-protein interactions on a low abundance pre-mRNA in living cells

RNA ◽  
2020 ◽  
pp. rna.077263.120
Author(s):  
Jodi L Bubenik ◽  
Melissa Hale ◽  
Ona McConnell ◽  
Eric Wang ◽  
Maurice S. Swanson ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Rna Structure ◽  
Living Cells ◽  
Structure Probing

scholarly journals Telomeric repeat-containing RNA structure in living cells

2010 ◽  
Vol 107 (33) ◽  
pp. 14579-14584 ◽  
Author(s):  
Y. Xu ◽  
Y. Suzuki ◽  
K. Ito ◽  
M. Komiyama
Keyword(s):  
Rna Structure ◽  
Living Cells ◽  
Telomeric Repeat

scholarly journals Progress and challenges for chemical probing of RNA structure inside living cells

2015 ◽  
Vol 11 (12) ◽  
pp. 933-941 ◽  
Author(s):  
Miles Kubota ◽  
Catherine Tran ◽  
Robert C Spitale
Keyword(s):  
Rna Structure ◽  
Living Cells ◽  
Chemical Probing

scholarly journals A novel SHAPE reagent enables the analysis of RNA structure in living cells with unprecedented accuracy

2021 ◽  
Author(s):  
Tycho Marinus ◽  
Adam B Fessler ◽  
Craig A Ogle ◽  
Danny Incarnato
Keyword(s):  
Rna Structure ◽  
Structure Prediction ◽  
Critical Role ◽  
Living Cells ◽  
Pathological Process ◽  
Rna Structures ◽  
Rna Molecules ◽  
Derived Data

Abstract Due to the mounting evidence that RNA structure plays a critical role in regulating almost any physiological as well as pathological process, being able to accurately define the folding of RNA molecules within living cells has become a crucial need. We introduce here 2-aminopyridine-3-carboxylic acid imidazolide (2A3), as a general probe for the interrogation of RNA structures in vivo. 2A3 shows moderate improvements with respect to the state-of-the-art selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) reagent NAI on naked RNA under in vitro conditions, but it significantly outperforms NAI when probing RNA structure in vivo, particularly in bacteria, underlining its increased ability to permeate biological membranes. When used as a restraint to drive RNA structure prediction, data derived by SHAPE-MaP with 2A3 yields more accurate predictions than NAI-derived data. Due to its extreme efficiency and accuracy, we can anticipate that 2A3 will rapidly take over conventional SHAPE reagents for probing RNA structures both in vitro and in vivo.

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