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Mod-seq: high-throughput sequencing for chemical probing of RNA structure

The functions of RNA molecules are intimately linked to their ability to fold into complex secondary and tertiary structures. Thus, understanding how these molecules fold is essential to determining how they function. Current methods for investigating RNA structure often use small molecules, enzymes...

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Autores principales: Talkish, Jason, May, Gemma, Lin, Yizhu, Woolford, John L., McManus, C. Joel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988572/
https://www.ncbi.nlm.nih.gov/pubmed/24664469
http://dx.doi.org/10.1261/rna.042218.113
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author Talkish, Jason
May, Gemma
Lin, Yizhu
Woolford, John L.
McManus, C. Joel
author_facet Talkish, Jason
May, Gemma
Lin, Yizhu
Woolford, John L.
McManus, C. Joel
author_sort Talkish, Jason
collection PubMed
description The functions of RNA molecules are intimately linked to their ability to fold into complex secondary and tertiary structures. Thus, understanding how these molecules fold is essential to determining how they function. Current methods for investigating RNA structure often use small molecules, enzymes, or ions that cleave or modify the RNA in a solvent-accessible manner. While these methods have been invaluable to understanding RNA structure, they can be fairly labor intensive and often focus on short regions of single RNAs. Here we present a new method (Mod-seq) and data analysis pipeline (Mod-seeker) for assaying the structure of RNAs by high-throughput sequencing. This technique can be utilized both in vivo and in vitro, with any small molecule that modifies RNA and consequently impedes reverse transcriptase. As proof-of-principle, we used dimethyl sulfate (DMS) to probe the in vivo structure of total cellular RNAs in Saccharomyces cerevisiae. Mod-seq analysis simultaneously revealed secondary structural information for all four ribosomal RNAs and 32 additional noncoding RNAs. We further show that Mod-seq can be used to detect structural changes in 5.8S and 25S rRNAs in the absence of ribosomal protein L26, correctly identifying its binding site on the ribosome. While this method is applicable to RNAs of any length, its high-throughput nature makes Mod-seq ideal for studying long RNAs and complex RNA mixtures.
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spelling pubmed-39885722015-05-01 Mod-seq: high-throughput sequencing for chemical probing of RNA structure Talkish, Jason May, Gemma Lin, Yizhu Woolford, John L. McManus, C. Joel RNA Methods The functions of RNA molecules are intimately linked to their ability to fold into complex secondary and tertiary structures. Thus, understanding how these molecules fold is essential to determining how they function. Current methods for investigating RNA structure often use small molecules, enzymes, or ions that cleave or modify the RNA in a solvent-accessible manner. While these methods have been invaluable to understanding RNA structure, they can be fairly labor intensive and often focus on short regions of single RNAs. Here we present a new method (Mod-seq) and data analysis pipeline (Mod-seeker) for assaying the structure of RNAs by high-throughput sequencing. This technique can be utilized both in vivo and in vitro, with any small molecule that modifies RNA and consequently impedes reverse transcriptase. As proof-of-principle, we used dimethyl sulfate (DMS) to probe the in vivo structure of total cellular RNAs in Saccharomyces cerevisiae. Mod-seq analysis simultaneously revealed secondary structural information for all four ribosomal RNAs and 32 additional noncoding RNAs. We further show that Mod-seq can be used to detect structural changes in 5.8S and 25S rRNAs in the absence of ribosomal protein L26, correctly identifying its binding site on the ribosome. While this method is applicable to RNAs of any length, its high-throughput nature makes Mod-seq ideal for studying long RNAs and complex RNA mixtures. Cold Spring Harbor Laboratory Press 2014-05 /pmc/articles/PMC3988572/ /pubmed/24664469 http://dx.doi.org/10.1261/rna.042218.113 Text en © 2014 Talkish et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Methods
Talkish, Jason
May, Gemma
Lin, Yizhu
Woolford, John L.
McManus, C. Joel
Mod-seq: high-throughput sequencing for chemical probing of RNA structure
title Mod-seq: high-throughput sequencing for chemical probing of RNA structure
title_full Mod-seq: high-throughput sequencing for chemical probing of RNA structure
title_fullStr Mod-seq: high-throughput sequencing for chemical probing of RNA structure
title_full_unstemmed Mod-seq: high-throughput sequencing for chemical probing of RNA structure
title_short Mod-seq: high-throughput sequencing for chemical probing of RNA structure
title_sort mod-seq: high-throughput sequencing for chemical probing of rna structure
topic Methods
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988572/
https://www.ncbi.nlm.nih.gov/pubmed/24664469
http://dx.doi.org/10.1261/rna.042218.113
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