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RNA–Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics

Cells frequently simultaneously express RNAs and cognate antisense transcripts without necessarily leading to the formation of RNA duplexes. Here, we present a novel transcriptome-wide experimental approach to ascertain the presence of accessible double-stranded RNA structures based on sequencing of...

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Autores principales: Bindewald, Eckart, Dai, Lisheng, Kasprzak, Wojciech K., Kim, Taejin, Gu, Shuo, Shapiro, Bruce A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321275/
https://www.ncbi.nlm.nih.gov/pubmed/30558267
http://dx.doi.org/10.3390/molecules23123329
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author Bindewald, Eckart
Dai, Lisheng
Kasprzak, Wojciech K.
Kim, Taejin
Gu, Shuo
Shapiro, Bruce A.
author_facet Bindewald, Eckart
Dai, Lisheng
Kasprzak, Wojciech K.
Kim, Taejin
Gu, Shuo
Shapiro, Bruce A.
author_sort Bindewald, Eckart
collection PubMed
description Cells frequently simultaneously express RNAs and cognate antisense transcripts without necessarily leading to the formation of RNA duplexes. Here, we present a novel transcriptome-wide experimental approach to ascertain the presence of accessible double-stranded RNA structures based on sequencing of RNA fragments longer than 18 nucleotides that were not degraded by single-strand cutting nucleases. We applied this approach to four different cell lines with respect to three different treatments (native cell lysate, removal of proteins, and removal of ribosomal RNA and proteins). We found that long accessible RNA duplexes were largely absent in native cell lysates, while the number of RNA duplexes was dramatically higher when proteins were removed. The majority of RNA duplexes involved ribosomal transcripts. The duplex formation between different non-ribosomal transcripts appears to be largely of a stochastic nature. These results suggest that cells are—via RNA-binding proteins—mostly devoid of long RNA duplexes, leading to low “noise” in the molecular patterns that are utilized by the innate immune system. These findings have implications for the design of RNA interference (RNAi)-based therapeutics by imposing structural constraints on designed RNA complexes that are intended to have specific properties with respect to Dicer cleavage and target gene downregulation.
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spelling pubmed-63212752019-01-14 RNA–Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics Bindewald, Eckart Dai, Lisheng Kasprzak, Wojciech K. Kim, Taejin Gu, Shuo Shapiro, Bruce A. Molecules Article Cells frequently simultaneously express RNAs and cognate antisense transcripts without necessarily leading to the formation of RNA duplexes. Here, we present a novel transcriptome-wide experimental approach to ascertain the presence of accessible double-stranded RNA structures based on sequencing of RNA fragments longer than 18 nucleotides that were not degraded by single-strand cutting nucleases. We applied this approach to four different cell lines with respect to three different treatments (native cell lysate, removal of proteins, and removal of ribosomal RNA and proteins). We found that long accessible RNA duplexes were largely absent in native cell lysates, while the number of RNA duplexes was dramatically higher when proteins were removed. The majority of RNA duplexes involved ribosomal transcripts. The duplex formation between different non-ribosomal transcripts appears to be largely of a stochastic nature. These results suggest that cells are—via RNA-binding proteins—mostly devoid of long RNA duplexes, leading to low “noise” in the molecular patterns that are utilized by the innate immune system. These findings have implications for the design of RNA interference (RNAi)-based therapeutics by imposing structural constraints on designed RNA complexes that are intended to have specific properties with respect to Dicer cleavage and target gene downregulation. MDPI 2018-12-15 /pmc/articles/PMC6321275/ /pubmed/30558267 http://dx.doi.org/10.3390/molecules23123329 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Bindewald, Eckart
Dai, Lisheng
Kasprzak, Wojciech K.
Kim, Taejin
Gu, Shuo
Shapiro, Bruce A.
RNA–Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics
title RNA–Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics
title_full RNA–Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics
title_fullStr RNA–Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics
title_full_unstemmed RNA–Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics
title_short RNA–Protein Interactions Prevent Long RNA Duplex Formation: Implications for the Design of RNA-Based Therapeutics
title_sort rna–protein interactions prevent long rna duplex formation: implications for the design of rna-based therapeutics
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321275/
https://www.ncbi.nlm.nih.gov/pubmed/30558267
http://dx.doi.org/10.3390/molecules23123329
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