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Quantifying RNA–protein interactions in situ using modified-MTRIPs and proximity ligation

The stabilization, translation and degradation of RNA are regulated by interactions between trans-acting factors, such as microRNA and RNA-binding proteins (RBP). In order to investigate the relationships between these events and their significance, a method that detects the localization of these in...

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Autores principales: Jung, Jeenah, Lifland, Aaron W., Zurla, Chiara, Alonas, Eric J., Santangelo, Philip J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592441/
https://www.ncbi.nlm.nih.gov/pubmed/22952158
http://dx.doi.org/10.1093/nar/gks837
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author Jung, Jeenah
Lifland, Aaron W.
Zurla, Chiara
Alonas, Eric J.
Santangelo, Philip J.
author_facet Jung, Jeenah
Lifland, Aaron W.
Zurla, Chiara
Alonas, Eric J.
Santangelo, Philip J.
author_sort Jung, Jeenah
collection PubMed
description The stabilization, translation and degradation of RNA are regulated by interactions between trans-acting factors, such as microRNA and RNA-binding proteins (RBP). In order to investigate the relationships between these events and their significance, a method that detects the localization of these interactions within a single cell, as well as their variability across a cell population, is needed. To visualize and quantify RNA–protein interactions in situ, we developed a proximity ligation assay (PLA) that combined peptide-modified, multiply-labelled tetravalent RNA imaging probes (MTRIPs), targeted to sequences near RBP binding sites, with proximity ligation and rolling circle amplification (RCA). Using this method, we detected and quantified, with single-interaction sensitivity, the localization and frequency of interactions of the human respiratory syncytial virus (hRSV) nucleocapsid protein (N) with viral genomic RNA (gRNA). We also described the effects of actinomycin D (actD) on the interactions of HuR with β-actin mRNA and with poly(A)+ mRNA at both native and increased HuR expression levels.
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spelling pubmed-35924412013-03-08 Quantifying RNA–protein interactions in situ using modified-MTRIPs and proximity ligation Jung, Jeenah Lifland, Aaron W. Zurla, Chiara Alonas, Eric J. Santangelo, Philip J. Nucleic Acids Res Methods Online The stabilization, translation and degradation of RNA are regulated by interactions between trans-acting factors, such as microRNA and RNA-binding proteins (RBP). In order to investigate the relationships between these events and their significance, a method that detects the localization of these interactions within a single cell, as well as their variability across a cell population, is needed. To visualize and quantify RNA–protein interactions in situ, we developed a proximity ligation assay (PLA) that combined peptide-modified, multiply-labelled tetravalent RNA imaging probes (MTRIPs), targeted to sequences near RBP binding sites, with proximity ligation and rolling circle amplification (RCA). Using this method, we detected and quantified, with single-interaction sensitivity, the localization and frequency of interactions of the human respiratory syncytial virus (hRSV) nucleocapsid protein (N) with viral genomic RNA (gRNA). We also described the effects of actinomycin D (actD) on the interactions of HuR with β-actin mRNA and with poly(A)+ mRNA at both native and increased HuR expression levels. Oxford University Press 2013-01 2012-09-05 /pmc/articles/PMC3592441/ /pubmed/22952158 http://dx.doi.org/10.1093/nar/gks837 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Jung, Jeenah
Lifland, Aaron W.
Zurla, Chiara
Alonas, Eric J.
Santangelo, Philip J.
Quantifying RNA–protein interactions in situ using modified-MTRIPs and proximity ligation
title Quantifying RNA–protein interactions in situ using modified-MTRIPs and proximity ligation
title_full Quantifying RNA–protein interactions in situ using modified-MTRIPs and proximity ligation
title_fullStr Quantifying RNA–protein interactions in situ using modified-MTRIPs and proximity ligation
title_full_unstemmed Quantifying RNA–protein interactions in situ using modified-MTRIPs and proximity ligation
title_short Quantifying RNA–protein interactions in situ using modified-MTRIPs and proximity ligation
title_sort quantifying rna–protein interactions in situ using modified-mtrips and proximity ligation
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592441/
https://www.ncbi.nlm.nih.gov/pubmed/22952158
http://dx.doi.org/10.1093/nar/gks837
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