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Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling

RNA-protein interactions have critical roles in gene regulation. However, high-throughput methods to quantitatively analyze these interactions are lacking. We adapted an Illumina GAIIx sequencer to make several million such measurements with a High-Throughput Sequencing – RNA Affinity Profiling (HiT...

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Autores principales: Tome, Jacob M., Ozer, Abdullah, Pagano, John M., Gheba, Dan, Schroth, Gary P., Lis, John T.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073888/
https://www.ncbi.nlm.nih.gov/pubmed/24809628
http://dx.doi.org/10.1038/nmeth.2970
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author Tome, Jacob M.
Ozer, Abdullah
Pagano, John M.
Gheba, Dan
Schroth, Gary P.
Lis, John T.
author_facet Tome, Jacob M.
Ozer, Abdullah
Pagano, John M.
Gheba, Dan
Schroth, Gary P.
Lis, John T.
author_sort Tome, Jacob M.
collection PubMed
description RNA-protein interactions have critical roles in gene regulation. However, high-throughput methods to quantitatively analyze these interactions are lacking. We adapted an Illumina GAIIx sequencer to make several million such measurements with a High-Throughput Sequencing – RNA Affinity Profiling (HiTS-RAP) assay. Millions of cDNAs are sequenced, bound by the E. coli replication terminator protein Tus, and transcribed in situ, whereupon Tus halts transcription leaving RNA stably attached to its template DNA. The binding of fluorescently-labeled protein is then quantified in the sequencer. We used HiTS-RAP to measure the affinity of mutagenized libraries of GFP-binding and NELF-E binding aptamers to their respective targets and thereby identified regions in both aptamers that are critical for their RNA-protein interaction. We show that mutations additively affect binding affinity of the NELF-E binding aptamer, whose interaction depends mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depends primarily on secondary structure.
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spelling pubmed-40738882014-12-01 Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling Tome, Jacob M. Ozer, Abdullah Pagano, John M. Gheba, Dan Schroth, Gary P. Lis, John T. Nat Methods Article RNA-protein interactions have critical roles in gene regulation. However, high-throughput methods to quantitatively analyze these interactions are lacking. We adapted an Illumina GAIIx sequencer to make several million such measurements with a High-Throughput Sequencing – RNA Affinity Profiling (HiTS-RAP) assay. Millions of cDNAs are sequenced, bound by the E. coli replication terminator protein Tus, and transcribed in situ, whereupon Tus halts transcription leaving RNA stably attached to its template DNA. The binding of fluorescently-labeled protein is then quantified in the sequencer. We used HiTS-RAP to measure the affinity of mutagenized libraries of GFP-binding and NELF-E binding aptamers to their respective targets and thereby identified regions in both aptamers that are critical for their RNA-protein interaction. We show that mutations additively affect binding affinity of the NELF-E binding aptamer, whose interaction depends mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depends primarily on secondary structure. 2014-05-08 2014-06 /pmc/articles/PMC4073888/ /pubmed/24809628 http://dx.doi.org/10.1038/nmeth.2970 Text en http://www.nature.com/authors/editorial_policies/license.html#terms Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms
spellingShingle Article
Tome, Jacob M.
Ozer, Abdullah
Pagano, John M.
Gheba, Dan
Schroth, Gary P.
Lis, John T.
Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling
title Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling
title_full Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling
title_fullStr Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling
title_full_unstemmed Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling
title_short Comprehensive Analysis of RNA-Protein Interactions by High Throughput Sequencing-RNA Affinity Profiling
title_sort comprehensive analysis of rna-protein interactions by high throughput sequencing-rna affinity profiling
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073888/
https://www.ncbi.nlm.nih.gov/pubmed/24809628
http://dx.doi.org/10.1038/nmeth.2970
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