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RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10
Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that influence gene expression in mitochondria and chloroplasts. PPR tracts can bind RNA via a modular one repeat–one nucleotide mechanism in which the nucleotide is specified by the identities of several amin...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cold Spring Harbor Laboratory Press
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5340921/ https://www.ncbi.nlm.nih.gov/pubmed/28108520 http://dx.doi.org/10.1261/rna.059568.116 |
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author | Miranda, Rafael G. Rojas, Margarita Montgomery, Michael P. Gribbin, Kyle P. Barkan, Alice |
author_facet | Miranda, Rafael G. Rojas, Margarita Montgomery, Michael P. Gribbin, Kyle P. Barkan, Alice |
author_sort | Miranda, Rafael G. |
collection | PubMed |
description | Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that influence gene expression in mitochondria and chloroplasts. PPR tracts can bind RNA via a modular one repeat–one nucleotide mechanism in which the nucleotide is specified by the identities of several amino acids in each repeat. This mode of recognition, the so-called PPR code, offers opportunities for the prediction of native PPR binding sites and the design of proteins to bind specified RNAs. However, a deep understanding of the parameters that dictate the affinity and specificity of PPR–RNA interactions is necessary to realize these goals. We report a comprehensive analysis of the sequence specificity of PPR10, a protein that binds similar RNA sequences of ∼18 nucleotides (nt) near the chloroplast atpH and psaJ genes in maize. We assessed the contribution of each nucleotide in the atpH binding site to PPR10 affinity in vitro by analyzing the effects of single-nucleotide changes at each position. In a complementary approach, the RNAs bound by PPR10 from partially randomized RNA pools were analyzed by deep sequencing. The results revealed three patches in which nucleotide identity has a major impact on binding affinity. These include 5 nt for which protein contacts were not observed in a PPR10–RNA crystal structure and 4 nt that are not explained by current views of the PPR code. These findings highlight aspects of PPR–RNA interactions that pose challenges for binding site prediction and design. |
format | Online Article Text |
id | pubmed-5340921 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Cold Spring Harbor Laboratory Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-53409212018-04-01 RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10 Miranda, Rafael G. Rojas, Margarita Montgomery, Michael P. Gribbin, Kyle P. Barkan, Alice RNA Article Pentatricopeptide repeat (PPR) proteins comprise a large family of helical repeat proteins that influence gene expression in mitochondria and chloroplasts. PPR tracts can bind RNA via a modular one repeat–one nucleotide mechanism in which the nucleotide is specified by the identities of several amino acids in each repeat. This mode of recognition, the so-called PPR code, offers opportunities for the prediction of native PPR binding sites and the design of proteins to bind specified RNAs. However, a deep understanding of the parameters that dictate the affinity and specificity of PPR–RNA interactions is necessary to realize these goals. We report a comprehensive analysis of the sequence specificity of PPR10, a protein that binds similar RNA sequences of ∼18 nucleotides (nt) near the chloroplast atpH and psaJ genes in maize. We assessed the contribution of each nucleotide in the atpH binding site to PPR10 affinity in vitro by analyzing the effects of single-nucleotide changes at each position. In a complementary approach, the RNAs bound by PPR10 from partially randomized RNA pools were analyzed by deep sequencing. The results revealed three patches in which nucleotide identity has a major impact on binding affinity. These include 5 nt for which protein contacts were not observed in a PPR10–RNA crystal structure and 4 nt that are not explained by current views of the PPR code. These findings highlight aspects of PPR–RNA interactions that pose challenges for binding site prediction and design. Cold Spring Harbor Laboratory Press 2017-04 /pmc/articles/PMC5340921/ /pubmed/28108520 http://dx.doi.org/10.1261/rna.059568.116 Text en © 2017 Miranda 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 | Article Miranda, Rafael G. Rojas, Margarita Montgomery, Michael P. Gribbin, Kyle P. Barkan, Alice RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10 |
title | RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10 |
title_full | RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10 |
title_fullStr | RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10 |
title_full_unstemmed | RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10 |
title_short | RNA-binding specificity landscape of the pentatricopeptide repeat protein PPR10 |
title_sort | rna-binding specificity landscape of the pentatricopeptide repeat protein ppr10 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5340921/ https://www.ncbi.nlm.nih.gov/pubmed/28108520 http://dx.doi.org/10.1261/rna.059568.116 |
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