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The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation

In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV’s largest subunit given t...

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Autores principales: Wendte, Jered M, Haag, Jeremy R, Pontes, Olga M, Singh, Jasleen, Metcalf, Sara, Pikaard, Craig S
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753486/
https://www.ncbi.nlm.nih.gov/pubmed/31329950
http://dx.doi.org/10.1093/nar/gkz615
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author Wendte, Jered M
Haag, Jeremy R
Pontes, Olga M
Singh, Jasleen
Metcalf, Sara
Pikaard, Craig S
author_facet Wendte, Jered M
Haag, Jeremy R
Pontes, Olga M
Singh, Jasleen
Metcalf, Sara
Pikaard, Craig S
author_sort Wendte, Jered M
collection PubMed
description In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV’s largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.
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spelling pubmed-67534862019-09-25 The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation Wendte, Jered M Haag, Jeremy R Pontes, Olga M Singh, Jasleen Metcalf, Sara Pikaard, Craig S Nucleic Acids Res Gene regulation, Chromatin and Epigenetics In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV’s largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci. Oxford University Press 2019-09-26 2019-07-22 /pmc/articles/PMC6753486/ /pubmed/31329950 http://dx.doi.org/10.1093/nar/gkz615 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Gene regulation, Chromatin and Epigenetics
Wendte, Jered M
Haag, Jeremy R
Pontes, Olga M
Singh, Jasleen
Metcalf, Sara
Pikaard, Craig S
The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation
title The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation
title_full The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation
title_fullStr The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation
title_full_unstemmed The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation
title_short The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation
title_sort pol iv largest subunit ctd quantitatively affects sirna levels guiding rna-directed dna methylation
topic Gene regulation, Chromatin and Epigenetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753486/
https://www.ncbi.nlm.nih.gov/pubmed/31329950
http://dx.doi.org/10.1093/nar/gkz615
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