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Functional analysis of the Pol IV DeCL domain

ABSTRACT: Functional analysis of the Pol IV DeCL domain

PROVIDER: PRJNA378527 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5320840.fastq.gz	Fastqsanger.gz
	SRR5320841.fastq.gz	Fastqsanger.gz
	SRR5320832_1.fastq.gz	Fastqsanger.gz
	SRR5320832_2.fastq.gz	Fastqsanger.gz
	SRR5320842.fastq.gz	Fastqsanger.gz

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CLSY docking to Pol IV requires a conserved domain critical for small RNA biogenesis and transposon silencing

Project description:RNA polymerase IV (Pol IV) transcribes transposable elements (TEs) into precursors for small interfering RNAs (siRNAs) that guide DNA methylation to silence these potentially dangerious mobile elements in plants. Pol IV is recruited to TEs via SNF2-like CLASSY (CLSY) proteins. How Pol IV evolved to partner with the CLSYs is unknown. Using phylogenetics, we identified a conserved CYC-YPMF motif that is specific to Pol IV’s largest subunit. Modeling predicts that this motif coordinates a zinc ion and is exposed near Pol IV’s surface in a domain contacting downstream DNA. Via immunoprecipitation and mass spectrometry we found that this motif is essential for the association of all four CLSYs with Pol IV and hereby designate it as a CLSY-docking motif. Consistent with these biochemical results, mutations in the CLSY-docking motif or in neighboring sites of the Pol IV clamp domain, phenocopy pol iv null mutants and display a near-total loss of siRNA biogenesis, resulting in defects in DNA methylation and TE silencing. Together, our findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing Pol IV to target, transcribe and limit the expression of TE regions, thereby protecting genome integrity.

2024-11-28 | PXD047743 | Pride

CLSY docking to Pol IV requires a conserved domain critical for small RNA biogenesis and transposon silencing.

Project description:Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a “one CLSY per Pol IV” model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null and clsy quadruple mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.

2024-08-22 | GSE242191 | GEO

CLSY docking to Pol IV requires a conserved domain critical for small RNA biogenesis and transposon silencing

Project description:Eukaryotes must balance the need for gene transcription by RNA polymerase II (Pol II) against the danger of mutations caused by transposable element (TE) proliferation. In plants, these gene expression and TE silencing activities are divided between different RNA polymerases. Specifically, RNA polymerase IV (Pol IV), which evolved from Pol II, transcribes TEs to generate small interfering RNAs (siRNAs) that guide DNA methylation and block TE transcription by Pol II. While the Pol IV complex is recruited to TEs via SNF2-like CLASSY (CLSY) proteins, how Pol IV partners with the CLSYs remains unknown. Here, we identified a conserved CYC-YPMF motif that is specific to Pol IV and is positioned on the complex exterior. Furthermore, we found that this motif is essential for the co-purification of all four CLSYs with Pol IV, but that only one CLSY is present in any given Pol IV complex. These findings support a "one CLSY per Pol IV" model where the CYC-YPMF motif acts as a CLSY-docking site. Indeed, mutations in and around this motif phenocopy pol iv null and clsy quadruple mutants. Together, these findings provide structural and functional insights into a critical protein feature that distinguishes Pol IV from other RNA polymerases, allowing it to promote genome stability by targeting TEs for silencing.

2024-10-01 | GSE278181 | GEO

Epigenomic profiles of Brachypodium distachyon mutants deficient in RNA polymerase IV (Pol IV)

Project description:Monocot grass species (Poaceae) express a diverse set of multisubunit RNA polymerase enzymes, including Pol II, Pol IV and Pol V. To better understand this functional diversity, we have charted Pol IV function in the model Brachypodium distachyon. Intriguingly, pol IV null mutations in Poaceae crops disrupt growth, reproductive development and seed set. In order to investigate how Pol IV controls vegetative growth and TE activity in these grasses, we have isolated B. distachyon mutant alleles for Pol IV’s largest subunit, NRPD1. We obtained the germplasm in which to screen for these pol IV mutations from the B. distachyon community's sodium azide (NaN) and T-DNA insertion collections.

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Assessment of the Pol IV largest subunit, NRPD1, DeCL domain deletion construct to rescue Pol IV-dependent siRNAs in the nrpd1-3 mutant (sRNA).

Project description:The Arabidopsis thaliana multi-subunit RNA Polymerase IV largest subunit DeCL domain was functionally assessed with an NRPD1 DeCL domain deletion construct.

2017-07-17 | GSE95824 | GEO

Assessment of the Pol IV largest subunit, NRPD1, DeCL domain deletion construct to rescue Pol IV-dependent DNA methylation in the nrpd1-3 mutant (Bisulfite-Seq).

Project description:The Arabidopsis thaliana multi-subunit RNA Polymerase IV largest subunit DeCL domain was functionally assessed with an NRPD1 DeCL domain deletion construct.

2017-07-17 | GSE95823 | GEO

Epigenetic memory mediated by HDA6, Pol IV and Pol V in Arabidopsis.

Project description:Multiple RNA-sequencing datasets were generated from wild-type Col-0 and hda6, pol IV or pol V null mutant samples to dissect genetic requirements for the maintenance of epigenetic memory and silent locus identity in Arabidopsis thaliana.

2014-03-27 | E-MTAB-4337 | biostudies-arrayexpress

Polymerases IV and V affect Pol II transcription termination

Project description:Nuclear run-on assays reveal that in the absence of Pol IV or Pol V, Pol II occupancy downstream of poly A sites increases for approximately 12% of protein-coding genes.

2017-12-10 | GSE101543 | GEO

RNA Pol IV induces antagonistic parent-of-origin effects on Arabidopsis endosperm [PareSeq]

Project description:Gene expression in endosperm – a seed tissue that mediates transfer of maternal resources to offspring – is under complex epigenetic control. We show here that plant-specific RNA Polymerase IV mediates parental control of endosperm gene expression. Pol IV is required for the production of small interfering RNAs that typically direct DNA methylation. We compared small RNAs, DNA methylation, and mRNAs in A. thaliana endosperm from reciprocal heterozygotes produced by crossing wild-type plants to Pol IV mutants. We find that maternally and paternally acting Pol IV have divergent effects on endosperm. Losses of maternal and paternal Pol IV impact sRNAs and DNA methylation at distinct genomic sites. Strikingly, maternally and paternally-acting Pol IV have antagonistic impacts on gene expression at some loci, divergently promoting or repressing endosperm gene expression. Antagonistic parent-of origin effects have only rarely been described and are consistent with a gene regulatory system evolving under parental conflict

2022-03-04 | GSE197715 | GEO

RNA Pol IV induces antagonistic parent-of-origin effects on Arabidopsis endosperm [bisulfite_sequencing]

2022-03-04 | GSE197712 | GEO