scholarly journals RNA polymerases IV and V influence the 3’ boundaries of Polymerase II transcription units in Arabidopsis

2017 ◽  
Author(s):  
Anastasia McKinlay ◽  
Ram Podicheti ◽  
Jered M. Wendte ◽  
Ross Cocklin ◽  
Douglas B. Rusch
Keyword(s):  
Cytosine Methylation ◽  
De Novo ◽  
Transcription Termination ◽  
Rna Polymerases ◽  
Protein Coding ◽  
Pol Ii ◽  
Protein Coding Genes ◽  
Pol Iv ◽  
Pol V ◽  
Genomic Repeats

AbstractNuclear multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved in plants as specialized forms of Pol II. Their functions are best understood in the context of RNA-directed DNA methylation (RdDM), a process in which Pol IV-dependent 24 nt siRNAs direct the de novo cytosine methylation of regions transcribed by Pol V. Pol V has additional functions, independent of Pol IV and 24 nt siRNA biogenesis, in maintaining the repression of transposons and genomic repeats whose silencing depends on maintenance cytosine methylation. Here we report that Pol IV and Pol V play unexpected roles in defining the 3’ boundaries of Pol II transcription units. 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. This effect is most pronounced for convergently transcribed gene pairs. Although Pols IV and V are detected near transcript ends of the affected Pol II – transcribed genes, their role in limiting Pol II read-through is independent of siRNA biogenesis or cytosine methylation. We speculate that Pols IV and V (and/or their associated factors) play roles in Pol II transcription termination by influencing polymerase bypass or release at collision sites for convergent genes.

scholarly journals Arabidopsis RNA Polymerases IV and V Are Required To Establish H3K9 Methylation, but Not Cytosine Methylation, on Geminivirus Chromatin

2016 ◽  
Vol 90 (16) ◽  
pp. 7529-7540 ◽  
Author(s):  
Jamie N. Jackel ◽  
Jessica M. Storer ◽  
Tami Coursey ◽  
David M. Bisaro
Keyword(s):  
Dna Methylation ◽  
Small Rnas ◽  
Cytosine Methylation ◽  
De Novo ◽  
Rna Polymerases ◽  
Epigenetic Marks ◽  
Content Type ◽  
Pol Iv ◽  
Pol V ◽  
Virus Genomes

ABSTRACTIn plants, RNA-directed DNA methylation (RdDM) employs small RNAs to target enzymes that methylate cytosine residues. Cytosine methylation and dimethylation of histone 3 lysine 9 (H3K9me2) are often linked. Together they condition an epigenetic defense that results in chromatin compaction and transcriptional silencing of transposons and viral chromatin. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was believed to be necessary to establish cytosine methylation, which in turn could recruit H3K9 methyltransferases. However, recent studies have revealed that a pathway involving Pol II and RNA-dependent RNA polymerase 6 (RDR6) (RDR6-RdDM) is likely responsible for establishing cytosine methylation at naive loci, while Pol IV-RdDM acts to reinforce and maintain it. We used the geminivirusBeet curly top virus(BCTV) as a model to examine the roles of Pol IV and Pol V in establishing repressive viral chromatin methylation. As geminivirus chromatin is formedde novoin infected cells, these viruses are unique models for processes involved in the establishment of epigenetic marks. We confirm that Pol IV and Pol V are not needed to establish viral DNA methylation but are essential for its amplification. Remarkably, however, both Pol IV and Pol V are required for deposition of H3K9me2 on viral chromatin. Our findings suggest that cytosine methylation alone is not sufficient to triggerde novodeposition of H3K9me2 and further that Pol IV-RdDM is responsible for recruiting H3K9 methyltransferases to viral chromatin.IMPORTANCEIn plants, RNA-directed DNA methylation (RdDM) uses small RNAs to target cytosine methylation, which is often linked to H3K9me2. These epigenetic marks silence transposable elements and DNA virus genomes, but how they are established is not well understood. Canonical RdDM, involving Pol IV and Pol V, was thought to establish cytosine methylation that in turn could recruit H3K9 methyltransferases, but recent studies compel a reevaluation of this view. We used BCTV to investigate the roles of Pol IV and Pol V in chromatin methylation. We found that both are needed to amplify, but not to establish, DNA methylation. However, both are required for deposition of H3K9me2. Our findings suggest that cytosine methylation is not sufficient to recruit H3K9 methyltransferases to naive viral chromatin and further that Pol IV-RdDM is responsible.

scholarly journals Catalytic properties of RNA polymerases IV and V: accuracy, nucleotide incorporation and rNTP/dNTP discrimination

2017 ◽  
Author(s):  
Michelle Marasco ◽  
Weiyi Li ◽  
Michael Lynch ◽  
Craig S. Pikaard
Keyword(s):  
Rna Synthesis ◽  
Catalytic Properties ◽  
Rna Polymerases ◽  
Catalytic Subunits ◽  
Pol Ii ◽  
Pol Iv ◽  
Pol V ◽  
Nucleotide Incorporation ◽  
In Trans ◽  
Conserved Amino Acids

AbstractCatalytic subunits of DNA-dependent RNA polymerases of bacteria, archaea and eukaryotes share hundreds of ultra-conserved amino acids. Remarkably, the plant-specific RNA silencing enzymes, Pol IV and Pol V differ from Pols I, II and III at ~140 of these positions, yet remain capable of RNA synthesis. Whether these amino acid changes in Pols IV and V alter their catalytic properties in comparison to Pol II, from which they evolved, is unknown. Here, we show that Pols IV and V differ from one another, and Pol II, in nucleotide incorporation rate, transcriptional accuracy and the ability to discriminate between ribonucleotides and deoxyribonucleotides. Pol IV transcription is notably error-prone, which may be tolerable, or even beneficial, for biosynthesis of siRNAs targeting transposon families in trans. By contrast, Pol V exhibits high fidelity transcription, suggesting a need for Pol V transcripts to faithfully reflect the DNA sequence of target loci in order to recruit siRNA-Argonaute protein silencing complexes.

scholarly journals A Null Allele of the Pol IV Second Subunit is Viable in Oryza sativa

2021 ◽  
Author(s):  
Tania Chakraborty ◽  
Joshua T Trujillo ◽  
Timmy Kendall ◽  
Rebecca A Mosher
Keyword(s):  
Dna Methylation ◽  
Small Rna ◽  
Null Allele ◽  
De Novo ◽  
Land Plants ◽  
Pol Ii ◽  
Pol Iv ◽  
Pol V ◽  
Grass Family ◽  
Adult Plants

All eukaryotes possess three DNA-dependent RNA polymerases, Pols I-III, while land plants possess two additional polymerases, Pol IV and Pol V. Derived through duplication of Pol II subunits, Pol IV produces 24-nt siRNAs that interact with Pol V transcripts to target de novo DNA methylation and silence transcription of transposons. Members of the grass family encode additional duplicated subunits of Pol IV and V, raising questions regarding the function of each paralog. In this study, we identify a null allele of the putative Pol IV second subunit, NRPD2, and demonstrate that NRPD2 is the sole subunit functioning with NRPD1 in small RNA production and CHH methylation in leaves. Homozygous nrpd2 mutants have neither gametophytic defects, nor embryo lethality, although adult plants are dwarf and sterile.

scholarly journals Chromosome-level assembly of Drosophila bifasciata reveals important karyotypic transition of the X chromosome

2019 ◽  
Author(s):  
Ryan Bracewell ◽  
Anita Tran ◽  
Kamalakar Chatla ◽  
Doris Bachtrog
Keyword(s):  
X Chromosome ◽  
Genome Assembly ◽  
De Novo ◽  
Pericentromeric Region ◽  
Species Group ◽  
Chromosome 15 ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Long Read ◽  
Chromosome Level

ABSTRACTThe Drosophila obscura species group is one of the most studied clades of Drosophila and harbors multiple distinct karyotypes. Here we present a de novo genome assembly and annotation of D. bifasciata, a species which represents an important subgroup for which no high-quality chromosome-level genome assembly currently exists. We combined long-read sequencing (Nanopore) and Hi-C scaffolding to achieve a highly contiguous genome assembly approximately 193Mb in size, with repetitive elements constituting 30.1% of the total length. Drosophila bifasciata harbors four large metacentric chromosomes and the small dot, and our assembly contains each chromosome in a single scaffold, including the highly repetitive pericentromere, which were largely composed of Jockey and Gypsy transposable elements. We annotated a total of 12,821 protein-coding genes and comparisons of synteny with D. athabasca orthologs show that the large metacentric pericentromeric regions of multiple chromosomes are conserved between these species. Importantly, Muller A (X chromosome) was found to be metacentric in D. bifasciata and the pericentromeric region appears homologous to the pericentromeric region of the fused Muller A-AD (XL and XR) of pseudoobscura/affinis subgroup species. Our finding suggests a metacentric ancestral X fused to a telocentric Muller D and created the large neo-X (Muller A-AD) chromosome ∼15 MYA. We also confirm the fusion of Muller C and D in D. bifasciata and show that it likely involved a centromere-centromere fusion.

scholarly journals Draft genome assembly data of Anoxybacillus sp. strain MB8 isolated from Tattapani hot springs, India

2021 ◽  
Author(s):  
VISHNU PRASOODANAN P K ◽  
Shruti S. Menon ◽  
Rituja Saxena ◽  
Prashant Waiker ◽  
Vineet K Sharma
Keyword(s):  
Hot Springs ◽  
De Novo ◽  
Draft Genome ◽  
Gc Content ◽  
Central India ◽  
Glycoside Hydrolases ◽  
Rrna Gene ◽  
Aerobic Bacterium ◽  
Protein Coding ◽  
Protein Coding Genes

Discovery of novel thermophiles has shown promising applications in the field of biotechnology. Due to their thermal stability, they can survive the harsh processes in the industries, which make them important to be characterized and studied. Members of Anoxybacillus are alkaline tolerant thermophiles and have been extensively isolated from manure, dairy-processed plants, and geothermal hot springs. This article reports the assembled data of an aerobic bacterium Anoxybacillus sp. strain MB8, isolated from the Tattapani hot springs in Central India, where the 16S rRNA gene shares an identity of 97% (99% coverage) with Anoxybacillus kamchatkensis strain G10. The de novo assembly and annotation performed on the genome of Anoxybacillus sp. strain MB8 comprises of 2,898,780 bp (in 190 contigs) with a GC content of 41.8% and includes 2,976 protein-coding genes,1 rRNA operon, 73 tRNAs, 1 tm-RNA and 10 CRISPR arrays. The predicted protein-coding genes have been classified into 21 eggNOG categories. The KEGG Automated Annotation Server (KAAS) analysis indicated the presence of assimilatory sulfate reduction pathway, nitrate reducing pathway, and genes for glycoside hydrolases (GHs) and glycoside transferase (GTs). GHs and GTs hold widespread applications, in the baking and food industry for bread manufacturing, and in the paper, detergent and cosmetic industry. Hence, Anoxybacillus sp. strain MB8 holds the potential to be screened and characterized for such commercially relevant enzymes.

scholarly journals Integrating healthcare and research genetic data empowers the discovery of 28 novel developmental disorders

2019 ◽  
Author(s):  
Joanna Kaplanis ◽  
Kaitlin E. Samocha ◽  
Laurens Wiel ◽  
Zhancheng Zhang ◽  
Kevin J. Arvai ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
De Novo ◽  
Genetic Data ◽  
Statistical Test ◽  
Integrated Healthcare ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Clinical Diagnostic ◽  
Simulation Based

SummaryDe novo mutations (DNMs) in protein-coding genes are a well-established cause of developmental disorders (DD). However, known DD-associated genes only account for a minority of the observed excess of such DNMs. To identify novel DD-associated genes, we integrated healthcare and research exome sequences on 31,058 DD parent-offspring trios, and developed a simulation-based statistical test to identify gene-specific enrichments of DNMs. We identified 285 significantly DD-associated genes, including 28 not previously robustly associated with DDs. Despite detecting more DD-associated genes than in any previous study, much of the excess of DNMs of protein-coding genes remains unaccounted for. Modelling suggests that over 1,000 novel DD-associated genes await discovery, many of which are likely to be less penetrant than the currently known genes. Research access to clinical diagnostic datasets will be critical for completing the map of dominant DDs.

scholarly journals Phylogenetic relationships and taxonomic position of genus Hyperacrius (Rodentia: Arvicolinae) from Kashmir based on evidences from analysis of mitochondrial genome and study of skull morphology

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10364
Author(s):  
Natalia I. Abramson ◽  
Fedor N. Golenishchev ◽  
Semen Yu. Bodrov ◽  
Olga V. Bondareva ◽  
Evgeny A. Genelt-Yanovskiy ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
De Novo ◽  
Phylogenetic Analyses ◽  
Complete Mitochondrial Genome ◽  
Morphological Characters ◽  
Molecular Data ◽  
Phylogenetic Position ◽  
Skull Morphology ◽  
Protein Coding ◽  
Protein Coding Genes

In this article, we present the nearly complete mitochondrial genome of the Subalpine Kashmir vole Hyperacrius fertilis (Arvicolinae, Cricetidae, Rodentia), assembled using data from Illumina next-generation sequencing (NGS) of the DNA from a century-old museum specimen. De novo assembly consisted of 16,341 bp and included all mitogenome protein-coding genes as well as 12S and 16S RNAs, tRNAs and D-loop. Using the alignment of protein-coding genes of 14 previously published Arvicolini tribe mitogenomes, seven Clethrionomyini mitogenomes, and also Ondatra and Dicrostonyx outgroups, we conducted phylogenetic reconstructions based on a dataset of 13 protein-coding genes (PCGs) under maximum likelihood and Bayesian inference. Phylogenetic analyses robustly supported the phylogenetic position of this species within the tribe Arvicolini. Among the Arvicolini, Hyperacrius represents one of the early-diverged lineages. This result of phylogenetic analysis altered the conventional view on phylogenetic relatedness between Hyperacrius and Alticola and prompted the revision of morphological characters underlying the former assumption. Morphological analysis performed here confirmed molecular data and provided additional evidence for taxonomic replacement of the genus Hyperacrius from the tribe Clethrionomyini to the tribe Arvicolini.

scholarly journals Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Todd Blevins ◽  
Ram Podicheti ◽  
Vibhor Mishra ◽  
Michelle Marasco ◽  
Jing Wang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Transferase Activity ◽  
Small Interfering Rnas ◽  
Pol Iv ◽  
Nuclear Rna ◽  
Rna Polymerase Iv ◽  
Nuclear Rna Polymerase

In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3’ termini. The 24 nt siRNAs primarily correspond to the 5’ or 3’ ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

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

2019 ◽  
Vol 47 (17) ◽  
pp. 9024-9036 ◽  
Author(s):  
Jered M Wendte ◽  
Jeremy R Haag ◽  
Olga M Pontes ◽  
Jasleen Singh ◽  
Sara Metcalf ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Catalytic Activity ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cytosine Methylation ◽  
Transcriptional Gene Silencing ◽  
Rna Dependent Rna Polymerase ◽  
Pol Ii ◽  
Pol Iv ◽  
Non Coding Rnas

Abstract 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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