RESUMO
In Arabidopsis, multisubunit RNA polymerases IV and V orchestrate RNA-directed DNA methylation (RdDM) and transcriptional silencing, but what identifies the loci to be silenced is unclear. We show that heritable silent locus identity at a specific subset of RdDM targets requires HISTONE DEACETYLASE 6 (HDA6) acting upstream of Pol IV recruitment and siRNA biogenesis. At these loci, epigenetic memory conferring silent locus identity is erased in hda6 mutants such that restoration of HDA6 activity cannot restore siRNA biogenesis or silencing. Silent locus identity is similarly lost in mutants for the cytosine maintenance methyltransferase, MET1. By contrast, pol IV or pol V mutants disrupt silencing without erasing silent locus identity, allowing restoration of Pol IV or Pol V function to restore silencing. Collectively, these observations indicate that silent locus specification and silencing are separable steps that together account for epigenetic inheritance of the silenced state.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/genética , Epigênese Genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Histona Desacetilases/genética , Interferência de RNA , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Elementos de DNA Transponíveis , RNA Polimerases Dirigidas por DNA/metabolismo , Loci Gênicos , Genótipo , Hereditariedade , Histona Desacetilases/metabolismo , Mutação , Fenótipo , RNA Interferente Pequeno/biossínteseRESUMO
This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).