Abstract
5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays.
Highlights
RNA silencing collectively refers to processes by which specialized RNA-protein complexes repress gene expression, guide repressive covalent modifications to histones or mediate DNA methylation within specific DNA regions [1,2,3,4]
Our results show that silencing of aberrant 5S rDNA transcripts depends on small-interfering RNAs (siRNA) biogenesis, in addition to DDM1
DDM1 limits accumulation of siRNAs derived from the 5S rDNA intergenic spacers (IGS)
Summary
RNA silencing collectively refers to processes by which specialized RNA-protein complexes repress gene expression, guide repressive covalent modifications to histones or mediate DNA methylation within specific DNA regions [1,2,3,4]. Most Arabidopsis siRNAs depend on RNA polymerase IV (Pol IV), a plant-specific enzyme composed of subunits homologous to RNA polymerases I, II and III [5,6,7,8,9,10]. The effector protein AGO4 preferentially binds heterochromatic siRNAs [20,21], which appear to guide RNAdirected DNA methylation (RdDM) [4]. A second plant-specific RNA polymerase, Pol V (previously known as Pol IVb), mediates production of non-coding transcripts critical for steps downstream of siRNA biogenesis [8,9,19,23]. Additional proteins have been implicated in these downstream steps [24,25]
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