Abstract
Plant mitochondrial genomes undergo frequent homologous recombination (HR). Ectopic HR activity is inhibited by the HR surveillance pathway, but the underlying regulatory mechanism is unclear. Here, we show that the mitochondrial RNase H1 AtRNH1B impairs the formation of RNA:DNA hybrids (R-loops) and participates in the HR surveillance pathway in Arabidopsis thaliana. AtRNH1B suppresses ectopic HR at intermediate-sized repeats (IRs) and thus maintains mitochondrial DNA (mtDNA) replication. The RNase H1 AtRNH1C is restricted to the chloroplast; however, when cells lack AtRNH1B, transport of chloroplast AtRNH1C into the mitochondria secures HR surveillance, thus ensuring the integrity of the mitochondrial genome and allowing embryogenesis to proceed. HR surveillance is further regulated by the single-stranded DNA-binding protein ORGANELLAR SINGLE-STRANDED DNA BINDING PROTEIN1 (OSB1), which decreases the formation of R-loops. This study uncovers a facultative dual targeting mechanism between organelles and sheds light on the roles of RNase H1 in organellar genome maintenance and embryogenesis.
Highlights
The R-loop is a 3-stranded nucleic acid structure consisting of an RNA–DNA hybrid strand and a single DNA strand [1,2,3]
To corroborate the subcellular localization of this protein, we generated stable transgenic Arabidopsis plants harboring AtRNH1B fused with the green fluorescent protein (GFP) gene expressed under its native promoter (AtRNH1Bpro:AtRNH1B-GFP atrnh1b) and examined GFP signal in both root tips and leaves of the transgenic plants by microscopy
We found that the accumulation of R-loops in mitochondria leads to genome instability by promoting ectopic mitochondrial DNA (mtDNA) homologous recombination (HR) at intermediate-sized repeat (IR) (Fig 10D)
Summary
The R-loop is a 3-stranded nucleic acid structure consisting of an RNA–DNA hybrid strand and a single DNA strand [1,2,3]. Genome-wide mapping studies showed that R-loops persist throughout the genomes of various species [2,3]. These common chromatin features participate in a number of physiological processes, such as gene expression, DNA replication, and DNA and histone modifications, and DNA damage repair and genome stability [1,2,3]. Many factors restrict R-loop formation, such as ribonucleases, helicases, topoisomerases, degradome components, and RNA binding and processing factors (reviewed in [2,4,5,6]).
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