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Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis

['Lingling Cheng', 'Center For Plant Biology', 'School Of Life Sciences', 'Tsinghua University', 'Beijing', 'Wenjie Wang', 'Tsinghua-Peking Center For Life Sciences', 'Yao Yao', 'Qianwen Sun']

Date: None

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.

Funding: This work was supported by grants from the Ministry of Science and Technology of China (2016YFA0500800 to Q.S.) and the National Natural Science Foundation of China (grants no. 31822028, 91740105, and 91940306 to Q.S.). The Sun Lab is supported by Tsinghua-Peking Center for Life Sciences, and WW is supported by the postdoctoral fellowship from Tsinghua-Peking Center for Life Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Cheng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Mitochondrial R-loops are important for development and mtDNA replication [ 11 ]. Here, to explore the biological functions of R-loop homeostasis in Arabidopsis mitochondria, we investigated the roles of the mitochondrial localized RNase H1 protein AtRNH1B. Mutants in AtRNH1B did not show any obvious phenotype. However, in a mutant with loss of function of both mitochondrial AtRNH1B and chloroplast AtRNH1C (atrnh1b/c double mutant), embryogenesis arrested at the transition stage, leading to embryo lethality. Unexpectedly, AtRNH1C localized to both the mitochondria and chloroplasts in atrnh1b mutants and compensated for the function of AtRNH1B in the mitochondria. This dual targeting capacity of AtRNH1C was inhibited in wild-type Arabidopsis by an unclear mechanism dependent on a fragment between the transit peptide and hybrid binding domain (HBD) of this protein. We explored the reasons for embryo arrest in the double mutant atrnh1b/c and found that it accumulated excessive R-loops and that extensive ectopic HR occurred in its mitochondrial genome. In addition, the copy number of mtDNA was dramatically reduced in the atrnh1b/c mutant. An analysis of the genetic interactions of AtRNH1B/1C with known components of the HR surveillance pathway suggested that the ssDNA binding protein OSB1 inhibits R-loop formation to restrict HR of mtDNA. We propose that a facultative dual targeting mechanism protects mitochondrial RNase H1 to maintain mitochondrial R-loop homeostasis and genome stability, thus safeguarding early embryogenesis in Arabidopsis.

By contrast, HR between intermediate-sized repeats (IRs, 50 to 500 bp) is infrequent and asymmetrical, and one of the 2 predicted DNA exchange products preferentially accumulates [ 17 , 20 , 21 , 23 ]. Because this recombination activity causes mtDNA rearrangements and genome instability that may affect plant fitness and survival, HR between IRs is rigorously restricted by the HR surveillance pathway. To date, only a few genes involved in this pathway have been identified in Arabidopsis, including MUTS HOMOLOG1 (MSH1) [ 20 , 24 ], ORGANELLAR SINGLE-STRANDED DNA BINDING PROTEIN1 (OSB1) [ 25 ], RecA homolog genes RECA2 and RECA3 [ 26 ], RECG1 [ 27 ], and SWI/SNF protein complex B (SWIB5) [ 28 ]. Disrupting these genes increases the ectopic HR frequency between IRs, but the precise regulatory mechanisms are unknown.

Compared to the compact circular mtDNA of mammals, which is only 15 to 17 kb in size, plant mitochondrial genomes are much larger, ranging from approximately 200 kb to over 10 Mb [ 16 ]. Plant mitochondrial genomes are more complex than their animal counterparts, comprising heterogeneous populations of circular, linear, and multibranched double- and single-stranded molecules [ 17 – 19 ]. This complexity is primarily due to frequent homologous recombination (HR) between repeated sequences, the efficiency of which largely depends on the lengths of the homologous sequences [ 17 , 20 , 21 ]. Large repeats (>1 kb) undergo high-frequency reciprocal recombination to generate equal isoforms, a process thought to participate in recombination-mediated replication [ 19 , 22 , 23 ].

Mitochondria and chloroplasts are endosymbiotic organelles that play essential roles in plant metabolism, cellular homeostasis, and environmental sensing. The vast majority of mitochondrial and chloroplast proteins are encoded in the nucleus and imported from the cytosol, primarily through the classical presequence pathway [ 14 , 15 ]. In this pathway, a precursor protein is biosynthesized in the cytosol as a larger preprotein with an N-terminal transit peptide, which directs the protein into the correct organelle/compartment. Mitochondria and chloroplasts contain their own genomes, which encode a limited set of proteins required for their activities. Thus, tight coordination between the nuclear and organellar genomes is important for full functionality.

Various factors increase the propensity for R-loop formation, such as specific DNA sequences, DNA topology, and regulatory proteins. To maintain balance, many factors restrict R-loop formation, such as ribonucleases, helicases, topoisomerases, degradome components, and RNA binding and processing factors (reviewed in [ 2 , 4 – 6 ]). Among these factors, the evolutionarily conserved RNase H proteins (H1 and H2) specifically digest the RNA moiety in RNA:DNA hybrids, thus directly removing R-loops from the genome [ 7 ]. Mammalian RNase H1 is dual localized to the nucleus and mitochondria. The loss of function of RNase H1 causes severe diseases in humans, and RNase H1 knockout mutants in mice are embryo lethal [ 8 , 9 ]. RNase H1 is essential for mammalian mitochondrial DNA (mtDNA) replication ([ 10 ]; reviewed in [ 11 ]). Arabidopsis thaliana contains 3 RNase H1s (AtRNH1A, B, and C) that localize to the nuclei, mitochondria, and chloroplasts, respectively [ 12 ]. AtRNH1C is important for chloroplast genome stability and plant development [ 12 , 13 ], while the biological functions of the 2 other Arabidopsis RNase H1 proteins remain enigmatic.

The R-loop is a 3-stranded nucleic acid structure consisting of an RNA–DNA hybrid strand and a single DNA strand [ 1 – 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 – 3 ]. Therefore, the formation and resolving of R-loops must be tightly regulated.

Results

Depletion of AtRNH1B promotes AtRNH1C expression In addition to the altered subcellular localization of AtRNH1C, AtRNH1C-GFP produced much stronger fluorescent signals in atrnh1b than in atrnh1c (Fig 4B), mainly in mitochondria, suggesting that AtRNH1C expression increased in the absence of AtRNH1B. To explore whether the expression of AtRNH1C changed when AtRNH1B was depleted, we performed RT-PCR, finding that AtRNH1C was expressed at significantly higher levels in atrnh1b compared to Col-0 (Fig 6A). We then generated AtRNH1Cpro:AtRNH1C-GUS (expressing AtRNH1C-GUS fusion under its own promoter) transgenic plants in the atrnh1b-1 and atrnh1c mutant backgrounds, respectively. GUS staining revealed very little AtRNH1C-GUS reporter activity in atrnh1c but extremely strong AtRNH1B-GUS reporter activity in atrnh1b. Consistent with our RT-PCR results, the expression of AtRNH1C was greatly enhanced in atrnh1b compared to atrnh1c (Figs 6B and S4C). Since AtRNH1B is specifically expressed in embryos (Fig 2C), we also observed significantly increased AtRNH1C-GUS expression in atrnh1b-1 versus atrnh1c embryos (Fig 6C). The increase of AtRNH1C-GUS in atrnh1b was confirmed through immunoblot analysis (Fig 6D). Consistent with previous observation (Fig 4B and 4C; there are much more immunogold labeled GFP particles detected in atrnh1b chloroplast than that in atrnh1c chloroplast), the accumulation of AtRNH1C in the mitochondria of atrnh1b was remarkable (Fig 6D). Moreover, the level of AtRNH1C was also slightly increased (Fig 6D). These results further support the notion that AtRNH1C makes up for the loss of AtRNH1B in the atrnh1b mutant potentially to compensate the mitochondrial RNase H1 function. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 6. The expression of AtRNH1C increases in atrnh1b. (A) A total of 28 RT-PCR cycles were used to detect the expression of AtRNH1B and AtRNH1C in Col-0, atrnh1b-1, and atrnh1c. GAPDH was used as the reference gene. (B, C) GUS staining of 2-week-old seedlings (B) and seeds at the globular, heart, torpedo, and mature embryo stages (C) from plants expressing AtRNH1Cpro:AtRNH1C-GUS in the atrnh1c and atrnh1b-1 backgrounds. (D) Characterize the localization of AtRNH1C protein by immunoblot. Intact chloroplasts and mitochondria were isolated from 3-week-old AtRNH1Cpro:AtRNH1C-GUS atrnh1c and AtRNH1Cpro:AtRNH1C-GUS atrnh1b transgenic plants. Anti-GUS polyclonal antibody was used to detect the AtRNH1C-GUS, and polyclonal antibodies anti-psbO, anti-IDH1, and anti-H3 were used to indicate chloroplast, mitochondria, and total protein fractions, respectively. Chl, proteins from isolated chloroplasts; Mito, proteins from isolated mitochondria; Total, total proteins from leaves. (E) A facultative dual targeting mechanism protects mitochondrial RNase H1. In the wild-type, AtRNH1C and AtRNH1B are predominately transported to the chloroplast and mitochondria, respectively, and the localization of AtRNH1C to the mitochondria is self-inhibited by an unknown mechanism. The level of AtRN1HB is much higher than that of AtRNH1C. When AtRNH1B is mutated (atrnh1b), AtRNH1C recovers its dual localization to both the chloroplast and mitochondria and its expression level increases, thus safeguarding mitochondrial function. The data underlying this figure can be found in S1 Raw Images. GUS, β-glucuronidase enzyme; H3, histone 3; IDH1, isocitrate dehydrogenase 1; psbO, photosystem II subunit O; RT-PCR, reverse transcription PCR; WT, wild type. https://doi.org/10.1371/journal.pbio.3001357.g006 Based on above results, we propose that a facultative dual targeting mechanism could ensure the mitochondria having RNase H1 function (Fig 6D). In the presence of functional AtRNH1B, the mitochondrial localization of AtRNH1C driven by the CTS is inhibited by another signal that prevents the overaccumulation of RNase H1 in mitochondria via an unknown mechanism. In cells lacking mitochondrial localized RNase H1 (e.g., when AtRNH1B is depleted, as in the current study), the inhibition of the mitochondrial localization of AtRNH1C is relieved and its expression increases, allowing sufficient amounts of AtRNH1C to be delivered into the mitochondria to ensure the proper functioning of mitochondrial RNase H1. This facultative dual targeting protective mechanism might ensure that plants properly respond to environmental stimuli.

Knocking down AtRNH1B in atrnh1c causes sterility As atrnh1b/c homozygous plants could not be generated for further functional analysis of plant mitochondrial RNase H1, we produced AtRNH1B knockdown transgenic plants in the atrnh1c mutant background by RNA interference (RNAi) (S5A Fig, hereafter referred to as AtRNH1BRNAi atrnh1c). Although the resulting individual transformants expressed AtRNH1B at different levels in atrnh1c (S5B Fig), all these plants displayed a yellowish phenotype like that of atrnh1c plants at the vegetative stage (S5C Fig), indicating that mitochondrial AtRNH1B is not involved in chloroplast development. The 3 most effective RNAi lines (AtRNH1BRNAi atrnh1c-#1, atrnh1c-#2, and atrnh1c-#3) with dramatically reduced AtRNH1B expression showed sterility (S5B, S5D and S5E Fig). Transmission electron microscopy of young siliques from these lines showed that the mitochondrial morphology was abnormal in sterile plants, in contrast to atrnh1c (S5F Fig), and was similar to that observed in atrnh1b/c (Fig 3D). Pollen viability assay and reciprocal cross indicated that both the male and female gametophytes are fertile in AtRNH1BRNAi atrnh1c (S5G and S5H Fig). These results confirm the notion that mitochondrial RNase H1 is important for reproduction.

[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001357

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