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Caenorhabditis elegans RMI2 functional homolog-2 (RMIF-2) and RMI1 (RMH-1) have both overlapping and distinct meiotic functions within the BTR complex

['Maria Velkova', 'Department Of Chromosome Biology', 'Max Perutz Labs', 'Vienna Biocenter', 'Vienna', 'Nicola Silva', 'Maria Rosaria Dello Stritto', 'Alexander Schleiffer', 'Research Institute Of Molecular Pathology', 'Campus Vienna Biocenter']

Date: 2021-09

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis.

Bloom syndrome is caused by mutations in proteins of the BTR complex (consisting of the Bloom helicase, topoisomerase 3, and the RMI1 and RMI2 scaffolding proteins) and the clinical characteristics are growth deficiency, short stature, skin photosensitivity, and increased cancer predisposition. At the cellular level, characteristic features are the presence of increased sister chromatid exchange on chromosomes; unresolved DNA recombination intermediates that eventually cause genome instability; and erroneous DNA repair by heterologous recombination (recombination between non-identical sequences, extremely rare in wild type animals), which can trigger translocations and chromosomal rearrangements. Identification of the Caenorhabditis elegans ortholog of RMI2 (called RMIF-2) allowed us to compare heterologous recombination in the germline of mutants of various BTR complex proteins. The heterologous recombination rate was several fold lower in rmif-2 mutants than in mutants of rmh-1 and him-6 (worm homologs of RMI1 and the Bloom helicase, respectively). Nevertheless, many phenotypic features point at RMIF-2 working together with RMH-1. If these germline functions of RMI2/RMIF-2 are conserved in humans, this might mean that individuals with RMI2 mutations have a lower risk of translocations and genome rearrangements than those with mutations in the other BTR complex genes.

Funding: The work was funded by FWF (Fonds zur Förderung der wissenschaftlichen Forschung, https://www.fwf.ac.at/en/ ) projects P 31275-B28 (to VJ) and F-34 (to MH). MV and MRDS were supported by the doctoral school "Chromosome Dynamics", FWF (Fonds zur Förderung der wissenschaftlichen Forschung, https://www.fwf.ac.at/en/ ) project W1238. MV is funded by a uni:doc fellowship from the University of Vienna. NS’s laboratory is funded by the Grant Agency of the Czech Republic, https://gacr.cz/en/ , (GA20–08819S) and a Start-Up grant from the Department of Biology, Masaryk University. Boehringer Ingelheim and the Austrian Academy of Sciences support AS. PB acknowledges financial support from the Centre national de la recherche scientifique. Some worm strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40OD010440), https://orip.nih.gov/ . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

So far, RMI2 orthologs have not been identified in yeast, Drosophila, or C. elegans. Here we report the identification of a novel C. elegans protein encoded by the open reading frame Y104H12D.4, which we found in RMH-1-containing protein complexes in the germline. Similar to RMI2 proteins, Y104H12D.4 contains an OB-fold domain. Based on its ability to stabilize RMH-1 (worm RMI1) and concentrate HIM-6 (Bloom helicase) and topoisomerase 3 into recombination foci, it qualifies as a functional homolog of RMI2. Thus, we named the protein RMI2 functional homolog-2 (RMIF-2). Similar to the other BTR complex proteins, RMIF-2 displays a dynamic localization pattern in recombination foci. Nevertheless, our detailed analysis of germline recombination revealed marked differences between rmh-1 and rmif-2 mutants, indicating that rmif-2 functions not just as RMH-1 stabilizer for all its activities in the germline as it is observed in mammalian cells. Our data suggest, that without RMIF-2 RMH-1 can function in some of the BTR related meiotic activities.

In mammalian cells, RMI2 was identified as an RMI1- or Bloom-interacting protein [ 27 , 28 ]. Like RMI1, it contains a characteristic OB-fold domain (OB, oligonucleotide/oligosaccharide binding), and RMI1 and RMI2 interact via their OB domains (OB2 in RMI1 and OB3 in RMI2). RMI2 is required to stabilize the other members of the complex within recombination foci, and is also suggested to function in governing post-translation modifications of other complex members [ 27 ]. Upon RMI2 depletion, elevated sister chromatid exchange and chromosome aberrations have been observed [ 28 ].

Overall, we know now from many model systems that the BTR complex plays separable roles in CO and NCO formation during meiosis and governs the number and placement of CO sites along the chromosomes [ 8 , 9 , 13 – 25 ]. In the C. elegans model system, pro-CO activity is particularly obvious in mutants, due to the presence of univalent chromosomes at diakinesis [ 19 , 22 , 26 ].

Detailed analyses of the homologous yeast STR complex (Sgs1 helicase–topoisomerase–Rmi1) have revealed that its meiotic functions include an important role in D-loop reversion in vivo [ 8 , 9 ], which has also been shown in vitro [ 10 ]. D-loop reversion prevents the generation of complex multi-joint molecules; in the absence of STR activity, multi-joint molecules can only be resolved by non-canonical resolvases, which generate a mix of COs and NCOs (i.e. additional COs are formed). It was also observed that Top3-Rmi1 form a sub complex that limits the accumulation of toxic recombination intermediates. Loss of function of both Rmi1 and topoisomerase 3 leads to meiotic catastrophe, due to persistent joint molecules that are resistant to cleavage by resolvases. It is conceivable that these DNA structures represent extended D-loops involving homologous and/or heterologous chromosomes or other branched structures (for a review, see [ 11 ]). The Caenorhabditis elegans Bloom ortholog HIM-6, similar to BRC-1/BRCA1, suppresses heterologous recombination in the germline, which could lead to translocations and genome rearrangements [ 12 ].

In mammals, the BTR complex consists of Bloom helicase, topoisomerase, and the RMI1 and RMI2 scaffolding proteins. In worms, the respective homologs are HIM-6, TOP-3, and RMH-1 and RMH-2 (both RMH proteins are RMI1 homologs—an RMI2 homolog has not been identified). Structural and biochemical analysis of RMI1 suggest its involvement in the strand passage–tyrosine transesterification reaction mediated by the topoisomerase. Based on these activities combined with the DNA unwinding activity of the helicase, the BTR complex has important roles in DNA metabolism [ 6 , 7 ].

The BTR complex dismantles joint DNA molecules via its decatenation activity, which has been reconstituted in vitro [ 3 ]. In decatenation, strand passage is achieved via cutting one DNA strand and then resealing the DNA break. Following HR in mitotically dividing cells, the BTR complex mostly mediates the NCO outcome since COs can have detrimental effects such as loss of heterozygosity [ 3 ]. For example, loss of heterozygosity of a tumor suppressor gene can lead to cancer development. Patients with mutations in components of the BTR complex show elevated rates of sister chromatid exchange and aberrant chromosomes [ 4 , 5 ].

Damage-induced DNA double-strand breaks (DSBs) pose a threat to genome integrity. High-fidelity repair via homologous recombination (HR) is employed during both mitotic and meiotic cell cycles. It involves the generation of 3′ overhang ends by DNA resection and their stabilization by the single-stranded DNA-binding protein RPA (replication protein A) (RPA-1 in worms). RPA-1 is subsequently exchanged with the RAD-51 recombinase to allow invasion of a homologous DNA strand, giving rise to a D-loop intermediate structure. After DNA synthesis and second-end capture, DNA joint molecules are generated. These can be processed to produce crossovers (COs), which result in the reciprocal exchange of large regions of chromosomes [ 1 ]. In meiosis, where Spo11-mediated DSBs are induced via a highly regulated program, crossing-over and cohesion establish a physical tether between homologous chromosomes, which greatly aids their correct segregation in meiotic anaphase I and drives genetic variability. In meiosis, one chromatid of the homologous chromosome is preferentially used as a repair template for HR. Joint DNA molecules must disengage in order to segregate, and this is achieved by redundant endonucleases (called resolvases) and the BTR complex [ 2 ]. Depending on the orientation of the resolvase-induced cut, the outcome is a CO or non-crossover (NCO) product. To ensure at least one CO per chromosome pair, excess DSBs are introduced and those that do not form the CO are repaired to form NCOs [ 2 ].

Results

RMIF-2 displays a dynamic localization throughout pachynema To gain insight into RMIF-2 expression and subcellular localization during meiotic prophase I, we added a 3′ HA-tag to the rmif-2 locus. The tagged line displayed wild type (WT) hatching rates and brood size, indicating that the strain is functional (Table 2). RMIF-2::HA localized into dynamic foci throughout pachynema (Fig 1C). The protein started to accumulate as foci in early pachytene nuclei (mean 5.6 (± 5.8 SD) foci per nucleus; Fig 1D). The number of RMIF-2 foci peaked in mid pachynema (range 2–24 foci per nucleus; mean 9.0 (± 4.6 SD)). Most late pachytene cells contained five RMIF-2::HA foci (range 0–7 foci per nucleus). This dynamic localization pattern strongly resembles that of RMH-1 [19]; in addition, RMH-1 and RMIF-2 foci extensively co-localized (Fig 1D and 1E).

RMIF-2 and RMH-1 may not always act interdependently in meiosis To investigate whether RMIF-2 acts as a BTR complex stabilizer by binding to and or stabilizing RMH-1, we generated the rmh-1; rmif-2 double mutant and compared its phenotype with those of the single mutants. First, we examined embryonic lethality and the number of males in the viable progeny. We observed that in the rmh-1; rmif-2 double mutant, levels of embryonic lethality were similar to those in rmh-1 mutants, 56% (± 37 SD) although with much higher variability between individual worms, with 5% (± 3 SD) of males in the progeny (Table 2; in comparison, embryonic lethality is 40% (± 4 SD) in rmif-2 and 68% (± 9 SD) in rmh-1 mutants). In the double mutant, the brood size was much smaller (mean 72 (± 83 SD)) than in the single mutants (Table 2). We next examined DAPI-stained diakinesis chromosomes in rmh-1; rmif-2 worms. There were an average of 6.7 (± 1.5 SD) DAPI bodies per nucleus, and most nuclei contained six aberrant bodies that differed markedly from the well-shaped bivalents in the WT (Fig 2A and 2B). We saw chromatin clumps together with DAPI bodies of differing sizes that resembled DNA fragments and/or univalents (Fig 2B). We wanted to examine whether these DAPI-stained bodies were the results of NHEJ activity and thus, constructed the triple mutant rmh-1; cku-70; rmif-2 (Fig 2A and 2B). Analysis of diakinesis nuclei in this mutant background revealed no difference in the number of DAPI bodies (7.45 (± 1.6 SD)) when compared to rmh-1; rmif-2 (6.7 (± 1.5 SD)). Therefore NHEJ does not cause the chromosome abnormalities observed at diakinesis stage. We also analyzed DSB induction and repair in the double mutant rmh-1; rmif-2. Assessment of RAD-51 loading/unloading on chromosomes revealed a striking accumulation of RAD-51 foci, which persisted throughout pachynema (statistically different from the single mutants; Fig 2C). Exacerbation of the meiotic phenotypes in the rmh-1; rmif-2 double mutant suggests that either RMIF-2 or RMH-1 may have an additional function outside of the mutually dependent formation of recombination foci and/or that RMIF-2 might not merely function as a RMH-1 stabilizer within the BTR complex. It could be that RMIF-2 has a less prominent role during meiotic DSB repair. We next generated the rmif-2 him-6 double mutant to test whether RMIF-2 was needed to support the activity of the HIM-6 helicase. In these worms, DAPI-body counts showed an increase to an average of 9.4 (± 2.0 SD) in comparison to the single mutants rmif-2, 6.9 (± 1.0 SD) and him-6, 7.5 (± 0.9 SD) (Fig 2A and 2B). In contrast, in the rmh-1; him-6 diakinesis DAPI-bodies were not statistically different in comparison to the him-6 single mutant (7.8 (± 1 SD) vs 7.3 (± 1.1 SD)) [19]. Moreover, in rmif-2 him-6 embryonic lethality strikingly increased to 99.2% (± 1.6 SD) from 41% (± 5.6 SD) in the him-6 single mutant (Table 2), while in the rmh-1; him-6 it was 68.22% [19]. The quantification of RAD-51 in the double mutant rmif-2 him-6 revealed accumulation of meiotic and mitotic recombination intermediates. RAD-51 was loaded earlier than in the WT and single mutants, from the mitotic zone (zone 1) onwards, with high and persisting numbers of unresolved breaks, which were not repaired by late pachynema (zone 7) (Fig 2C). Taken together, RAD-51 quantification revealed high levels of genomic instability in the rmif-2 him-6 animals arising from mitotic and meiotic defects. The severe phenotype of rmif-2 him-6 double mutants suggests that RMIF-2 and HIM-6 might act in independent parallel pathways. Analysis of the accumulation of recombination intermediates in the rmh-1; him-6 revealed differences in comparison to the rmif-2 him-6 double mutant. Here, RAD-51 was also observed in significantly higher numbers throughout pachynema, with accumulation of unrepaired breaks until late pachynema (Fig 2C), however no significant amount of RAD-51 signals was quantified in the mitotic zone (zones 1–2).

[END]

[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1009663

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