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

Abstract

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.

The topoisomerase 3 zinc finger domain cooperates with the RMI1 scaffold to promote stable association of the BTR complex to recombination intermediates in the Caenorhabditis elegans germline

“…We will refer to the new top-3 allele, jf153 , as top-3-ZnF . We also inserted an internal HA tag into both the wt and top-3-ZnF alleles, as previously described ( 16 ). Western blot analysis showed that the truncated protein is expressed at similar levels to the wt (Figure 1B ).…”

“…The recombination frequency and CO number and position in wt, top-3-ZnF and rmh-2(jf168) were determined as previously described ( 16 ) using the C. elegans Hawaiian and Bristol hybrid strains. Specifically, we injected the mutation of interest into the Hawaiian strain and subsequently crossed it with the same mutation in the Bristol strain to generate the Hawaiian/Bristol hybrid.…”

“…F2 generation worms heterozygous for visible marker expression, were lysed and differences between Bristol and Hawaiian were detected by PCR and subsequently SNP analysis of chromosomes V and IV. Oligonucleotide sequences and chromosome positions were published previously ( 16 ).…”

“…Sgs1, the yeast Bloom helicase, is also involved in reversing strand invasion events into homeologous sequences during mitosis ( 14 ). Moreover, the germline activities of C. elegans BTR complex proteins suppress heterologous recombination to prevent chromosome translocations and genome rearrangements ( 15 , 16 ). Therefore, the BTR complex participates in multiple steps during meiotic recombination in many organisms.…”

“…Analysis of C. elegans mutants showed that the BTR complex proteins HIM-6 (Bloom helicase), RMH-1 (one of two RMI1 homologs) and RMIF-2 (the RMI2 homolog) provide both CO and NCO activities and prevent CO formation at chromosome centers ( 16 , 21 , 23 , 24 , 31 ), see Supplementary Figure S1A for a summary table for the C. elegans BTR complex protein nomenclature). The second RMI1 homolog, RMH-2, makes little contribution to meiosis by itself, as evidenced by the high hatch rates of embryos and lack of univalent chromosomes in the rmh-2 mutant, in contrast to the him-6 , rmh-1 and rmif-2 mutants.…”