A new partial loss of function allele of rad-54.L

Abstract

RAD-54.L is required for the repair of meiotic double-strand DNA breaks (DSBs), playing an essential role in promoting removal of recombinase RAD-51 and normal completion of meiotic recombination. Failure to complete meiotic DSB repair leads to 100% lethality of embryos produced by rad-54.L null mutant mothers. Here we report a new partial loss of function allele, rad-54.L(me139) , that may prove useful for investigating meiotic mechanisms by providing a sensitized genetic background that reduces but does not eliminate the essential functions of RAD-54.L.

Figure 1. Nature of the rad-54.L(me139) mutation .

A. Schematic of the rad-54.L gene structure taken as a screenshot from WormBase WS285, indicating the position of the 102 nt deletion in the me139 mutant. B. Sequence context of the me139 deletion. UTR and intron sequences are indicated in lower case, exon sequences in upper case. The nucleotides deleted in the me139 mutant are indicated by cyan highlighting, and potential cryptic splice donor sequences that could be used to generate modified proteins retaining key conserved protein domains are indicated as depicted on figure.

Description

The rad-54.L(me139) mutation was isolated in a modified version of the previously-described “Green Eggs & Him” EMS screen for meiotic mutants based on mis-segregation of X chromosomes (Kelly et al. 2000). The causative mutation was mapped to chromosome I by outcrossing with worms carrying the tmC18 balancer chromosome (marked with the red pharynx marker tmIs1236 (Dejima et al. 2018)). Homozygous me139 mutant worms exhibit a variable number of DAPI-stained bodies in diakinesis oocytes and produce many inviable embryos (“maternal-effect lethal” or Mel phenotype), but they also produce some viable progeny that survive to adulthood (15.2% survivors, n = 961), many of which are males (16% males, n = 146; the “high incidence of males” or Him phenotype). Three-factor mapping of the Him/Mel phenotype using dpy-5 and unc-29 markers indicated a map position near or to the right of unc-29 (11/11 Dpy non-Unc recombinants picked up the Him/Mel mutation; 7/7 Unc non-Dpy recombinants did not). Consistent with this mapping, whole genome sequencing of DNA from pooled homozygous mutant recombinants revealed a candidate deletion in the rad-54.L gene (formerly called rad-54 (Mets and Meyer 2009)). This deletion was confirmed by PCR and Sanger sequencing, which identified the lesion as a 102 nt deletion that removes nucleotide residues I..9065146 - I..9065247, thereby truncating the first coding exon and eliminating the adjacent splice donor sequence and a portion of the adjacent intron. Finally, rad-54.L(me139) was confirmed to be the causative mutation based on failure to complement the Mel phenotype of rad-54.L(me98) , a rad-54.L null allele (Roelens et al. 2019), as no viable progeny resulted from the embryos produced by me139 / me98 heterozygous hermaphrodites. As worms homozygous for the rad-54L null allele produce 100% dead embryos (Roelens et al. 2019), we infer that rad-54.L(me139) does not eliminate RAD-54.L function but instead results in a partial loss of function. Perusal of the mutant sequence indicates a capacity to produce several potential modified versions of the RAD-54.L protein that could retain partial function. For example, there are several potential cryptic splice donor sequences that could be used to generate in-frame translatable mRNAs encoding proteins missing only amino acids 21-35 of the normal RAD-54.L protein (with 43, 60, or 62 extra amino acids inserted). As the affected portion of the protein is N-terminal to the highly conserved ATP binding and enzymatic domains that define the protein superfamily, it is plausible that such altered proteins could be at least partially functional. This partial loss of function allele could prove useful for investigating mechanisms of meiotic chromosome inheritance.

Reagents

Strain	Genotype	Available from
AV1138	rad-54.L(me139) / tmC18[dpy-5(tmIs1236)] I NOTE: Two transgene insertions from the original screening strain are floating in the background of this strain: ieSi11[EmGFP::syp-3] II and ieSi21[sun-1::mRuby] IV	AV lab
AV1238	rad-54.L(me139) / tmC18[dpy-5(tmIs1236)] I; ieSi21[sun-1::mRuby] IV NOTE: rad-54.L(me139) homozygotes derived from this strain were used to quantify progeny viability and male frequency	AV lab