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RPA complexes in Caenorhabditis elegans meiosis; unique roles in replication, meiotic recombination and apoptosis.

ABSTRACT: Replication Protein A (RPA) is a critical complex that acts in replication and promotes homologous recombination by allowing recombinase recruitment to processed DSB ends. Most organisms possess three RPA subunits (RPA1, RPA2, RPA3) that form a trimeric complex critical for viability. The Caenorhabditis elegans genome encodes RPA-1, RPA-2 and an RPA-2 paralog RPA-4. In our analysis, we determined that RPA-2 is critical for germline replication and normal repair of meiotic DSBs. Interestingly, RPA-1 but not RPA-2 is essential for somatic replication, in contrast to other organisms that require both subunits. Six different hetero- and homodimeric complexes containing permutations of RPA-1, RPA-2 and RPA-4 can be detected in whole animal extracts. Our in vivo studies indicate that RPA-1/4 dimer is less abundant in the nucleus and its formation is inhibited by RPA-2. While RPA-4 does not participate in replication or recombination, we find that RPA-4 inhibits RAD-51 filament formation and promotes apoptosis of a subset of damaged nuclei. Altogether these findings point to sub-functionalization and antagonistic roles of RPA complexes in C. elegans.

SUBMITTER: Hefel A

PROVIDER: S-EPMC7913698 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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RPA complexes in Caenorhabditis elegans meiosis; unique roles in replication, meiotic recombination and apoptosis.

Hefel Adam A Honda Masayoshi M Cronin Nicholas N Harrell Kailey K Patel Pooja P Spies Maria M Smolikove Sarit S

Nucleic acids research 20210201 4

Replication Protein A (RPA) is a critical complex that acts in replication and promotes homologous recombination by allowing recombinase recruitment to processed DSB ends. Most organisms possess three RPA subunits (RPA1, RPA2, RPA3) that form a trimeric complex critical for viability. The Caenorhabditis elegans genome encodes RPA-1, RPA-2 and an RPA-2 paralog RPA-4. In our analysis, we determined that RPA-2 is critical for germline replication and normal repair of meiotic DSBs. Interestingly, RP ...[more]

PMID: 33476370

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Project description:Expression data from Caenorhabditis elegans let-418(RNAi), mep-1(RNAi) and gfp(RNAi) L1 larvae. The C. elegans genome encodes two homologs of the human protein Mi-2, namely LET-418 and CHD-3. LET-418 plays an essential role during development; its depletion leads to a pleiotropic and lethal phenotype that includes larval arrest, an everted vulva and sterility. Without maternal contribution, let-418 mutants stop their development at the L1 larval stage (von Zelewsky et al., 2000). We further characterized this arrest and showed that it is very similar to the L1 diapause induced by starvation; both germline and somatic cells remain in a quiescent state in let-418 L1 arrested larvae, indicating that LET-418 activity is required to bypass the L1 arrest in presence of food. The let-418 L1 larvae express ectopically the P granule component PGL-1 in somatic cells (Unhavaithaya et al., 2002). Interestingly, the phenotype of mep-1 mutants is remarkably similar to that of let-418: RNAi targeting mep-1 also induced an L1 arrest phenotype; furthermore, MEP-1 and LET-418 have been shown to physically interact (Unhavaithaya et al., 2002 and M. Passannante). The null allele mep-1(q660) is temperature sensitive and shows a more severe phenotype at higher temperatures. At 20Â°C, about 10% of mep-1 homozygotes derived from heterozygous mothers arrest as young larvae, whereas the remaining 90% develop into sterile adults (Belfiore et al., 2002). Later in development, the somatic gonad is affected in mep-1(q660) mutants. This results in an abnormal and disorganized gonad, a phenotype also observed in let-418(s1617) mutants. Both let-418 and mep-1 mutants produce a very limited number of oocytes and have pseudovulvae derived from P8.p (Belfiore et al., 2002; von Zelewsky et al., 2000 and C. Wicky, personal communication). Preliminary quantitative real-time PCR revealed that the expression of genes coding for P granule components was deregulated in both mep-1(RNAi) and let-418(RNAi) L1 larvae (data not shown). To further investigate this issue, we performed a complete gene expression analysis. Given the fact that mep-1(q660) mutants are sterile, we used RNA interference to generate mep-1 depleted worms. Bacteria expressing gfp dsRNA (pPE128.110 in HT115) were used as reference, since RNA interference may induce gene expression changes by itself. C. elegans L1 larvae treated with RNA interference were selected for RNA extraction and hybridization on Affymetrix microarrays. Synchronized wild type L4 animals were grown at 25Â° on bacteria expressing either gfp, let-418 or mep-1 dsRNA. Eggs were collected by bleaching gravid adults and allowed to hatch in the absence of food at 25Â°C. Newly hatched L1 larvae were fed on bacteria expressing the different dsRNA for three hours to recover from starvation. Three replicates per RNAi.

Dataset Information

RPA complexes in Caenorhabditis elegans meiosis; unique roles in replication, meiotic recombination and apoptosis.

Publications

RPA complexes in Caenorhabditis elegans meiosis; unique roles in replication, meiotic recombination and apoptosis.

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