COM-1 promotes homologous recombination during Caenorhabditis elegans meiosis by antagonizing Ku-mediated non-homologous end joining.

Successful completion of meiosis requires the induction and faithful repair of DNA double-strand breaks (DSBs). DSBs can be repaired via homologous recombination (HR) or non-homologous end joining (NHEJ), yet only repair via HR can generate the interhomolog crossovers (COs) needed for meiotic chromosome segregation. Here we identify COM-1, the homolog of CtIP/Sae2/Ctp1, as a crucial regulator of DSB repair pathway choice during Caenorhabditis elegans gametogenesis. COM-1-deficient germ cells repair meiotic DSBs via the error-prone pathway NHEJ, resulting in a lack of COs, extensive chromosomal aggregation, and near-complete embryonic lethality. In contrast to its yeast counterparts, COM-1 is not required for Spo11 removal and initiation of meiotic DSB repair, but instead promotes meiotic recombination by counteracting the NHEJ complex Ku. In fact, animals defective for both COM-1 and Ku are viable and proficient in CO formation. Further genetic dissection revealed that COM-1 acts parallel to the nuclease EXO-1 to promote interhomolog HR at early pachytene stage of meiotic prophase and thereby safeguards timely CO formation. Both of these nucleases, however, are dispensable for RAD-51 recruitment at late pachytene stage, when homolog-independent repair pathways predominate, suggesting further redundancy and/or temporal regulation of DNA end resection during meiotic prophase. Collectively, our results uncover the potentially lethal properties of NHEJ during meiosis and identify a critical role for COM-1 in NHEJ inhibition and CO assurance in germ cells.

A role for the malignant brain tumour (MBT) domain protein LIN-61 in DNA double-strand break repair by homologous recombination.

Malignant brain tumour (MBT) domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR) for the repair of DNA double-strand breaks (DSBs). lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT-deficient tumours may also have defective DSB repair.

The impact of COVID-19 on pulmonary function and airway reactivity after recovery in college-aged adults.

The purpose of this study was to determine (1) whether pulmonary function is reduced, and airway reactivity is increased after recovery from COVID-19 in individuals who did not have severe illness, and (2) whether physical activity levels had any impact on pulmonary function or airway reactivity. An exploratory aim of the study was also to assess whether number of symptoms was associated with pulmonary function outcomes. The maximal flow volume loop was used to measure pulmonary function in individuals who had previously tested positive for COVID-19 (COV; n = 20, 23.0 ± 5.4 years) and those who had not (CON; n = 20, 23.7 ± 5.5 years) before and after a hypertonic saline challenge (HSC) designed to increase airway reactivity. Self-reported symptoms and physical activity levels (MET (min/week)) were collected to examine their correlation with pulmonary outcomes. There were no significant differences in any pulmonary function outcomes between the COV and CON groups before or after the HSC. There were also no associations between physical activity and pulmonary function outcomes. However, among participants who reported greater than four symptoms, there was a larger decline in forced expiratory volume in 1 s divided by forced vital capacity following HSC (p = 0.035). Pulmonary function and airway reactivity are not impacted after recovery from COVID-19 in young individuals; however, it appears that the number of symptoms reported may be associated with increased airway reactivity even after recovery in young adults who were not hospitalized with the virus.

pWormgatePro enables promoter-driven knockdown by hairpin RNA interference of muscle and neuronal gene products in Caenorhabditis elegans.

Recent advances in genome research and RNA interference (RNAi) technology have accelerated the adoption of genome-wide experimental approaches for determining gene function in the model organism Caenorhabditis elegans. Despite recent successes, the application of RNAi is limited when gene knockdown causes complex phenotypes or embryonic lethality. Recently, the high-throughput pWormgate cloning system has been introduced as a tool to efficiently generate heat-shock-inducible hairpin RNA constructs using the Gateway recombination technology. We have modified pWormgate into a versatile hairpin cloning plasmid, pWormgatePro, which facilitates temporally and spatially inducible hairpin RNAi using constitutively active, tissue-specific promoters. To demonstrate its utility we knocked down unc-22 in body wall muscles as well as the axon guidance gene unc-5 in the nervous system indicating that promoter-driven hairpins can overcome the neuronal resistance to RNAi. Using pWormgatePro we also show that RNAi in the nervous system of C. elegans is non-autonomous and that spreading of the RNAi signal from neurons to muscle is substantially reduced but not abolished in spreading-defective sid-1 mutant animals. Our findings illustrate the effectiveness of pWormgatePro for gene silencing in muscle cells and neurons and bring forward the possibility of applying tissue-specific RNAi on a genome-wide scale.

Heritable and inducible gene knockdown in C. elegans using Wormgate and the ORFeome.

Double-stranded RNA (dsRNA) mediated gene silencing (RNA interference; RNAi) is a powerful tool for investigating gene function. It is usually performed in Caenorhabditis elegans via the injection or oral delivery of dsRNA, but an alternative approach, the expression of RNA hairpins from introduced DNA (hairpin RNAi; hpRNAi) has several advantages: (1) it can be induced systemically or in a tissue-specific manner; (2) because it is heritable, it allows consistent RNAi silencing across a whole population of genetically identical animals; and (3) it can be applied in refractory tissue such as neurons. hpRNAi has not been widely used to investigate gene function because a number of steps are relatively inefficient and labour-intensive. We describe Wormgate, a new cloning system, which facilitates the efficient high-throughput production of hpRNAi constructs using clones from the C. elegans ORFeome library. The combined use of pWormgate2 and the ORFeome library, with a recently developed particle bombardment transformation system, expedites hpRNAi gene silencing. This will be particularly useful for studying those genes that are refractory to the effects of injected or fed dsRNA, such as neural genes. We report the efficient production of hpRNAi constructs using pWormgate2 and also the knockdown of selected genes, including neurally expressed genes that have previously been refractory to RNAi. Further, when combined with the rrf-3 RNAi hypersensitive strain, the Wormgate approach delivered a highly penetrant knockdown phenotype in nearly 100% of worms for a gene that was completely refractory to other RNAi delivery methods.

VHA-19 is essential in Caenorhabditis elegans oocytes for embryogenesis and is involved in trafficking in oocytes.

There is an urgent need to develop new drugs against parasitic nematodes, which are a significant burden on human health and agriculture. Information about the function of essential nematode-specific genes provides insight to key nematode-specific processes that could be targeted with drugs. We have characterized the function of a novel, nematode-specific Caenorhabditis elegans protein, VHA-19, and show that VHA-19 is essential in the germline and, specifically, the oocytes, for the completion of embryogenesis. VHA-19 is also involved in trafficking the oocyte receptor RME-2 to the oocyte plasma membrane and is essential for osmoregulation in the embryo, probably because VHA-19 is required for proper eggshell formation via exocytosis of cortical granules or other essential components of the eggshell. VHA-19 may also have a role in cytokinesis, either directly or as an indirect effect of its role in osmoregulation. Critically, VHA-19 is expressed in the excretory cell in both larvae and adults, suggesting that it may have a role in osmoregulation in C. elegans more generally, probably in trafficking or secretion pathways. This is the first time a role for VHA-19 has been described.

Draft genome of the filarial nematode parasite Brugia malayi.

Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the approximately 90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict approximately 11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during approximately 350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.