Nuclear cGAS restricts L1 retrotransposition by promoting TRIM41-mediated ORF2p ubiquitination and degradation.

Cyclic GMP-AMP synthase (cGAS), initially identified as a cytosolic DNA sensor, detects DNA fragments to trigger an innate immune response. Recently, accumulating evidence reveals the presence of cGAS within the nucleus. However, the biological functions of nuclear cGAS are not fully understood. Here, we demonstrate that nuclear cGAS represses LINE-1 (L1) retrotransposition to preserve genome integrity in human cells. Mechanistically, the E3 ligase TRIM41 interacts with and ubiquitinates ORF2p to influence its stability, and cGAS enhances the association of ORF2p with TRIM41, thereby promoting TRIM41-mediated ORF2p degradation and the suppression of L1 retrotransposition. In response to DNA damage, cGAS is phosphorylated at serine residues 120 and 305 by CHK2, which promotes cGAS-TRIM41 association, facilitating TRIM41-mediated ORF2p degradation. Moreover, we show that nuclear cGAS mediates the repression of L1 retrotransposition in senescent cells induced by DNA damage agents. We also identify several cancer-associated cGAS mutations that abolish the suppressive effect on L1 retrotransposition by disrupting the CHK2-cGAS-TRIM41-ORF2p regulatory axis. Together, these findings indicate that nuclear cGAS exhibits an inhibitory function in L1 retrotransposition which could provide avenues for future interventions in both aging and tumorigenesis.

Androgen signaling stabilizes genomes to counteract senescence by promoting XRCC4 transcription.

Aging is accompanied by a decreased DNA repair capacity, which might contribute to age-associated functional decline in multiple tissues. Disruption in hormone signaling, associated with reproductive organ dysfunction, is an early event of age-related tissue degeneration, but whether it impacts DNA repair in nonreproductive organs remains elusive. Using skin fibroblasts derived from healthy donors with a broad age range, we show here that the downregulation of expression of XRCC4, a factor involved in nonhomologous end-joining (NHEJ) repair, which is the dominant pathway to repair somatic double-strand breaks, is mediated through transcriptional mechanisms. We show that the androgen receptor (AR), whose expression is also reduced during aging, directly binds to and enhances the activity of the XRCC4 promoter, facilitating XRCC4 transcription and thus stabilizing the genome. We also demonstrate that dihydrotestosterone (DHT), a powerful AR agonist, restores XRCC4 expression and stabilizes the genome in different models of cellular aging. Moreover, DHT treatment reverses senescence-associated phenotypes, opening a potential avenue to aging interventions in the future.

The cAMP-PKA signalling pathway regulates hyphal growth, conidiation, trap morphogenesis, stress tolerance, and autophagy in Arthrobotrys oligospora.

The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signalling pathway is evolutionarily conserved in eukaryotes and plays a crucial role in defending against external environmental challenges, which can modulate the cellular response to external stimuli. Arthrobotrys oligospora is a typical nematode-trapping fungus that specializes in adhesive networks to kill nematodes. To elucidate the biological roles of the cAMP-PKA signalling pathway, we characterized the orthologous adenylate cyclase AoAcy, a regulatory subunit (AoPkaR), and two catalytic subunits (AoPkaC1 and AoPkaC2) of PKA in A. oligospora by gene disruption, transcriptome, and metabolome analyses. Deletion of Aoacy significantly reduced the levels of cAMP and arthrobotrisins. Results revealed that Aoacy, AopkaR, and AopkaC1 were involved in hyphal growth, trap morphogenesis, sporulation, stress resistance, and autophagy. In addition, Aoacy and AopkaC1 were involved in the regulation of mitochondrial morphology, thereby affecting energy metabolism, whereas AopkaC2 affected sporulation, nuclei, and autophagy. Multi-omics results showed that the cAMP-PKA signalling pathway regulated multiple metabolic and cellular processes. Collectively, these data highlight the indispensable role of cAMP-PKA signalling pathway in the growth, development, and pathogenicity of A. oligospora, and provide insights into the regulatory mechanisms of signalling pathways in sporulation, trap formation, and lifestyle transition.

Characterization and functional analysis of calcium/calmodulin-dependent protein kinases (CaMKs) in the nematode-trapping fungus Arthrobotrys oligospora.

Ca2+/calmodulin-dependent protein kinases (CaMKs) are unique second-messenger molecules that impact almost all cellular processes in eukaryotes. In this study, five genes encoding different CaMKs were characterized in the nematode-trapping fungus Arthrobotrys oligospora. These CaMKs, which were retrieved from the A. oligospora genome according to their orthologs in fungi such as Aspergillus nidulans and Neurospora crassa, were expressed at a low level in vitro during mycelial growth stages. Five deletion mutants corresponding to these CaMKs led to growth defects in different media and increased sensitivity to several environmental stresses, including H2O2, menadione, SDS, and Congo red; they also reduced the ability to produce conidia and traps, thus causing a deficiency in nematicidal ability as well. In addition, the transcriptional levels of several typical sporulation-related genes, such as MedA, VelB, and VeA, were down-regulated in all ΔCaMK mutants compared with the wild-type (WT) strain. Moreover, these mutants exhibited hypersensitivity to heat shock and ultraviolet-radiation stresses compared with the WT strain. These results suggest that the five CaMKs in A. oligospora are involved in regulating multiple cellular processes, such as growth, environmental stress tolerance, conidiation, trap formation, and virulence.

Two Rab GTPases play different roles in conidiation, trap formation, stress resistance, and virulence in the nematode-trapping fungus Arthrobotrys oligospora.

Rab GTPases are the largest group of the small GTPases family, which play a pivotal role in the secretion of proteins. Arthrobotrys oligospora is a representative nematode-trapping fungus that can produce adhesive networks to capture nematodes. In this study, the roles of two Rab GTPases AoRab-7A and AoRab-2 were characterized by gene knockout in the fungus A. oligospora. The disruption of AoRab-7A hindered the mycelial growth in different media, the conidiation of ΔAoRab-7A transformants was almost abolished, and the transcription of four sporulation-related genes (AbaA, FluG, Hyp1, and VosA) was downregulated compared to the wild-type strain (WT). Furthermore, the tolerance of the ΔAoRab-7A mutants to sodium dodecyl sulfate (SDS) and H2O2 was also significantly reduced compared to the WT, and the transcription of several genes related to environmental resistance, such as genes for catalase and trehalose synthase, was downregulated. Similarly, the extracellular proteolytic activity was decreased. Importantly, the ΔAoRab-7A mutants were unable to produce traps and capture nematodes. However, the disruption of gene AoRab-2 only affected the conidiation slightly but non-significantly, while other phenotypic traits were unaffected. Moreover, the gene AoRab-7A was also involved in the autophagy induced by nitrogen deprivation in A. oligospora. Our results revealed for the first time that the Rab GTPases are involved in the regulation of mycelial growth, conidiation, trap formation, stress resistance, and pathogenicity in the nematode-trapping fungus A. oligospora.

MAP kinase Slt2 orthologs play similar roles in conidiation, trap formation, and pathogenicity in two nematode-trapping fungi.

Mitogen-activated protein (MAP) kinase Slt2 is a key player in the cell-wall integrity pathway of budding yeast. In this study, we functionally characterized Slt2 orthologs AoSlt2 and MhSlt2 from the nematode-trapping fungi Arthrobotrys oligospora and Monacrosporium haptotylum, respectively. We found that disruption of AoSlt2 and MhSlt2 led to reduced mycelial growth, increased sensitivity to environmental stresses such as sodium dodecyl sulfate, Congo red, and H2O2, and an inability to produce conidia and nematode-trapping structures. Real-time polymerase chain reaction-based analyses showed that the transcription of sporulation-related (AbaA, Sep2, and MedA) and cell wall synthesis-related (Chs, Glu, and Gfpa) genes was down-regulated in the mutants compared with the wild-type strains. Moreover, the mutant strains showed reduced extracellular proteolytic activity and decreased transcription of three homologous serine protease-encoding genes. These results show for the first time that MAP kinase Slt2 orthologs play similar roles in regulating mycelial growth, conidiation, trap formation, stress resistance, and pathogenicity in the divergent nematode-trapping fungal species A. oligospora and M. haptotylum.