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The amino acid cysteine is critical for many aspects of life, yet excess cysteine is toxic. Therefore, animals require pathways to maintain cysteine homeostasis. In mammals, high cysteine activates cysteine dioxygenase, a key enzyme in cysteine catabolism. The mechanism by which cysteine dioxygenase is regulated remains largely unknown. We discovered thatC. eleganscysteine dioxygenase (cdo-1) is transcriptionally activated by high cysteine and the hypoxia inducible transcription factor (hif-1).hif-1-dependent activation ofcdo-1occurs downstream of an H2S-sensing pathway that includesrhy-1, cysl-1, andegl-9. cdo-1transcription is primarily activated in the hypodermis where it is sufficient to drive sulfur amino acid metabolism. EGL-9 and HIF-1 are core members of the cellular hypoxia response. However, we demonstrate that the mechanism of HIF-1-mediated induction ofcdo-1functions largely independent of EGL-9 prolyl hydroxylation and the von Hippel-Lindau E3 ubiquitin ligase; classical hypoxia signaling pathway components. We propose that the intersection ofhif-1andcdo-1reveals a negative feedback loop for maintaining cysteine homeostasis. High cysteine stimulates the production of an H2S signal. H2S then activates therhy-1/cysl-1/egl-9signaling pathway, increasing HIF-1-mediated transcription ofcdo-1, promoting degradation of cysteine via CDO-1.

Surveillance of

Surveillance of Caenorhabditis elegans mitochondrial status is coupled to defense responses such as drug detoxification, immunity, antiviral RNA interference (RNAi), and regulation of life span. A cytochrome p540 detoxification gene, cyp-14A4 , is specifically activated by mitochondrial dysfunction. The nuclear hormone receptor NHR-45 and the transcriptional Mediator component MDT-15/MED15 are required for the transcriptional activation of cyp-14A4 by mitochondrial mutations, gene inactivations, or toxins. A genetic screen for mutations that fail to activate this cytochrome p450 gene upon drug or mutation-induced mitochondrial dysfunction identified a DNA helicase ARIP-4 that functions in concert with the NHR-45 transcriptional regulatory cascade. In response to mitochondrial dysfunction, ARIP-4 and NHR-45 protein interaction is enhanced, and they relocalize from the nuclear periphery to the interior of intestinal nuclei. NHR-45/ARIP-4 also regulates the transcriptional activation of the eol-1 gene that encodes a decapping enzyme required for enhanced RNAi and transgene silencing of mitochondrial mutants. In the absence of arip-4 , animals were more susceptible to the mitochondrial inhibitor antimycin. Thus, ARIP-4 serves as a transcriptional coactivator of NHR-45 to promote this defense response. A null mutation in arip-4 extends the life span and health span of both wild type and a mitochondrial mutant, suggesting that the activation of detoxification pathways is deleterious to health when the mitochondrial dysfunction is caused by mutation that cannot be cytochrome p450-detoxified. Thus, arip-4 acts in a pathway that couples mitochondrial surveillance to the activation of downstream immunity, detoxification, and RNAi responses.

The National Aeronautics and Space Administration (NASA) and the European Space Agency (ESA) are studying how samples might be brought back to Earth from Mars safely. Backward planetary protection is key in this complex endeavour, as it is required to prevent potential adverse effects from returning materials to Earth's biosphere. As the question of whether or not life exists on Mars today or whether it ever did in the past is still unanswered, the effort to return samples from Mars is expected to be categorized as a ‘Restricted Earth Return’ mission, for which NASA policy requires the containment of any unsterilized material returned to Earth. NASA is investigating several solutions to contain Mars samples and sterilize any uncontained Martian particles. This effort has significant implications for both NASA's scientific mission, and the Earth's environment; and so special care and vigilance are needed in planning and execution in order to assure acceptance of safety to Earth's biosphere. To generate a technically acceptable sterilization process across a wide array of scientific and other stakeholders, on 30–31 January 2019, 10–11 June 2019 and 19–20 February 2020, NASA informally convened a Sterilization Working Group (SWG) composed of experts from industry, academia and government to assess methods for sterilization and inactivation, to identify future work needed to verify these methods against biological challenges, and to determine their feasibility for implementation on robotic spacecraft in deep space. The goals of the SWG were:(1)Understand what it means to sterilize and/or inactivate Martian materials and how that understanding can be applied to the Mars Sample Return (MSR) mission.(2)Assess methods for sterilization and inactivation, and identify future work needed to verify these methods.(3)Provide an effective plan for communicating with other agencies and the public.This paper provides a summary of the discussions and conclusions of the SWG over these three workshops. It reflects a consensus position based on qualitative discussion of how agencies might approach the problem of sterilization of Mars material. The SWG reached a consensus that sterilization options can be considered on the basis of biology as we know it, and that sterilization modalities that are effective on terrestrial materials and organisms should be part of the MSR planetary protection strategy. Conclusions pointed to several industry standards for sterilization to include heat, chemical, UV radiation and low-heat plasma. Technical trade-offs for each sterilization modality were discussed while simultaneously considering the engineering challenges and limitations for spaceflight. Future work includes more in-depth discussions on technical trade-offs of sterilization modalities, identifying and testing Earth analogue challenge organisms and proteinaceous molecules against chosen modalities, and executing collaborative agreements between NASA and external working group partners to help close data gaps, and to establish strong, scientifically grounded sterilization and inactivation standards for MSR.

Caenorhabditis elegans consumes bacteria, which can supply essential vitamins and cofactors, especially for mitochondrial functions that have a bacterial ancestry. Therefore, we screened the Keio Escherichia coli knockout library for mutations that induce the C. elegans hsp-6 mitochondrial damage response gene, and identified 45 E. coli mutations that induce hsp-6::gfp We tested whether any of these E. coli mutations that stress the C. elegans mitochondrion genetically interact with C. elegans mutations in mitochondrial functions. Surprisingly, 4 E. coli mutations that disrupt the import or removal of iron from the bacterial siderophore enterobactin were lethal in combination with a collection of C. elegans mutations that disrupt particular iron-sulfur proteins of the electron transport chain. Bacterial mutations that fail to synthesize enterobactin are not synthetic lethal with these C. elegans mitochondrial mutants; it is the enterobactin-iron complex that is lethal in combination with the C. elegans mitochondrial mutations. Antioxidants suppress this inviability, suggesting that reactive oxygen species (ROS) are produced by the mutant mitochondria in combination with the bacterial enterobactin-iron complex.

Many viral proteins, including multiple SARS-CoV-2 proteins, are secreted via the endoplasmic reticulum, and viral particles are assembled and exported in ER-associated replication compartments. Viral coat proteins such as the SARS-CoV-2 Spike protein are N-glycosylated at NxS/T sites as they enter the ER. N-glycosylated sites in many eukaryotic proteins are deglycosylated by the NGLY1/PNG-1 deglycosylation enzyme which also deamidates the N-glycosylated asparagine to aspartic acid, thus editing the target protein sequence. Proteomic analysis of mammalian cell lines has revealed deamidation of many host N-glycosylated asparagines to aspartic acid by NGLY1/PNG-1 on peptides that are presented by mammalian HLA for immune surveillance. The key client protein for NGLY1/PNG-1 deglycosylation and N to D protein editing was revealed by genetic analysis ofC. elegansproteasome regulation to be the intact endoplasmic reticulum-transiting SKN-1A transcription factor. Strikingly, an analysis of cancer cell genetic dependencies for growth revealed that the mammalian orthologue of SKN-1A, NRF1 (also called NFE2L1) is required by a highly correlated set of cell lines as NGLY1/PNG-1, supporting that NGLY1/PNG-1 and NRF1 act in the same pathway. NGLY1/PNG-1 edits N-glycosylated asparagines on the intact SKN-1 protein as it is retrieved by ERAD from the ER to in turn activate the transcription of target proteasomal genes. The normal requirement for NGLY1/PNG-1 editing of SKN-1A can be bypassed by a genomic substituion of N to D in four NxS/T N-glycosylation motifs of SKN-1A. Thus NGLY1/PNG-1-mediated N to D protein editing is more than a degradation step for the key client protein for proteasomal homeostasis inC. elegansor tumor growth in particular mammalian cell lines, SKN-1A/NRF1. In addition, such N to D substitutions in NxS/T N-glycosylation motifs occur in evolution: N to D substitutions are observed in phylogenetic comparisons of SKN-1A between nematode species that diverged hundreds of millions of years ago or of the vertebrate NRF1 between disparate vertebrates. Genomic N to D mutations bypass the many steps in N-glycosylation in the ER and deglycosylation-based editing of N to D, perhaps based on differences in the competency of divergent species for various N-glycosylation or deglycosylation steps.We surveyed the N-glycosylation sites in coronavirus proteins for such phylogenetic evidence for N to D protein editing in viral life cycles, and found evidence for preferential N to D residue substitutions in NxS/T N-glycosylation sites in comparisons of the genome sequences of hundreds of coronaviruses. This suggests that viruses use NGLY1/PNG-1 in some hosts, for example humans, to edit particular N-glycosylated residues to aspartic acid, but that in other hosts, often in bats, an N to D substitution mutation in the virus genome is selected. Single nucleotide mutations in Asp or Asn codons can produce viruses with N to D or D to N substitutions that might be selected in different animal hosts from the population of viral variants produced in any previous host. NGLY1/PNG-1 has been implicated in viral immunity in mammalian cell culture, favoring this hypothesis.Because of the phylogenetic evidence that the NGLY1/PNG-1 editing of protein sequences has functional importance for SKN-1A/NRF1 and viruses, and because most immunization protocols do not address the probable editing and functional importance of N-glycosylated aspargines to aspartic acid in normal viral infections, we suggest that immunization with viral proteins engineered to substitute D at genomically encoded NxS/T sites of N-glycosylated viral proteins that show a high frequency of N to D substitution in viral phylogeny may enhance immunological response to peptide antigens. Such genomically-edited peptides would not require ER-localization for N-glycosylation or other cell compartment localization for NGLY1/PNG-1 N to D protein editing. In addition, such N to D edited protein vaccines could be produced in bacteria since N-glycosylation and deglycosylation which do not occur in bacteria would no longer be required to immunize with a D-substituted peptide. Bacterially-expressed vaccines would be much lower cost and with fewer failure modes than attenuated viral vaccines or recombinant animal viruses produced in chicken eggs, mammalian tissue culture cells, or delivered by mRNA vectors to the patient directly. Because N to D edited peptides are clearly produced by NGLY1/PNG-1, and may be and presented by mammalian HLA, such peptides may more robustly activate T-cell killing or B-cell maturation to mediate more robust viral immunity.

Nrf1 is a member of the nuclear erythroid 2-like family of transcription factors that regulate stress-responsive gene expression in animals. Newly synthesized Nrf1 is targeted to the endoplasmic reticulum (ER) where it is

Habitable regions of Europa may include a subsurface ocean and transient liquid environments within its icy shell. Ocean-surface communication may occur on 1–2 million-year (My) timescales or even ...

In their natural habitat of rotting fruit, the nematode Caenorhabditis elegans feeds on the complex bacterial communities that thrive in this rich growth medium. Hundreds of diverse bacterial strains cultured from such rotting fruit allow C. elegans growth and reproduction when tested individually. In screens for C. elegans responses to single bacterial strains associated with nematodes in fruit, we found that Rhizobium causes a genome instability phenotype; we observed abnormally long or fragmented intestinal nuclei due to aberrant nuclear division, or defective karyokinesis. The karyokinesis defects were restricted to intestinal cells and required close proximity between bacteria and the worm. A genetic screen for C. elegans mutations that cause the same intestinal karyokinesis defect followed by genome sequencing of the isolated mutant strains identified mutations that disrupt DNA damage repair pathways, suggesting that Rhizobium may cause DNA damage in C. elegans intestinal cells. We hypothesized that such DNA damage is caused by reactive oxygen species produced by Rhizobium and found that hydrogen peroxide added to benign Escherichia coli can cause the same intestinal karyokinesis defects in WT C. elegans. Supporting this model, free radical scavengers suppressed the Rhizobium-induced C. elegans DNA damage. Thus, Rhizobium may signal to eukaryotic hosts via reactive oxygen species, and the host may respond with DNA damage repair pathways.

Animals have evolved critical mechanisms to maintain cellular and organismal proteostasis during development, disease, and exposure to environmental stressors. The Unfolded Protein Response (UPR) is a conserved pathway that senses and responds to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen. We have previously demonstrated that the IRE-1-XBP-1 branch of the UPR is required to maintain Caenorhabditis elegans ER homeostasis during larval development in the presence of pathogenic Pseudomonas aeruginosa. In this study, we identify loss-of-function mutations in four conserved transcriptional regulators that suppress the larval lethality of xbp-1 mutant animals caused by immune activation in response to infection by pathogenic bacteria: FKH-9, a forkhead family transcription factor; ARID-1, an ARID/Bright domain-containing transcription factor; HCF-1, a transcriptional regulator that associates with histone modifying enzymes; and SIN-3, a subunit of a histone deacetylase complex. Further characterization of FKH-9 suggests that loss of FKH-9 enhances resistance to the ER toxin tunicamycin and results in enhanced ER-associated degradation (ERAD). Increased ERAD activity of fkh-9 loss-of-function mutants is accompanied by a diminished capacity to degrade cytosolic proteasomal substrates and a corresponding increased sensitivity to the proteasomal inhibitor bortezomib. Our data underscore how the balance between ER and cytosolic proteostasis can be influenced by compensatory activation of ERAD during the physiological ER stress of infection and immune activation.

Enterococcus faecalis is a multidrug resistant, opportunistic human pathogen and a leading cause of hospital acquired infections. Recently, isolates have been recovered from the air and surfaces onboard the International Space Station (ISS). Pangenomic and functional analyses were carried out to assess their potential impact on astronaut health. Genomes of each ISS isolate, and both clinical and commensal reference strains, were evaluated for their core and unique gene content, acquired antibiotic resistance genes, phage, plasmid content, and virulence traits. In order to determine their potential survival when outside of the human host, isolates were also challenged with three weeks of desiccation at 30% relative humidity. Finally, pathogenicity of the ISS strains was evaluated in the model organism Caenorhabditis elegans. At the culmination of this study, there were no defining signatures that separated known pathogenic strains from the more commensal phenotypes using the currently available resources. As a result, the current reliance on database information alone must be shifted to experimentally evaluated genotypic and phenotypic characteristics of clinically relevant microorganisms.

The molybdenum cofactor (Moco) is a 520 dalton prosthetic group that is synthesized in a multi-step enzymatic pathway present in most Archaea, Bacteria, and Eukarya. In animals, four oxidases (among them sulfite oxidase) use Moco as a prosthetic group. Moco is essential in animals; humans with mutations in genes that encode Moco-biosynthetic enzymes display lethal neurological and developmental defects. Moco supplementation seems a logical therapy, however the instability of Moco has precluded biochemical and cell biological studies of Moco transport and bioavailability. The nematode Caenorhabditis elegans can take up Moco from its bacterial diet and transport it to cells and tissues that express Moco-requiring enzymes, suggesting a system for Moco uptake and distribution. Here we show that protein-bound Moco is the stable, bioavailable species of Moco taken up by C. elegans from its diet and is an effective dietary supplement in a C. elegans model of Moco deficiency. Diverse purified Moco:protein complexes from bacteria, bread mold, green algae, and dairy cows were able to support the growth of otherwise Moco-deficient C. elegans mutants grown on Moco-deficient E. coli. We show that these Moco:protein complexes are very stable, suggesting they may provide a strategy for the production and delivery of therapeutically active Moco to treat human Moco deficiency.

Mitochondrial fission and fusion are highly regulated by energy demand and physiological conditions to control the production, activity, and movement of these organelles. Mitochondria are arrayed in a periodic pattern in Caenorhabditis elegans muscle, but this pattern is disrupted by mutations in the mitochondrial fission component dynamin DRP-1. Here we show that the dramatically disorganized mitochondria caused by a mitochondrial fission-defective dynamin mutation is strongly suppressed to a more periodic pattern by a second mutation in lysosomal biogenesis or acidification. Vitamin B12 is normally imported from the bacterial diet via lysosomal degradation of B12-binding proteins and transport of vitamin B12 to the mitochondrion and cytoplasm. We show that the lysosomal dysfunction induced by gene inactivations of lysosomal biogenesis or acidification factors causes vitamin B12 deficiency. Growth of the C. elegans dynamin mutant on an Escherichia coli strain with low vitamin B12 also strongly suppressed the mitochondrial fission defect. Of the two C. elegans enzymes that require B12, gene inactivation of methionine synthase suppressed the mitochondrial fission defect of a dynamin mutation. We show that lysosomal dysfunction induced mitochondrial biogenesis, which is mediated by vitamin B12 deficiency and methionine restriction. S-adenosylmethionine, the methyl donor of many methylation reactions, including histones, is synthesized from methionine by S-adenosylmethionine synthase; inactivation of the sams-1 S-adenosylmethionine synthase also suppresses the drp-1 fission defect, suggesting that vitamin B12 regulates mitochondrial biogenesis and then affects mitochondrial fission via chromatin pathways.

The C. elegans genome encodes nineteen functional Argonaute proteins that utilize 22G-RNAs, 26G-RNAs, miRNAs, or piRNAs to regulate their target transcripts. Only one Argonaute is essential under normal laboratory conditions: CSR-1. While CSR-1 has been studied widely, nearly all studies have overlooked the fact that the csr-1 locus encodes two isoforms. These isoforms differ by an additional 163 amino acids present in the N-terminus of CSR-1a. Using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG into the long (CSR-1a) and short (CSR-1b) isoforms, we found that CSR-1a is expressed during spermatogenesis and in several somatic tissues, including the intestine. CSR-1b is expressed constitutively in the germline. small RNA sequencing of CSR-1 complexes shows that they interact with partly overlapping sets of 22G-RNAs. Phenotypic analyses reveal that the essential functions of csr-1 described in the literature coincide with CSR-1b, while CSR-1a plays tissue specific functions. During spermatogenesis, CSR-1a integrates into an sRNA regulatory network including ALG-3, ALG-4, and WAGO-10 that is necessary for fertility at 25°C. In the intestine, CSR-1a silences immunity and pathogen-responsive genes, and its loss results in improved survival from the pathogen Pseudomonas aeruginosa. Our findings functionally distinguish the CSR-1 isoforms and highlight the importance of studying each AGO isoform independently.

RNA interference (RNAi) is an antiviral pathway common to many eukaryotes that detects and cleaves foreign nucleic acids. In mammals, mitochondrially localized proteins such as MAVS, RIG-I, and MDA5 mediate antiviral responses. Here, we report that mitochondrial dysfunction inCaenorhabditis elegansactivates RNAi-directed silencing via induction of a pathway homologous to the mammalian RIG-I helicase viral response pathway. The induction of RNAi also requires the conserved RNA decapping enzyme EOL-1/DXO. The transcriptional induction ofeol-1requires DRH-1 as well as the mitochondrial unfolded protein response (UPRmt). Upon mitochondrial dysfunction, EOL-1 is concentrated into foci that depend on the transcription of mitochondrial RNAs that may form dsRNA, as has been observed in mammalian antiviral responses. The enhanced RNAi triggered by mitochondrial dysfunction contributes to the increase in longevity that is induced by mitochondrial dysfunction.

Mitochondrial fission and fusion are highly regulated by energy demand and physiological conditions to control the production, activity, and movement of these organelles. Mitochondria are arrayed in a periodic pattern inCaenorhabditis elegansmuscle, but this pattern is disrupted by mutations in the mitochondrial fission component dynamin. Here we show that the dramatically disorganized mitochondria caused by a mitochondrial fission-defective dynamin mutation is strongly suppressed to a more periodic pattern by a second mutation in lysosomal biogenesis or acidification. Vitamin B12 is normally imported from the bacterial diet via lysosomal degradation of B12-binding proteins and transport of vitamin B12 to the mitochondrion and cytoplasm. We show that the lysosomal dysfunction induced by gene inactivations of lysosomal biogenesis or acidification factors causes vitamin B12 deficiency. Growth of theC. elegansdynamin mutant on anE. colistrain with low vitamin B12 also strongly suppressed the mitochondrial fission defect. Of the twoC. elegansenzymes that require B12, gene inactivation of methionine synthase suppressed the mitochondrial fission defect of a dynamin mutation. We show that lysosomal dysfunction induced mitochondrial biogenesis which is mediated by vitamin B12 deficiency and methionine restriction. S-adenosylmethionine, the methyl donor of many methylation reactions, including histones, is synthesized from methionine by S-adenosylmethionine synthase; inactivation of thesams-1S-adenosylmethionine synthase also suppresses thedrp-1fission defect, suggesting that vitamin B12 regulates mitochondrial biogenesis and then affects mitochondrial fission via chromatin pathways.SIGNIFICANCE STATEMENTThe balance of mitochondrial fission and fusion, two aspects of mitochondrial dynamics, is important for mitochondrial function. Here we show thatCaenorhabditis eleganslysosomal activity regulates mitochondrial dynamics by affecting mitochondrial fission through interfering the metabolism of a micronutrient, vitamin B12. Vitamin B12 is exclusively obtained from diets in animals includingC. elegansand humans, and its uptake is mediated by the lysosome. We show that lysosomal dysfunction causes vitamin B12 deficiency that leads to reduction of methionine and S-adenosylmethionine to in turn increase mitochondrial biogenesis and fission. Our study provides an insight on the interactions between mitochondrial function and micronutrient metabolism.

Nanopore sequencing, as represented by Oxford Nanopore Technologies’ MinION, is a promising technology for in situ life detection and for microbial monitoring including in support of human space exploration, due to its small size, low mass (∼100 g) and low power (∼1W). Now ubiquitous on Earth and previously demonstrated on the International Space Station (ISS), nanopore sequencing involves translocation of DNA through a biological nanopore on timescales of milliseconds per base. Nanopore sequencing is now being done in both controlled lab settings as well as in diverse environments that include ground, air and space vehicles. Future space missions may also utilize nanopore sequencing in reduced gravity environments, such as in the search for life on Mars (Earth-relative gravito-inertial acceleration (GIA) g = 0.378), or at icy moons such as Europa (g = 0.134) or Enceladus (g = 0.012). We confirm the ability to sequence at Mars as well as near Europa or Lunar (g = 0.166) and lower g levels, demonstrate the functionality of updated chemistry and sequencing protocols under parabolic flight, and reveal consistent performance across g level, during dynamic accelerations, and despite vibrations with significant power at translocation-relevant frequencies. Our work strengthens the use case for nanopore sequencing in dynamic environments on Earth and in space, including as part of the search for nucleic-acid based life beyond Earth.

Microbial toxins and virulence factors often target the eukaryotic translation machinery. Caenorhabditis elegans surveils for such microbial attacks by monitoring translational competence, and if a deficit is detected, particular drug detoxification and bacterial defense genes are induced. The bacteria Kocuria rhizophila has evolved countermeasures to animal translational surveillance and defense pathways. Here, we used comprehensive genetic analysis of Kocuria rhizophila to identify the bacterial genetic pathways that inhibit C. elegans translational toxin surveillance and defense. Kocuria rhizophila mutations that disrupt its ability to disable animal immunity and defense map to multiple steps in the biosynthesis of a 50-carbon bacterial carotenoid from 5 carbon precursors. Extracts of the C50 carotenoid from wild type K. rhizophila could restore this bacterial anti-immunity activity to K. rhizophila carotenoid biosynthetic mutant. Corynebacterium glutamicum, also inhibits the C. elegans translation detoxification response by producing the C50 carotenoid decaprenoxanthin, and C. glutamicum carotenoid mutants are defective in this suppression of C. elegans detoxification. Consistent with the salience of these bacterial countermeasures to animal drug responses, bacterial carotenoids sensitize C. elegans to drugs that target translation and inhibit food aversion behaviors normally induced by protein translation toxins or mutations. The surveillance and response to toxins is mediated by signaling pathways conserved across animal phylogeny, suggesting that these bacterial carotenoids may also suppress such human immune and toxin responses.

Significance Silencing of transposable elements and viruses is critical for the maintenance of genome integrity, cellular homeostasis, and organismal health. Here we describe multiple factors that control different types of transposable elements, providing insight into how they are regulated. We also identify stress response pathways that are triggered upon misregulation of these transposable elements. The conservation of these factors and pathways in human suggests that our studies in Caenorhabditis elegans can provide general insight into the regulation of and response to transposable elements and viruses., Endogenous retroviruses and long terminal repeat (LTR) retrotransposons are mobile genetic elements that are closely related to retroviruses. Desilenced endogenous retroviruses are associated with human autoimmune disorders and neurodegenerative diseases. Caenorhabditis elegans and related Caenorhabditis spp. contain LTR retrotransposons and, as described here, numerous integrated viral genes including viral envelope genes that are part of LTR retrotransposons. We found that both LTR retrotransposons and endogenous viral elements are silenced by ADARs [adenosine deaminases acting on double-stranded RNA (dsRNA)] together with the endogenous RNA interference (RNAi) factor ERI-6/7, a homolog of MOV10 helicase, a retrotransposon and retrovirus restriction factor in human. siRNAs corresponding to integrated viral genes and LTR retrotransposons, but not to DNA transposons, are dependent on the ADARs and ERI-6/7. siRNAs corresponding to palindromic repeats are independent of the ADARs and ERI-6/7, and are in fact increased in adar- and eri-6/7–defective mutants because of an antiviral RNAi response to dsRNA. Silencing of LTR retrotransposons is dependent on downstream RNAi factors and P granule components but is independent of the viral sensor DRH-1/RIG-I and the nuclear Argonaute NRDE-3. The activation of retrotransposons in the ADAR- and ERI-6/7/MOV10–defective mutant is associated with the induction of the unfolded protein response (UPR), a common response to viral infection. The overlap between genes induced upon viral infection and infection with intracellular pathogens and genes coexpressed with retrotransposons suggests that there is a common response to different types of foreign elements that includes a response to proteotoxicity presumably caused by the burden of replicating pathogens and expressed retrotransposons.

RNAi pathways detect and silence foreign nucleic acids such as viruses as well as endogenous genes in many species. The phylogenetic profile across eukaryotes of proteins that mediate key steps in RNAi is correlated with the profiles of multiple mRNA splicing proteins and with intron number, suggesting that RNAi may surveil mRNA splicing to detect the divergent or absent introns of viruses. Here we examine the role of mRNA splicing in Caenorhabditis elegans RNAi. We found that viable null mutations in U1 and U2 small nuclear ribonucleic protein (snRNP)-specific splicing factor genes cause defects in RNAi. The U1A ortholog rnp-2 is required for normal ERGO-1 Argonaute class 26G siRNA biogenesis, trans-splicing of the eri-6/7 transcript, and targeting of poorly conserved gene transcripts by WAGO Argonaute class 22G siRNAs. We found that gene transcripts engaged by the siRNA-generating machinery are poorly conserved, possess few introns, and often have introns that are divergent from introns with strong consensus splicing sites found in highly conserved genes. We present biochemical evidence that RNAi targeted transcripts are tightly bound to spliceosomes. These findings suggest multiple layers of regulation by the spliceosome at early steps of small RNA-mediated gene silencing.

C. elegansconsumes bacteria which can supply essential vitamins and cofactors especially for mitochondrial functions ancestrally related to bacteria. Therefore, we screened the KeioE. coliknockout library for mutations that induce aC. elegansmitochondrial damage response gene. We identified 45E. colimutations that induce a theC. elegans hsp-6::gfpresponse gene. Surprisingly, four of theseE. colimutations that disrupt the import or removal of iron from the bacterial siderophore enterobactin were lethal in combination withC. elegansmutations that disrupt particular iron-sulfur proteins of the electron transport chain. Bacterial mutations that fail to synthesize enterobactin are not synthetic lethal with theseC. elegansmitochondrial mutants; it is the enterobactin-iron complex that is lethal in combination with theC. elegansmitochondrial mutations. Antioxidants suppress this inviability, suggesting that reactive oxygen species (ROS) are produced by the mutant mitochondria in combination with the bacterial enterobactin-iron complex.Significance StatementThe animal mitochondrion has a bacterial origin and continues to have a dialogue with the bacterial metabolisms of their microbiome. We identified 45E. coligene disruptions that induce aC. elegansmitochondrial damage response gene. Four of theseE. colimutations that disrupt the import or retrieval of iron from the siderophore enterobactin were synthetic lethal withC. elegansmitochondrial mutants. Antioxidants strongly suppressed the inviability ofC. elegansmitochondrial mutants grown on theE. colienterobactin siderophore utilization or import mutants. We hypothesize that reactive oxygen species are produced by C. elegans mitochondrial mutations and that this non-lethal ROS triggers ferric-chelated enterobactin to induce dramatically increased ROS, which leads to lethality.

Many neurons display characteristic patterns of synaptic connections that are under genetic control. The Caenorhabditis elegans DA cholinergic motor neurons form synaptic connections only on their dorsal axons. We explored the genetic pathways that specify this polarity by screening for gene inactivations and mutations that disrupt this normal polarity of a DA motorneuron. A RAB-3::GFP fusion protein that is normally localized to presynaptic terminals along the dorsal axon of the DA9 motorneuron was used to screen for gene inactivations that disrupt the DA9 motorneuron polarity. This screen identified heterochronic genes as major regulators of DA neuron presynaptic polarity. In many heterochronic mutants, presynapses of this cholinergic motoneuron are mislocalized to the dendrite at the ventral side: inactivation of the blmp-1 transcription factor gene, the lin-29/Zn finger transcription factor, lin-28/RNA binding protein, and the let-7miRNA gene all disrupt the presynaptic polarity of this DA cholinergic neuron. We also show that the dre-1/F box heterochronic gene functions early in development to control maintenance of polarity at later stages, and that a mutation in the let-7 heterochronic miRNA gene causes dendritic misplacement of RAB-3 presynaptic markers that colocalize with muscle postsynaptic terminals ectopically. We propose that heterochronic genes are components in the UNC-6/Netrin pathway of synaptic polarity of these neurons. These findings highlight the role of heterochronic genes in postmitotic neuronal patterning events.