Rodrigues, Pedro R. et al. (2019) International Worm Meeting "A Draft Metabolome for C. elegans."

Fox, Bennett, Rodrigues, Pedro R., Schroeder, Frank C., Helf, Maximilian J.

[International Worm Meeting, 2019]

Progress in ascribing phenotypic characteristics to genetic identity has moved at a fast pace since the annotation of genomes. However, a comprehensive annotation of metabolomes has moved at a woefully slow pace. Recent advances made in high resolution accurate mass spectrometry (HRAM) now provide the opportunity for small molecule research to adopt that same discovery rate that has been seen for genomes. While traditional metabolomic analysis measures dozens or, at best, hundreds of metabolites, common HRAM analysis detects tens of thousands of features with largely unknown identities. In this work, we are pursuing a comprehensive annotation of the entire C. elegans metabolome. By using Furrier Transform Mass Spectrometry (FTMS), we have developed the methods to systematically annotate molecular formulae and structural characteristics of the C. elegans metabolome across all developmental stages. We have evidence for the presence of more than 10,000 different compounds, and thus genome and metabolome of C. elegans may be of similar numerical sizes. Among several thousand novel molecules we have identified or partially characterized, some represent compound classes that have not previously been described in C. elegans but are known from other organisms, and others are entirely novel. Many of the detected compounds suggest the presence of yet unexplored biosynthetic networks. For example, we identified over 80 N-acyl amino acids, a class of endogenous molecules not previously described in worms, whereas in mammals these compounds are well known for their anti-inflammatory and analgesic functions. Biosynthesis and function of these small molecules and many other new compound families in C. elegans we will present on is currently unknown and remains a focus of this work. In parallel, we are developing a timeline for the production of all metabolites throughout larval stages and adulthood. We believe that our comprehensive structural and functional annotation of the C. elegans metabolome will become an invaluable resource to the worm community and beyond, allowing researchers to connect metabolomic information with genetic tractability. Lastly, the first draft annotation of the C. elegans metabolome may become an example for other metazoan model systems, akin to the sequencing of the C. elegans genome over 20 years ago.

Rodrigues, Pedro R. et al. (2011) International Worm Meeting "Proteomic Analysis of Age-dependent Changes in Protein Solubility Identifies Genes that Modulate Lifespan."

Czerwieniec, Gregg, Gaman, Emily A., Lithgow, Gordon J., Hughes, Robert E., Rodrigues, Pedro R., Evani, Uday S., Peters, Theodore, Lucanic, Mark, Alavez, Silvestre, Mooney, Sean D., Gibson, Bradford W.

[International Worm Meeting, 2011]

While it is generally recognized that misfolding of specific proteins can cause late-onset disease states, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry-based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging. SDS Insoluble proteins purified from young and aged C. elegans were chemically labelled by isobaric tagging for relative and absolute quantitation (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS-insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS-insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Transgenic nematodes expressing three proteins identified in the insoluble fraction, DAF-21, RPS-0 and EFT-3 fused to Green Fluorescent Protein (GFP) showed the formation of visible aggregates by fluorescence microscopy. In the case of RPS-0 and EFT-3, these aggregates appeared immobile as measured by Fluorescence Recovery after Photobleaching (FRAP). Expression of genes encoding insoluble proteins observed in aged nematodes was knocked-down in using RNAi and effects on lifespan were measured. Forty of 100 genes tested were shown to extend lifespan after RNAi. These data indicate that genes encoding proteins that become insoluble with age are modifiers of lifespan. These data also demonstrate that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that aggregation of a diverse range of proteins may be a general feature of aging.

Rodrigues, Pedro R. et al. (2013) International Worm Meeting "Isolation of N-acylethanolamine resistant mutants using a forward genetic screen for resistance to synthetic cannabinoids."

Mishra, Jitendra, Bannister, Thomas, Rodrigues, Pedro R., Harrison, Neale, Gill, Matthew S.

[International Worm Meeting, 2013]

In mammals, endocannabinoids (EC) and synthetic cannabinoids (CB) mediate their effects through CB receptors. We have previously demonstrated that eicosapentaenoyl ethanolamine (EPEA), an N-acylethanolamine (NAE) which is structurally most similar to the mammalian EC anandamide, prevents dauer formation in dauer constitutive mutants. Despite the biological activity of endocannabinoids in worms, there is abundant evidence that they do not possess orthologs of the canonical CB receptors. We have therefore performed a forward genetic screen to identify components of NAE / cannabinoid signaling pathways in the worm. EPEA prevents dauer formation in daf-c mutants at semi-permissive temperatures, but it is not fully penetrant, thus limiting its utility in a forward genetic screen. However, we have found that the CB receptor inverse agonist AM251 rescues dauer formation in daf-2(e1368) mutants at the fully restrictive temperature (25 deg C) and acts synergistically with EPEA. Conversely, the CB1 receptor agonist O-2545 induces dauer formation in daf-2(e1368) at semi-permissive temperatures and competes with AM251 and EPEA at the restrictive temperature, suggesting shared molecular targets. While performing structure activity studies, we identified an AM251 derivative, SR10589, which not only prevents dauer entry at lower doses but also causes a fully penetrant L1 arrest phenotype at higher concentrations. Importantly, the L1 arrest is competed by O-2545, and SR10589 at low doses acts synergistically with AM251 and EPEA to prevent dauer formation. We have conducted an F1 forward mutagenesis screen and isolated mutations that suppress the L1 arrest of SR10589 treated animals. The mutants have different degrees of resistance to SR10589, and, interestingly, all the mutants characterized so far also display resistance to AM251 and EPEA, indicating that these molecules act through common factors. We are currently cloning the mutations and believe that their identification will help understand the non-canonical actions of N-acylethanolamines and cannabinoids in worms and higher organisms.

Pereirinha, Joana et al. (2021) International Worm Meeting "Investigation of the role of the protein complex PETISCO in C. elegans embryonic viability"

Cordeiro Rodrigues, Ricardo, Ketting, Rene, Pereirinha, Joana

[International Worm Meeting, 2021]

The PIWI/piRNA pathway is a highly conserved small RNA pathway. Central to the pathway is an RNA silencing complex made of an Argonaute protein of the PIWI clade and PIWI-interacting RNAs (piRNAs), which target transposons in animal germlines. In C. elegans, piRNAs also target endogenous protein-coding genes, making the PIWI/piRNA pathway an important regulator of gene expression. C. elegans piRNAs are referred to as 21U RNAs since they are 21 nucleotides long and have a 5' bias for uridine monophosphate. We recently identified a protein complex driving 21U RNA biogenesis, which we named PETISCO (Cordeiro Rodrigues et al., 2019). However, we found that PETISCO can have different functions depending on whether it interacts with the proteins PID-1 (PiRNA Induced silencing Defective) or TOST-1 (Twenty-One U antagoniST). PETISCO:PID-1 complex participates in the 21U RNA biogenesis by interaction and stabilization of the 21U RNA precursors. While being important for gene regulation, the loss of PID-1 is not embryonic lethal. In contrast, PETISCO:TOST-1 is not involved in 21U RNA biogenesis but is nevertheless essential for embryonic development of the subsequent generation (Cordeiro Rodrigues et al., 2019; Zeng et al., 2019). The molecular function of this complex as well as the observed lethality effect is not yet understood. Based on preliminary data, we hypothesize that the PETISCO:TOST-1 complex regulates replication-dependent histones mRNA expression. In this study we want to characterize the PETISCO::TOST-1 complex and dissect its role in embryogenesis. This study would highlight the role of 21U RNA pathway factors in gene expression and C. elegans embryonic development.

Placentino, M. et al. (2017) International Worm Meeting "Dissecting 21U RNA biogenesis and activity in the germline and the early embryo."

Placentino, M., Butter, F., Rodrigues, R., Ketting, R., Dietz, S., Domingues, A.

[International Worm Meeting, 2017]

Many different small RNA pathways exist in C. elegans. Some of these affect the expression of 'regular' genes, while others are more geared towards the silencing of repetitive elements in the genome. In general, they have a large impact on fertility. Most of these pathways, with the notable exception of miRNAs, act in a two-step manner. First, so-called primary Argonaute proteins, such as PRG-1 that binds to 21U RNAs, recognize a target transcript based on homology between small RNA and target RNA. This is followed by a second step in which an RNA-dependent RNA polymerase (RdRP) enzyme is recruited to the target, resulting in 22G RNA production. As the target-RNA is used as template by the RdRP, 22G RNAs are anti-sense with respect to the target of the primary small RNA. 22G RNAs are bound by secondary Argonaute proteins that induce silencing. In case of 21U RNAs, the secondary phase can become self-sustainable and independent of PRG-1, in which case the target is said to be under control of RNAe (RNA induced epigenetic silencing). The molecular differences between a PRG-1 target that is under control of RNAe and one that is not are presently completely unclear. We are further dissecting both the biogenesis of 21U RNAs as well as how and when 21U RNAs induce RNAe. Based on IP-mass spec experiments on PID-1, a factor we previously showed to be essential for 21U RNA production, we found evidence for a complex that drives the production of 21U RNA. The identity of the PID-1 interactors has implications for 21U RNA precursor recognition and the stabilization and/or transfer of processing intermediates. We established that 21U RNA-mediated silencing most likely takes place during early embryogenesis, since maternally provided 21U RNAs are both required and sufficient to induce an RNAe-like state. We then identified a novel protein, PID-2, that is required for PRG-1-mediated silencing activity, but is dispensable for RNAe. In pid-2 mutants, PRG-1 is normally loaded with 21U RNAs. Therefore, PID-2 acts downstream of PRG-1 loading, but upstream of the secondary RNAe pathway that is PRG-1 independent. Consistent with PRG-1 silencing activity in the early embryo, zygotic PID-2 is sufficient to induce silencing. We are currently further dissecting PID-2 activity, but it is already clear from our analyses that PID-2 defines an as yet poorly understood step in between target recognition by PRG-1 and establishment of a stably inherited RNAe-state.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.

Hicks, Tara et al. (2021) International Worm Meeting "R-loop-induced irreparable DNA damage in C. elegans meiosis"

Hicks, Tara, Koury, Emily, Williams, Cameron, Smolikove, Sarit

[International Worm Meeting, 2021]

Most DNA-RNA hybrids are formed naturally during transcription and are composed of a nascent RNA strand hybridized to DNA as part of R-loops. The accumulation of these structures in S-phase can result in replication-transcription conflict, an outcome which can lead to the formation of double strand breaks (DSBs). While R-loops' role in mitotically dividing cells has been characterized, there are only a handful of studies describing the effect of R-loops in meiosis and these studies present a complex picture of the outcome of R-loop formation on germ cells. Here we show that DSBs formed by R-loops trigger an altered cellular response to DNA damage. RNase H is an enzyme responsible for degradation of the RNA strand in DNA-RNA hybrids and plays an essential role in preventing this outcome and its deleterious consequences. Using null mutants for the two Caenorhabditis elegans genes encoding for RNase H1 and RNase H2 (hereby rnh mutants), our studies explore the effects of replication stress-induced DNA-RNA hybrid accumulation on meiosis. As expected, rnh mutants exhibit an increase in R-loop formation. Consequently, an elevation of DSBs in germline nuclei is evidenced by the accumulation of RAD-51 foci. Despite no repair mechanism abrogation, rnh mutants fail to repair all DSBs generated, leading to a fragmentation of chromosomes in diakinesis oocytes. By combining our double mutant with a spo-11 null mutation, we show that although replicative defects are the main contributor to the phenotype, R-loops formed in meiosis are likely contributors as well. We present evidence that while rnh mutants accumulate DNA-RNA hybrids and subsequent DSBs may signal a degree of checkpoint activation in mitosis, some damaged nuclei prevail past the checkpoint, enter into meiosis, and remain unrepaired throughout. Moreover, we find no evidence of an increase in apoptosis, which indicates that DNA damage generated by R-loops remain undetected by an apoptotic checkpoint. This data altogether points to DSBs initiated by R-loops representing an irreparable type of DNA damage that evades cellular machineries designed for damage recognition.