Jamie R Konwerski et al. (2005) International Worm Meeting "A comparative approach to understanding P granule function in Caenorhabditis"

Jennifer A Schisa, Megan Senchuk, Jamie R Konwerski, David LaHaie, Emily Petty

[International Worm Meeting, 2005]

Proper germline formation is important for propagation of species. P granules, an organelle found only in the germline, may be important for germ cell identity and function in Caenorhabditis . Though the biochemical function of P granules is unknown, several lines of evidence suggest they may function in the regulation of germline mRNA. All proteins known to associate with P granules contain putative RNA-binding domains and several mRNAs are known which preferentially associate with P granules. In addition, P granules in immature germ cells of the adult are associated with nuclear pores; therefore, many of the newly transcribed RNAs in germ cells contact P granules as they are exported out of the nucleus. Thus, we and others hypothesize that P granules function to regulate the translation of maternal mRNAs. We are taking a comparative approach to better understand P granule function by determining whether selected P granule characteristics are conserved in C. briggsae and C. remanei. Preliminary data using TEM suggest that P granules are associated with clusters of nuclear pores in the immature germ cells of C. remanei. Thus it appears P granules in C. remanei interact with many of the newly transcribed RNAs as in C. elegans. Since this association appears to be conserved, it may be important to P granule function. We have also focused on understanding the significance of the association of RNA with P granules. In C. elegans, pos-1 RNA associates with P granules and is translationally regulated. In order to identify sequences that are important for pos-1 RNA to associate with P granules and/or be appropriately translationally regulated, we have cloned orthologs of pos-1 from C. briggsae and C. remanei. We have identified one novel amino acid domain that is highly conserved among the three species. We have also identified two regions in the 3 UTR that are highly conserved. In addition, we have shown that pos-1 RNA is expressed in the germline of C. briggsae and C. remanei and that regulation of pos-1 RNA translation is conserved. POS-1 is not expressed at high levels in C. briggsae until the 2-cell stage of embryogenesis. We conclude that pos-1 appears to be translationally regulated in C. briggsae as it is in C. elegans and speculate the conserved 3 UTR sequences may be involved. We plan to disrupt the conserved 3UTR sequences of pos-1 in C. elegans and determine any effects on the association of the RNA to P granules or proper translational regulation of the RNA.

Emily L Petty et al. (2005) International Worm Meeting "Analysis of protein aggregates in oocytes of aging C. elegans"

Emily L Petty, Jamie R Konwerski, Jennifer A Schisa

[International Worm Meeting, 2005]

As C. elegans hermaphrodites age, sperm become depleted, oogenesis arrests, and oocytes accumulate in the gonad arm. Certain proteins in arrested oocytes, such as MEX-3, undergo reorganization into large aggregates. Within hours of mating with a male, MEX-3 aggregates in oocytes dissociate and fertilization commences. The majority of arrested oocytes with MEX-3 aggregates result in viable embryos upon mating, suggesting that aggregates are not deleterious. Our data suggest that absence of normal sperm, as in fem-1, and fer-1, is sufficient but not necessary to stimulate MEX-3 aggregation; mutants with normal sperm but interrupted ovulation (mup-2) or improper oocyte development (emo-1) can form MEX-3 aggregates. Aggregates of MEX-3 protein have also been observed in the arrested oocytes of unmated C. remanei females. The components of aggregates in both C. elegans and C. remanei include nuclear pore proteins, putative RNA-binding domain proteins, as well as high levels of RNA. These components suggest that aggregates in oocytes have an RNA-related function. One attractive hypothesis is that the RNA-binding proteins in aggregates protect maternal mRNA from either degradation or precocious translation while the oocytes wait to be fertilized. To elucidate the function of aggregates in oocytes we are examining two classes of mutants for the ability to properly form and dissociate aggregates. MAP Kinase regulates the progression of oocytes through meiosis in an MSP-dependent fashion. When sperm are absent, MAPK is not activated by MSP and this correlates with the formation of aggregates in oocytes. Therefore, we are investigating several MAPK mutants, including kgb-1 (gift of K. Bennett), for defective MEX-3 aggregation. We are also examining chaperone or heat shock protein (hsp) mutants. The function of hsp's is to assist proteins in folding and unfolding and they have been implicated in regulating protein aggregate formation in neurodegenerative diseases. We are investigating the chaperone protein mutants ooc-3, ooc-5, daf-21, and crt-1 to determine if they have defective MEX-3 aggregation or dissociation. RNA interference is being used to knock down expression of the chaperone proteins HSP-1, SIP-1, and D2030 in a fem-1;MEX-3::GFP strain. This strain allows us to visualize MEX-3 in animals before and after mating to observe both aggregate formation and dissociation. By identifying a mutant that either cannot form or dissociate MEX-3 aggregates, we expect to learn whether aggregate formation is important for the viability of oocytes that are delayed in being fertilized.

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.

Birsoy B et al. (2010) Biology of the C. elegans Male, Madison, WI "Novel Left-Right Asymmetries of Male C.elegans"

Rothman J H, Choi S, Birsoy B, Downes J C, Bloss T A

[Biology of the C. elegans Male, Madison, WI, 2010]

While the mechanism that breaks left-right (L-R) anatomical asymmetry has been described in C. elegans, we have found that at least one additional mechanism must exist to generate L-R differences in the deployment of alternative cell death pathways in the male tail, male mating behavior and patterning of the male gonad. Upon recognizing a hermaphrodite, males initiate backward locomotion and continuously scan along the hermaphrodite's body. When their tails reach either the head or tail of the hermaphrodite, males turn to maintain contact and then continue backward locomotion on the opposite side of the hermaphrodite. We found that this turning behavior shows a distinct right-handed bias: when individual males were allowed to mate with lin-2(e1309) vulvaless hermaphrodites, >70% of the turns when made over the hermaphrodites' body (away from the agar surface) and >55% of turns when made under the hermaphrodites' body (toward the agar surface) are right-handed. To determine whether this bias in turning direction correlated with L-R asymmetry in the male mating structure, which results from stochastic EGL-1-dependent loss of sensory rays, we examined the turning bias in egl-1(n1084n3082) mutants. Wild-type males lack rays more frequently on the right and egl-1 mutant males have no ray loss but still display the right-hand turning bias. To assess whether this L-R behavioral bias is dependent on anatomical asymmetry, we analyzed gpa-16(it143) mutants in which the L-R asymmetry of the internal organs is reversed as a result of the reversal in the early embryonic symmetry break. Males with reversed anatomical asymmetry showed a virtually identical right hand bias in turning, demonstrating that an asymmetry-determining system that is independent of the previously described L-R symmetry breaking event must control this behavior. During the characterization of the gonad reversals of males, we also observed a previously unreported temperature sensitive reversal of L-R asymmetry of the male gonads in N2 (Bristol) and a Hawaiian wild isolate. We will describe our recent findings on these novel L-R asymmetries of the C.elegans male anatomy and behavior.

L C Bowen et al. (2002) European Worm Meeting "The C. elegans cyclic AMP-dependent protein kinase (PK-A) catalytic subunit gene (kin-1)"

L C Bowen, M Tabish, M J Fisher, H H Rees, R A Clegg

[European Worm Meeting, 2002]

PK-A is one of the major multifunctional ser/thr protein kinases found in eukaryotic organisms. The holoenzyme is comprised of two catalytic (C) and two regulatory (R) subunits. Both R and C subunits occur in multiple isoforms encoded by distinct genes. In many organisms, the genes that encode the C subunits also show alternative splicing behaviour. This generates an even broader array of C subunit heterogeneity. The diversity of C subunit polypeptides may enable this protein kinase to selectively target substrate polypeptides for phosphorylation.

Martyna W Pastok et al. (2007) International Worm Meeting "Studies on the C. elegans Protein Kinase A (PK-A) Catalytic and Regulatory Subunit Genes."

Huw H Rees, Martyna W Pastok, Michael J Fisher, Caroline Dart, Patrica A Murray

[International Worm Meeting, 2007]

The cyclic AMP-dependent protein kinase (protein kinase A; PK-A) holoenzyme consists of two catalytic (C) subunits bound to two regulatory (R) subunits, forming an inactive heterotetramer (R2C2). Binding of cyclic AMP to the R subunits results in the dissociation of catalytically active monomeric C subunits. These C subunits catalyse the protein phosphorylation events underlying the diverse range of cellular processes triggered by alterations in cyclic AMP concentration. In C. elegans, C and R subunits are encoded by the kin-1 and kin-2 genes respectively. We have undertaken both bioinformatic and molecular studies on the structure and expression of these genes, in C. elegans. We have also undertaken comparative bioinformatic studies on orthologus genes, and gene products, in C. briggsae and C. remanei. These studies have highlighted the potential significance of alternative splicing processes, as a means of generating multiple isoforms of both the C and R subunits in C. elegans. The possible significance of such multiple isoforms is discussed in the context of the subcellular distribution of PK-A and in relation to the targeting of specific phosphorylation events, in responses to changes in intracellular cyclic AMP concentration.