Olson, Andrew et al. (2017) International Worm Meeting "The Role of Post-Translational Modifications in the Regulation of Serotonin Signaling."

Koelle, Michael, Kumar, Santosh, Olson, Andrew

[International Worm Meeting, 2017]

C. elegans uses serotonin as a neurotransmitter to slow locomotion, and we have used this model system to discover that post-translational modifications appear to regulate serotonin signaling. Through large-scale genetic screens for mutants that fail to paralyze in response to exogenous serotonin, we found that C. elegans mutants for either of two subunits of the ELPC Elongator Protein Complex are defective for serotonin signaling. Conversely, transgenic animals overexpressing ELPC are hypersensitive to the effects of exogenous serotonin. ELPC is conserved from C. elegans to humans and functions as a cytoplasmic lysine acetylase to reversibly modify other proteins. This is the first time that ELPC or lysine acetylation has been implicated in regulating serotonin signaling. We used two-dimensional gel electrophoresis to show that in C. elegans lysates, Gao, a neural G protein encoded by the goa-1 gene through which serotonin signals to slow locomotion, exists as a complex series of species of differing charge. Acetylation eliminates the positive charge on a lysine side chain, and differential acetylation on several Lys could produce the complex pattern of Gao species we see. Our preliminary results suggest that the series of differentially charged Gao species in wild-type lysates shifts to a less complex and more positively charged set of Gao species in lysates of ELPC mutants, as would be predicted if Gao is acetylated by ELPC. We isolated Gao from both mouse brain and C. elegans lysates and analyzed the purified proteins for post-translational modifications using mass spectrometry. In both species, Gao is acetylated on several conserved Lys residues near the N-terminus. We note that the signaling defects in ELPC mutants are much more restricted than those of Gao null mutants. Both ELPC and Gao mutants are defective for response to exogenous serotonin, but Gao null mutants have additional defects not seen in ELPC mutants, including defects in response to exogenous dopamine as well as in many behavioral assays. Thus we hypothesize that ELPC may reversibly acetylate Gao to specifically regulate its ability to be activated by serotonin receptors, while not strongly affecting its ability mediate signaling by other receptors. We are using CRISPR-Cas9 technology to mutate the acetylated Lys residues of Gao to the similar but non-acetylatable amino acid arginine to determine if acetylation at specific positions is responsible for regulating serotonin signaling.

Hofler, Catherine et al. (2011) International Worm Meeting "AGS-3 alters C. elegans behavior after food deprivation via RIC-8 activation of the neural G protein Gao."

Hofler, Catherine, Koelle, Michael

[International Worm Meeting, 2011]

Like all animals, C. elegans modifies a number of behaviors upon food deprivation in order to seek food. These behavioral changes depend on neurotransmitters and peptides that typically signal through the major neural G protein Gao, suggesting that signaling through this G protein ultimately mediates responses to food deprivation. Proteins containing the G Protein Regulator (GPR) domain bind to Gao in vitro, but the biological functions of GPR proteins in neurons and the mechanism by which they might affect G protein signaling has remained unclear. We found that the conserved GPR domain protein AGS-3 activates Gao signaling in vivo to allow C. elegans to alter several behaviors after food deprivation. These behaviors include octanol avoidance, area-restricted search, and egg laying. AGS-3 protein in whole worm lysates undergoes a progressive change in its biochemical fractionation pattern upon food deprivation, demonstrating that food deprivation changes the physical state of the protein. Cell-specific rescue and cell-specific inactivation experiments show that AGS-3 and Gao act together in the ASH chemosensory neurons to allow food deprivation to modify response to octanol. Genetic epistasis experiments show that AGS-3 activation of Gao in the ASHs requires the guanine nucleotide exchange factor RIC-8. Conversely, RIC-8 function in the ASHs also requires AGS-3. Using purified recombinant proteins, we characterized interactions of the proteins consistent with the genetic epistasis results. AGS-3 forms a complex with the the inactive, GDP-bound form of Gao, and RIC-8 can act on this complex to promote nucleotide exchange and dissociation of all the proteins, generating active Gao-GTP. These results identify a biological role for AGS-3 in response to food deprivation and indicate the mechanism for its activation of Gao signaling in vivo.

Heather A Hess et al. (2004) East Coast Worm Meeting "RGS-7 Completes a Receptor-independent Heterotrimeric G Protein Signaling Cycle to Regulate Mitotic Spindle Positioning in C. elegans"

Heather A Hess, Michael R Koelle

[East Coast Worm Meeting, 2004]

Heterotrimeric G proteins promote astral microtubule forces on centrosomes to position mitotic spindles properly during asymmetric cell division in C. elegans embryos. While all previously studied G protein functions require activation by seven-transmembrane receptors, this function appears to be receptor-independent, and the active form of the G proteins remains unclear. We obtained mutants for all 13 C. elegans regulators of G protein signaling and found that one, RGS-7, decreases the speed and magnitude of centrosome movements. The effects of RGS-7 require two redundant Gao-related G proteins and their non-receptor activators. Using recombinant proteins, we found that the non-receptor activator RIC-8 stimulates GTP binding by Gao, and the RGS domain of RGS-7 stimulates GTP hydrolysis by Gao. These results demonstrate that the active species in the receptor-independent G protein cycle is GaoGTP, and that RGS-7 completes the cycle by driving Gao to its inactive, GDP-bound form.

Huitema K et al. (2004) EMBO J "Identification of a family of animal sphingomyelin synthases."

van den Dikkenberg J, Holthuis JC, Brouwers JF, Huitema K

[EMBO J, 2004]

Sphingomyelin (SM) is a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively. SM synthesis occurs in the lumen of the Golgi as well as on the cell surface. As no gene for SM synthase has been cloned so far, it is unclear whether different enzymes are present at these locations. Using a functional cloning strategy in yeast, we identified a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase. Strikingly, human, mouse and Caenorhabditis elegans genomes each contain at least two different SM synthase (SMS) genes. Whereas human SMS1 is localised to the Golgi, SMS2 resides primarily at the plasma membrane. Collectively, these findings open up important new avenues for studying sphingolipid function in animals.

Barbee SA et al. (2006) Dev Biol "The Sm proteins regulate germ cell specification during early C. elegans embryogenesis."

Barbee SA, Evans TC

[Dev Biol, 2006]

Sm and Sm-like proteins are core components of the splicesome but have other functions distinct from pre-mRNA processing. Here, we show that Sm proteins also regulate germ cell specification during early C. elegans embryogenesis. SmE and SmG were required to maintain transcriptional quiescence in embryonic germ cell precursors. In addition, depletion of SmE inhibited expression of the germ lineage-specific proteins PIE-1, GLD-1, and NOS-2, but did not affect maintenance of several maternal mRNAs. PIE-1 had previously been shown to activate transcriptional silencing and NOS-2 expression. We found that PIE-1 also promotes GLD-1 expression by a process that is independent of transcriptional silencing. Thus, Sm proteins could control transcriptional silencing and maternal protein expression by regulating PIE-1. However, loss of SmE function also caused defects in P granule localization and premature division in early germline blastomeres, processes that are independent of PIE-1 function. Therefore, the Sm proteins control multiple aspects of germ cell precursor development. Because depletion of several other core splicing factors did not affect these events, these Sm functions are likely distinct from pre-mRNA splicing. Sm family proteins assemble into ribonucleoprotein complexes (RNPs) that control RNA activities. We suggest that novel Sm RNPs directly or indirectly influence posttranscriptional control of maternal mRNAs to promote germ cell specification in the early C. elegans embryo.

Peg MacMorris et al. (2007) International Worm Meeting "A novel family of C. elegans snRNPs contains proteins associated with trans-splicing."

Erika Lasda, Alison Larsen, Brian Kraemer, Tom Blumenthal, Madhur Kumar, Peg MacMorris

[International Worm Meeting, 2007]

In approximately 70% of C. elegans pre-mRNAs, the RNA sequence between the 5 cap and the first 3 splice site is replaced by trans-splicing a short spliced leader (SL) from the Sm snRNP, SL1. C. elegans also utilizes a similar Sm snRNP, SL2, to trans-splice at sites between genes in polycistronic pre-mRNAs from operons. How do SL1 and SL2 snRNPs function in different contexts? Here we show that the SL1 snRNP contains a complex of SL75p and SL21p, homologs of novel proteins previously reported in the Ascaris SL snRNP. Interestingly, the SL2 snRNP does not contain either of these proteins. However, SL75p and SL26p, a paralog of SL21p, are components of another Sm snRNP that contains a novel snRNA species, Sm Y. Knockdown of SL75p is lethal. However, knockdown of either SL21p or SL26p alone leads to cold-sensitive sterility, whereas knockdown of both SL21p and SL26p is lethal. This suggests that these two proteins have overlapping functions even though they are associated with different classes of snRNP. These phenotypic relationships, along with the association of SL26p with SL75p imply that, like the SL1 RNA/Sm/SL75p/SL21p complex, the Sm Y/Sm/SL75p/SL26p complex is associated with trans-splicing. We hypothesize that the Sm Y snRNP is somehow associated with SL2-specific trans-splicing. This idea is supported by the fact that the sequences of SL2 RNA and Sm Y allow base pairing of the two snRNPs through their second stem/loops. We are currently testing for this base pairing interaction using psoralen/UV crosslinking.

Macmorris M et al. (2007) RNA "A novel family of C. elegans snRNPs contains proteins associated with trans-splicing."

Kumar M, MacMorris M, Lasda E, Kraemer B, Blumenthal T, Larsen A

[RNA, 2007]

In many Caenorhabditis elegans pre-mRNAs, the RNA sequence between the 5'' cap and the first 3'' splice site is replaced by trans-splicing a short spliced leader (SL) from the Sm snRNP, SL1. C. elegans also utilizes a similar Sm snRNP, SL2, to trans-splice at sites between genes in polycistronic pre-mRNAs from operons. How do SL1 and SL2 snRNPs function in different contexts? Here we show that the SL1 snRNP contains a complex of SL75p and SL21p, which are homologs of novel proteins previously reported in the Ascaris SL snRNP. Interestingly, we show that the SL2 snRNP does not contain these proteins. However, SL75p and SL26p, a paralog of SL21p, are components of another Sm snRNP that contains a novel snRNA species, Sm Y. Knockdown of SL75p is lethal. However, knockdown of either SL21p or SL26p alone leads to cold-sensitive sterility, whereas knockdown of both SL21p and SL26p is lethal. This suggests that these two proteins have overlapping functions even though they are associated with different classes of snRNP. These phenotypic relationships, along with the association of SL26p with SL75p, imply that, like the SL1 RNA/Sm/SL75p/SL21p complex, the Sm Y/Sm/SL75p/SL26p complex is associated with trans-splicing.

Scott A Barbee et al. (2001) International C. elegans Meeting "The splicesomal Sm proteins play a role in P granule localization during early embryogenesis."

Scott A Barbee, Thomas C Evans, Alex L Lublin

[International C. elegans Meeting, 2001]

In early C. elegans embryos, development is initiated by asymmetric cell division, which leads to the precise localization cell fate regulators to specific cells. One feature of early asymmetry is the localization of cytoplasmic RNP particles, the P granules, to the germ cell precursors at each of several polarized cell divisions. The composition and function of P-granules is not well understood. Further, the mechanisms that control the localization of P-granules and other factors in the embryo are poorly understood. We used RNA interference (RNAi) to screen a cDNA library for new genes involved in early polarity. Surprisingly, one of the genes identified is the ortholog of human SmE. SmE is one of several Sm proteins that form a multisubunit complex required for snRNP assembly, nuclear import, and mRNA splicing. RNAi of Sm subunits in C. elegans caused mislocalization of P granules to somatic sisters of germ cells after the 4-8 cell stages in the early embryo. In addition, Sm RNAi disrupted the subcellular distribution of PGL-1 (a P-granule component) in both mature germ cells and in germ cell precursors after the 8-cell stage. P-granules in wild type germ cells are primarily attached to the nucleus, but in Sm (RNAi) germ cells and embryos, the nuclear attachment of many P-granules is lost. At lower penetrance, GLP-1 protein was inappropriately expressed in posterior cells following Sm RNAi. By contrast, RNAi of the core splicing factors U1 70K and U2AF65, or of RNA polymerase II, had no effect on P granule segregation, subcellular distribution, or GLP-1 asymmetry, although all caused severe embryonic defects. These data suggest that the P-granule and GLP-1 asymmetry phenotypes from Sm RNAi are not likely to result from a general defect in splicing. Therefore, Sm proteins may have a role in P-granule localization that is independent of splicing. Interestingly, antibodies against the Sm complex stain P granules at all stages of development. Sm RNAi attenuates this staining. Therefore, Sm proteins may be P granule components that affect the localization of P granules by controlling their integrity or nuclear attachment in germ cells and their precursors. The Sm proteins are also found in the nucleus of most cells. However, nuclear localization of the Sm’s is dynamically regulated during oogenesis and germ cell precursor formation, suggesting that regulation of the Sm complex or snRNPs may be important for germ cell development.

Zhang W et al. (2016) Cell Mol Neurobiol "Shengmai Formula Ameliorates Pathological Characteristics in AD C.elegans."

Zhang Z, Zhu H, Fei D, Li H, Ren H, Wang D, Wang X, Zhang W, Zhi D

[Cell Mol Neurobiol, 2016]

Shengmai (SM) formula, a classical traditional Chinese medicine formula, is composed of Panax ginseng (Pg), Ophiopogon japonicus (Oj), and Schisandra Chinesis (Sc). SM has been clinically used to treat heart failure and ischemic heart disease. Although SM formula has been reported to be potential for fighting against Alzheimer's disease (AD) by previous works, there are many gaps in our knowledge on its usage in AD treatment on an organism level and will then need to be further clarified. In this study, transgenic Caenorhabditis elegans expressing human A1-42 are used to evaluate SM formula efficacy to treat AD phenotype and to investigate its underlying mechanism. The results showed that SM formula ameliorated AD pathological characteristics of paralysis behavior and chemotaxis defect in transgenic C.elegans. With SM treatment, the number of A deposits decreased, the levels of gene expressions of hsp16-2, hsp16-41, ace-1, ace-2, and TNFA1P1 homolog genes were down-regulated. Our results also showed that Oj exhibited more stronger effect on delaying paralysis in worms than Pg and Sc did, and synergistic action was observed between Pg and Oj, and Sc further enhanced the activity of Pg/Oj combination on delaying paralysis behavior. Further, SM with herbs of Pg, Oj, and Sc at a dose proportion of 9:9:6 exhibited superior therapeutic efficacy in comparison with herbs at other dose proportions. After SM formula extracted by ethanol, it delayed AD symptoms on a wider dose from 0.2 to 10.0mg/mL with no toxic effect. These results provided more evidence for SM formula being potential to be used to treat AD.

Henderson ST et al. (1994) Worm Breeder's Gazette "The lag-2 Gene Encodes a Protein With Homology to Drosophila Delta and is Expressed in the Distal Tip Cell"

Gao DL, Henderson ST, Kimble JE, Lambie EJ

[Worm Breeder's Gazette, 1994]

The lag-2 gene encodes a protein with homology to Drosophila Delta and is expressed in the distal tip cell. Sam Henderson, Dali Gao, Eric Lambie#, and Judith Kimble, University of Wisconsin, Madison WI 53706, Dartmouth College, Hanover, NH 03755