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[
Worm Breeder's Gazette,
1995]
ELAV is a Drosophila protein that has been linked genetically to the differentiation of neuroblasts to neurons and is also responsible for the maintenance of terminally differentiated neurons. ELAV like proteins contain three RNA Recognition Motifs (RRMs), the first two RRMs are usually found at the N terminus of the protein separated by one or two amino acids. The third RRM is separated from the first two by a variable hinge region that appears to be alternatively spliced with the presence of miniexons that may influence the function of the protein. Several labs have successfully cloned homologs of the ELAV protein from other organisms such as rat, humans, and Xenopus. The human counterparts of ELAV have been shown to bind to AU rich stretches in the 3' untranslated regions of certain mRNAs, such as c-myc, c-fos, and gm-csf. Analysis of the Xenopus clones have shown that three of the ELAV homologs are expressed sequentially during the development of the frog embryo. While screening libraries for a C. elegans counterpart of ELAV, we found a cDNA that encoded a putative ORF encoding the first ELAV homolog. This clone corresponded to a predicted polypeptide on chromosome II sequenced by the genome sequencing project. Our screening has produced a full length clone corresponding to the predicted nucleotide sequence of the putative ORF. A western was performed using anti Human ELAV-Like Neuronal protein 1 (HEL-N1) rabbit polyclonal antisera against a protein extract of a mixed population of N2 worms and revealed a band, of the predicted size of the CEL-1 protein. Among other things we intend to determine the developmental regulation of this protein, attempt to rescue preexisting mutants, generate mutants if none now exist, determine the localization of the protein, elucidate the in vivo mRNA targets of the protein, and use the genetics of C. elegans to uncover the mechanism of action of this family of proteins.
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[
International C. elegans Meeting,
1995]
ELAV is a Drosophila protein that has been linked genetically to the differentiation of neuroblasts to neurons and is also responsible for the maintenance of terminally differentiated neurons. It has been shown that ELAV like proteins bind to the 3' UTR of selected transcription factors and cytokines. It is hypothesized that the expression of the ELAV family of proteins help take proliferating cells out of the cell cycle and enhance differentiation. I am using both biochemical approaches and genetic analysis to study the mechanism and function of the ELAV family of proteins in C. elegans. Using antibodies to an ELAV like protein, I have detected a protein in C. elegans mixed-stage extracts that migrates at 45 kDa on an SDS-PAGE gel. By screening of lambda zap libraries, I have obtained clones of Cel-1(C. elegans ELAV Like protein 1) that correspond to a putative ORF found by the genomic sequencing project, and appears to correspond to the 45kDa protein detected by immunoblotting. Given the striking homology among the vertebrate and invertebrate forms of the ELAV family within the RRM regions, it is clear that these are important cellular RNA-binding proteins that likely target homologous, key mRNAs regulated in growth and differentiation from worms to humans. I am in the process of expressing the Cel-1 clone in E. coli to determine if the protein is cross reactive to the Hel-N1 antibodies, to perform in vitro RNA binding experiments, make rabbit polyclonal sera, and to utilize random RNA selection. I am also attempting to determine if the Cel-1 protein is developmentally regulated, if there are pre existing mutants, where the protein is localized, and am collaborating with Dr. Plasterk, to isolate Tc1 insertions in the Cel -1 gene for genetic analysis.
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[
International C. elegans Meeting,
1997]
ELAV is a Drosophila protein that has been linked genetically to the differentiation of neuroblasts to neurons and is also responsible for the maintenance of terminally differentiated neurons. It has been shown that ELAV like proteins bind to the 3' UTR of selected transcription factors and cytokines. It is hypothesized that the expression of the ELAV family of proteins help take proliferating cells out of the cell cycle and enhance differentiation. I am using both biochemical approaches and genetic analysis to study the mechanism and function of the ELAV family of proteins in C. elegans. Using antibodies to human ELAV like protein, I have detected a protein in C. elegans mixed-stage extracts that migrates at 50 kDa on an SDS-PAGE gel. By screening of lambda zap libraries, I have obtained clones of Cel-1(C. elegans ELAV Like protein 1) that correspond to a putative ORF found by the genomic sequencing project, and appears to correspond to the 50 kDa protein detected by immunoblotting. Given the striking homology among the vertebrate and invertebrate forms of the ELAV family within the RRM regions, it is clear that these are important cellular RNA-binding proteins that likely target a subset of key mRNAs regulated in growth and differentiation from worms to humans. I have expressed the Cel-1 protein in E. coli and determined the protein is cross reactive to the Hel-N1 antibodies. This protein is currently being used to perform in vitro RNA binding experiments to identify its target mRNAs and as an antigen in the production of rabbit polyclonal sera. Further, I have attempted to determine if there are pre existing mutants by using microinjection techniques with cosmids and antisense RNA. Preliminary results from both the DNA rescue and the RNA antisense experiments have suggested that the preexisting mutants located in the region do not correspond to the phenotype that I have observed. Due to these observations I have begun a Tc1 deletion screen a worm line containing a Tc1 insertion isolated Dr. Plasterk's laboratory to isolate a mutant in the Cel-1 gene.
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[
East Coast Worm Meeting,
1996]
We have obtained a cDNA clone encoding a C. elegans RNA-binding protein, Cel-1, that is 67% identical to proteins ELAV and Hel-N1, both of which are implicated in neuronal differentiation. ELAV is a Drosophila protein that is essential genetically for the differentiation of neuroblasts to neurons and is also required for the maintenance of terminally differentiated neurons. Our laboratory has studied Hel-N1 (human elav like neuronal protein-1) and other mammalian counterparts of ELAV which are localized to certain neurons of the CNS. We have shown that Hel-N1 binds to the 3' UTR of characteristically unstable mRNAs encoding proto-oncoproteins and cytokines, all of which are implicated in growth regulation. We have hypothesized that the expression of Hel-N1, Cel-1 and other members of this family help take proliferating cells out of the cell cycle and enhance differentiation by altering protein expression levels. Recently, we have obtained a worm line with an insertion of the transposable element TC1 into an intron of the Cel-1 gene, and are deriving gene knockouts by excision of the element. This should allow genetic characterization of Cel-1 mutants and allow us to identify a phenotype. We are also examining known mutants of C. elegans which map in the chromosomal region near Cel-1 as candidate phenotypes. In addition, we have used biochemical approaches to examine the mechanism and function of Cel-1. Using anti-Hel-N1 antibodies, we have detected a Cel-1 band that appears to migrate at 50 kDa by immunoblotting of mixed stage extracts of C. elegans. By microinjection of cDNA constructs expressing b-galactosidase, we have found that Cel-1 is developmentally expressed in eggs and in larval stage 1 embryos, a time at which the nervous system is differentiating. Given the striking sequence homology among the vertebrate and invertebrate forms of the ELAV family, it is clear that these are important cellular RNA-binding proteins likely to target key mRNAs regulated in growth and differentiation.
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[
Worm Breeder's Gazette,
1996]
We have recently cloned a C. elegans gene, we call cevh, that expresses an RNA-binding protein of unknown function. So far we have a full-length cDNA clone and also a cosmid that contains the complete gene. The cevh gene physically maps near the end of the left arm of chromosome I, however we have been unable to identify any existing mutants thus far. The CEVH protein is about 65 kD and is a member of the RRM (RNA Recognition Motif) protein superfamily. The protein contains 3 RRMs, two adjacent to one another in the middle of the protein followed by a 19 amino acid linker and then a third RRM. Previous work in our laboratory indicates that each RRM has the potential to bind to a specific RNA sequence, so CEVH may bind several RNAs or may bind the same RNA in several places. Preliminary in vitro selection experiments suggest the protein may prefer RNA that contains a UGGGC/U sequence. We have overexpressed and purified the CEVH protein in order to produce antibodies for immunoprecipitation of natural ligands. The CEVH antibody will also be used to look for tissue specific and developmental expression of the protein by immunofluorescence in worms. In an effort to uncover the function of this protein we will continue to characterize the gene and the protein it expresses.
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[
International C. elegans Meeting,
1995]
We have recently cloned a C. elegans gene, we call CEVH, that expresses an RNA-binding protein of unknown function. So far we have a full-length cDNA clone and also a cosmid that we believe contains the complete gene. The cevh gene physically maps near the end of the left arm of chromosome I, however we have been unable to identify any existing mutants thus far. The CEVH protein is about 65 kD and is a member of the RRM (RNA Recognition Motif) protein superfamily. The protein contains 3 RRMs, two adjacent to one another in the middle of the protein followed by a 19 amino acid linker and then a third RRM. Previous work in our laboratory indicates that each RRM has the potential to bind to a specific RNA sequence, so CEVH may bind several RNAs or may bind the same RNA in several places. Preliminary in vitro selection experiments suggest the protein may prefer RNA that contains a TGGGC/T sequence. We have overexpressed and purified the CEVH protein in order to produce antibodies for immunoprecipitation of natural ligands. The CEVH antibody will also be used to look for tissue-specific and developmental expression of the protein by immunofluorescence in worms. In an effort to uncover the function of this protein we will continue to characterize the gene and the protein it expresses.
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Relini A, Airoldi C, Regonesi ME, Gatta E, Tortora P, Bonanomi M, Vertemara J, De Gioia L, Pellistri F, Visentin C, Natalello A, Penco A
[
Hum Mol Genet,
2017]
The protein ataxin-3 (ATX3) triggers an amyloid-related neurodegenerative disease when its polyglutamine stretch is expanded beyond a critical threshold. We formerly demonstrated that the polyphenol epigallocatechin-3-gallate (EGCG) could redirect amyloid aggregation of a full-length, expanded ATX3 (ATX3-Q55) towards non-toxic, soluble, SDS-resistant aggregates. Here, we have characterized other related phenol compounds, although smaller in size, i.e., (-)-epigallocatechin gallate (EGC), and gallic acid (GA). We analyzed the aggregation pattern of ATX3-Q55 and of the N-terminal globular Josephin domain (JD) by assessing the time course of the soluble protein, as well its structural features by FTIR and AFM, in the presence and the absence of the mentioned compounds. All of them redirected the aggregation pattern towards soluble, SDS-resistant aggregates. They also prevented the appearance of ordered side-chain hydrogen bonding in ATX3-Q55, which is the hallmark of polyQ-related amyloids. Molecular docking analyses on the JD highlighted three interacting regions, including the central, aggregation-prone one. All three compounds bound to each of them, although with different patterns. This might account for their capability to prevent amyloidogenesis. Saturation transfer difference NMR experiments also confirmed EGCG and EGC binding to monomeric JD. ATX3-Q55 pre-incubation with any of the three compound prevented its calcium-influx-mediated cytotoxicity towards neural cells. Finally, all the phenols significantly reduced toxicity in a transgenic Caenorhabditis elegans strain expressing an expanded ATX3. Overall, our results show that the three polyphenols act in a substantially similar manner. GA, however, might be more suitable for antiamyloid treatments due to its simpler structure and higher chemical stability.
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Bonanomi, M., Airoldi, C., Natalello, A., Visentin, C., Tortora, P., Regonesi, M.E., Sciandrone, B., Amigoni, L.
[
International Worm Meeting,
2019]
Ataxin-3 (ATX3) is the protein that triggers the neurodegenerative disorder spinocerebellar ataxia type 3 (SCA3) when its polyglutamine stretch exceeds the critical length of 55 residues. This expansion results in misfolding and structural rearrangements, leading to aberrant interactions and the consequent formation of fibrillar amyloid-like aggregates. Currently, no effective treatment is available for SCA3 disease. In order to find natural compounds able to prevent ATX3 aggregation, we studied the effect of tetracycline (TET) and epigallocatechin-3-gallate (EGCG) on ATX3 fibrillogenesis. We displayed that TET increased the solubility of the different aggregated species, without affecting the secondary structures of the final aggregates. Otherwise, EGCG interfered with the early steps of aggregation, accelerating the Josephin Domain (JD) misfolding, and leading to the formation of off-pathway, non-amyloid, final aggregates. By NMR analyses, we proved that the different behavior could be related to the capability of the sole EGCG to bind the monomeric JD. Successively, among a small library of tetracycline, we identified methacycline (MET) as the most effective compound, which exerts the same mechanism of action of TET. The pharmacological efficacy of EGCG, TET and MET has been evaluated on a SCA3 C. elegans model and we demonstrated that all compounds induced a significant increase in mobility without extending the lifespan. MET was proven to be the most effective in relieving the locomotion defects. Even more remarkably, MET treatment also resulted in an ameliorated motility even in wild-type worms suggesting that MET fulfills its action via diverse mechanisms, besides interfering with amyloid aggregation. Furthermore, the drug did not significantly modify the life span in any of the strains investigated, which supports the expectation that it may be devoid of toxic effects in long-term administration.
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[
International C. elegans Meeting,
1999]
To investigate genes potentially involved in gut development and differentiation, we are developing a genetic screen for gut obstructed (gob) mutants. The gut specific
elt-2 GATA factor appears necessary for correct gut cell development.
elt-2 null mutants arrest as L1s with a blocked intestinal lumen and abnormal brush border (1). When
elt-2 null mutants are fed on a mixture of fluorescent beads and Escherichia coli , the beads collect as a large "gob" in the posterior pharynx and anterior gut that can be easily detected under the dissecting microscope. We are now using this gut obstructed phenotype to screen for mutants that should potentially identify genes involved in gut development. Preliminary results of this screen are promising, as several candidates have already been isolated. 1) Fukushige T, Hawkins MG and McGhee JD (1998) Dev Biol 198(2):286-302
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[
International Worm Meeting,
2015]
Environmentally induced changes in neuronal gene expression are a critical feature underlying behavioral plasticity. We have previously shown that transcription of the neuromodulator TGF-beta/DAF-7 is rapidly activated in the ASJ chemosensory neuron pair in response to the pathogenic bacteria Pseudomonas aeruginosa and that this expression change promotes avoidance behavior (Meisel, JD et. al., Cell, 2014). Using a genetically encoded calcium indicator we have also shown that specific secondary metabolites produced by P. aeruginosa can activate the ASJ neurons. To further understand the molecular mechanisms and genetic pathways responsible for transducing an environmental sensory input into a transcriptional response we performed a forward genetic screen to identify genes necessary for the activation of
daf-7 transcription in response to P. aeruginosa. We have identified conserved genes involved in G protein-coupled receptor signaling and calcium signaling that are required for
daf-7 activation in the ASJ neuron pair. We have also identified mutants with constitutive
daf-7 expression in the ASJ neurons when C. elegans are propagated on non-pathogenic E. coli. Analysis of neuronal calcium dynamics in these mutant backgrounds will provide insight into how sensory activity and calcium signaling are coupled to rapid changes in transcription that modulate behavior.