Nicolas Chartier et al. (2008) Development & Evolution Meeting "Defining the PAR-4/LKB1 Signalling Pathway in Cell Polarity by Identification of par-4 Suppressors."

Diana Paulina Salazar, Nicolas Chartier, Amy Maddox, Jean-Claude Labbe

[Development & Evolution Meeting, 2008]

Our goal is to understand the mechanisms of polarity establishment during early development the C. elegans embryo. Among the seven PAR proteins required for early polarity events, the serine-threonine kinase PAR-4 is of great interest as mutations in the gene that encodes its human homologue LKB1 are directly responsible for the Peutz-Jeghers syndrome, a condition in which patients display a high risk of developing epithelial cancers. Like other par genes, par-4 mutations induce polarity defects during early embryogenesis, including synchronous division at the two-cell stage and lack of P-granule segregation, resulting in embryonic lethality. However, the mechanism by which par-4 regulates cell polarity is still unclear and information is missing about potential partners of the PAR-4 signalling pathway in C. elegansIn order to identify genes that function with par-4 in the establishment of cell polarity, we are completing an RNAi-based, genome-wide screen to uncover genes whose disruption can suppress par-4(it57ts) lethality. Interestingly, one of the confirmed suppressors of par-4 is predicted to function in the regulation of the acto-myosin cytoskeleton, a process that was previously shown to participate in the polarization of the one-cell embryo. We found that disrupting the function of this suppressor by RNAi can partially revert the par-4 phenotype such as synchronous early divisions at the two-cell stage and P-granule segregation. We are investigating the link between this suppressor and PAR-4 as well as with other PAR proteins at the molecular level and details on our progress will be presented.Overall, this analysis will provide a better understanding of PAR-4/LKB1 function in the early embryo and, more largely, a better comprehension of the signalling pathway leading to LKB1-dependent polarity establishment in various cell types.

Vlachos IS et al. (2016) Nucleic Acids Res "DIANA-mirExTra v2.0: Uncovering microRNAs and transcription factors with crucial roles in NGS expression data."

Vlachos IS, Hatzigeorgiou AG, Chatzopoulos S, Lykokanellos F, Paraskevopoulou MD, Georgiou P, Karagkouni D, Vergoulis T, Georgakilas G, Christodoulou F, Dalamagas T

[Nucleic Acids Res, 2016]

Differential expression analysis (DEA) is one of the main instruments utilized for revealing molecular mechanisms in pathological and physiological conditions. DIANA-mirExTra v2.0 (http://www.microrna.gr/mirextrav2) performs a combined DEA of mRNAs and microRNAs (miRNAs) to uncover miRNAs and transcription factors (TFs) playing important regulatory roles between two investigated states. The web server uses as input miRNA/RNA-Seq read count data sets that can be uploaded for analysis. Users can combine their data with 350 small-RNA-Seq and 65 RNA-Seq in-house analyzed libraries which are provided by DIANA-mirExTra v2.0.The web server utilizes miRNA:mRNA, TF:mRNA and TF:miRNA interactions derived from extensive experimental data sets. More than 450 000 miRNA interactions and 2 000 000 TF binding sites from specific or high-throughput techniques have been incorporated, while accurate miRNA TSS annotation is obtained from microTSS experimental/in silico framework. These comprehensive data sets enable users to perform analyses based solely on experimentally supported information and to uncover central regulators within sequencing data: miRNAs controlling mRNAs and TFs regulating mRNA or miRNA expression. The server also supports predicted miRNA:gene interactions from DIANA-microT-CDS for 4 species (human, mouse, nematode and fruit fly). DIANA-mirExTra v2.0 has an intuitive user interface and is freely available to all users without any login requirement.

Maragkakis M et al. (2011) Nucleic Acids Res "DIANA-microT Web server upgrade supports Fly and Worm miRNA target prediction and bibliographic miRNA to disease association."

Alexiou P, Reczko M, Vergoulis T, Kourtis K, Maragkakis M, Plomaritou K, Koziris N, Hatzigeorgiou AG, Dalamagas T, Gousis M

[Nucleic Acids Res, 2011]

microRNAs (miRNAs) are small endogenous RNA molecules that are implicated in many biological processes through post-transcriptional regulation of gene expression. The DIANA-microT Web server provides a user-friendly interface for comprehensive computational analysis of miRNA targets in human and mouse. The server has now been extended to support predictions for two widely studied species: Drosophila melanogaster and Caenorhabditis elegans. In the updated version, the Web server enables the association of miRNAs to diseases through bibliographic analysis and provides insights for the potential involvement of miRNAs in biological processes. The nomenclature used to describe mature miRNAs along different miRBase versions has been extensively analyzed, and the naming history of each miRNA has been extracted. This enables the identification of miRNA publications regardless of possible nomenclature changes. User interaction has been further refined allowing users to save results that they wish to analyze further. A connection to the UCSC genome browser is now provided, enabling users to easily preview predicted binding sites in comparison to a wide array of genomic tracks, such as single nucleotide polymorphisms. The Web server is publicly accessible in www.microrna.gr/microT-v4.

Hsu TY et al. (2018) Methods Mol Biol "The Use of Caenorhabditis elegans to Study Progranulin in the Regulation of Programmed Cell Death and Stress Response."

Kao AW, Hsu TY, Butler VJ

[Methods Mol Biol, 2018]

The nematode Caenorhabditis elegans (C. elegans) has proven to be a powerful model organism for the study of many biological processes, with major implications for human health and disease. As progranulin is a pleiotropic, secreted protein with both cell autonomous and non-autonomous roles, a multicellular organism such as C. elegans is ideal for the investigation of its normal function and pathological effects. The C. elegans genome contains a progranulin-like gene known as pgrn-1. The nematode pgrn-1 encodes a protein with three cysteine-rich granulin domains, compared to the seven and a half granulins in the human protein. We have shown that C. elegans mutants lacking pgrn-1 appear grossly normal, but exhibit accelerated apoptotic cell engulfment as well as a stress resistance phenotype (Kao et al., Proc Natl Acad Sci U S A 108:4441-4446, 2011; Judy et al., PLoS Genet 9:e1003714, 2013). In addition, the roles of individual granulins can also be dissected in C. elegans (Salazar et al., J Neurosci 35:9315-9328, 2015). Here, we describe methods for studying apoptosis and stress response in C. elegans.

Furda AM et al. (2012) Methods Mol Biol "Analysis of DNA damage and repair in nuclear and mitochondrial DNA of animal cells using quantitative PCR."

Meyer JN, Van Houten B, Bess AS, Furda AM

[Methods Mol Biol, 2012]

This chapter was written as a guide to using the long-amplicon quantitative PCR (QPCR) assay for the measurement of DNA damage in mammalian as well as nonmammalian species such as Caenorhabditis elegans (nematodes), Drosophila melanogaster (fruit flies), and two species of fish (Fundulus heteroclitus and Danio rerio). Since its development in the early 1990s (Kalinowski et al., Nucleic Acids Res 20:3485-3494, 1992; Salazar and Van Houten, Mutat Res 385:139-149, 1997; Yakes and Van Houten, Proc Natl Acad Sci USA 94:514-519, 1997), the QPCR assay has been widely used to measure DNA damage and repair kinetics in nuclear and mitochondrial genomes after genotoxin exposure (Yakes and Van Houten, Proc Natl Acad Sci USA 94:514-519, 1997; Santos et al., J Biol Chem 278:1728-1734, 2003; Mandavilli et al., Mol Brain Res 133:215-223, 2005). One of the main strengths of the assay is that the labor-intensive and artifact-generating step of mitochondrial isolation is not needed for the accurate measurement of mitochondrial DNA copy number and damage. Below we present the advantages and limitations of using QPCR to assay DNA damage in animal cells and provide a detailed protocol of the QPCR assay that integrates its usage in newly developed animal systems.

Gilchrist EJ et al. (1995) International C. elegans Meeting "A C. ELEGANS MYOTONIC DYSTROPHY GENE HOMOLOGUE."

Gilchrist EJ, Baillie DL

[International C. elegans Meeting, 1995]

Myotonic Dystrophy is an inherited, autosomal dominant disease that results in a progressive wasting of the skele- tal muscles (and sometimes heart and smooth muscles) in humans. The gene has been cloned and sequenced (R. Korneluk, personal communication) and encodes a large protein that has been localised to neuromuscular and myotendinous junctions. The N-terminus domain bears similarity to cAMP-dependent serine/threonine protein kinases. Yuji Kohara, in his cDNA sequencing project, identified a C. elegans gene that is 75% similar (if conserved amino acids are considered) to the kinase domain of the human myotonic dystrophy gene. He mapped the cDNA to LG I (personal communication), and, through Southern analysis, we have placed the cDNA on cosmid K06G1. This cosmid was injected into wild-type N2 hermaphrodites by Diana Collins, and we are currently trying to rescue some of the genes in this region with the extrachromosomal array. One candidate gene was unc-57. Animals homozygous for mutations in unc-57 show no obvious structural muscle defects. This is consistent with unc-57 being a muscle, or neuro- muscular kinase. However, the Unc-57 phenotype is not rescued in animals carrying the K06G1 extrachromo- somal array. We are now focusing on some of the lethal mutations in this region that have been isolated in the laboratory of Ann Rose at UBC.

Stewart HI et al. (1996) West Coast Worm Meeting "Rescue of New Essential Genes on LGIII(left), in the Nematode Caenorhabditis elegans."

Stewart HI, Franz NW, Barbazuk BW, Baillie DL, Ha TT

[West Coast Worm Meeting, 1996]

We utilized trangenic strains containing cosmids, sequenced by the sequencing consortium, to rescue the lethal phenotype of several essential genes (See presentation by Diana Janke et al, this session). The rescued genes are positioned on LGIII(left) balanced by the duplication sDp3(see poster; Stewart et al, this session). A set of overlapping deficiencies was utilized to define areas within which to focus our rescue attempts). PCR analysis was utilized to further define the end points of these deficiencies, facilitating our analysis ( see poster ; Norm Franz et al, this session). We concentrated our analysis on areas to the right of dpy-17, in the interval between dpy-17 to dpy-19. Lethals mapping to sDf125 that were not rescued by sDp8 are presented in a poster by Nigel O'Niel et al, this session. We have rescued several essential genes. Our rescues will facilitate the functional analysis of these new genes. These rescues have also helped us to align and correlate the physical and genetic maps. Our estimates are that there will be more than two essential genes per cosmid. A total of 202 recessive lethal mutations have been generated, using EMS as a mutagen. We intend to make molecular assignments of all of these elements, through our cosmid rescue experiments. As we have estimated that there is only a 28% saturation of the area for essential genes, we will be generating more recessive lethal genes, to facilitate this analysis. This work has been funded by grants from the National Institute of Health (N.I.H.), U. S.A. to Ann Rose; D. Riddle and David Baillie and Canadian Genome and Advanced Technologies (C.G.A.T.), Canada to Ann Rose and David Baillie.

Stewart HI et al. (1995) International C. elegans Meeting "CAENORHABDITIS ELEGANS: BALANCERS, RECESSIVE LETHAL MUTATIONS AND DEFICIENCIES ON LGIII(LEFT) AND LGV(RIGHT)."

Ha TT, Stewart HI, Baillie DL, Rose AM, Collins D

[International C. elegans Meeting, 1995]

The new balancers sC1 and sDp3 along with the well documented eT1 balancer (Rosenbluth et al, 1981) completely balance LGIII. We have validated sC1 and sDp3 by isolating recessive lethal mutations and have generated a set of overlapping deficiencies spanning these balancer systems. A set of 91 new essential genes were isolated, while validating sDp3, as a balancer. These recessive lethal mutations were also utilized in the initial mapping of the new set of deficiencies. We have 11 confirmed deficiencies and have several other putative deficiencies, balanced by sDp3. Work validating the new balancer sC1 is also well underway. We have isolated over 70 recessive mutations and have also generated a set of deficiencies, in this region, two of which have been confirmed. As the sequencing of LGIII is rapidly being completed by the sequencing consortium, work on the correlation of the genetic and physical maps is also progressing. About 100 transgenic strains, using sequenced cosmids, have been constructed to aid in this work (See poster Diana Collins et al, this session). LGV(Right) is currently balanced by nT1 and sC4. sC4 is a new balancer, unc-76 rol-9/ sC4[dpy-21], which is not homozygous viable. It incompletly balances the right end of LGV. Work is in progress to develop a new balancer for the far right of LGV., using a derivative of nT1. The genetic tools provided by this reasearch are available to the C. elegans genetic community on request. Information on how to access these mutations, through the www site, is available at http: This research is being funded by The National Institutes of Health (NIH) U.S.A.

Butler, V.J. et al. (2017) International Worm Meeting "A novel stress and age-responsive lysosomal protease inhibitor affecting proteostasis in neurodegeneration."

Ashrafi, K., Chand, S., Miller, B.L., Jacobson, M.P., Gao, F., Pierce, O.M., Coppola, G., Butler, V.J., Caballero, B., Oses-Prieto, J.A., Burlingame, A.L., Kao, A.W., Seeley, W.W., Argouarch, A.R., Vohra, M., Cuervo, A.M., Mohan, S.

[International Worm Meeting, 2017]

Progressive impairment of protein homeostasis is a universal hallmark of aging, stress and disease. One potential site for this cellular dysfunction is the lysosome, which is responsible for macromolecular breakdown and recycling. We previously showed in C. elegans that granulin peptides, the cleavage products of the neurodegenerative disease protein progranulin, selectively enhance TAR DNA-binding protein 43 (TDP-43) toxicity and impair its degradation (Salazar J. Neurosci 2015). Now, we provide genetic, biochemical, computational modeling and behavioral evidence supporting a role for C. elegans granulin 3 as a stress and age-responsive inhibitor of the lysosomal aspartyl protease, ASP-3/CTSD. C. elegans progranulin contains three granulins. We show that granulin production increases with age and stress. Transgenic expression of individual granulins impairs stress response and learning and memory. Granulins localize to the lysosome where they stimulate movement of the autophagy-promoting transcription factor HLH-30/TFEB from the cytosol to the nucleus. Co-immunoprecipitation experiments show that C. elegans granulin 3 interacts with the lysosomal aspartyl protease, ASP-3, which has previously been implicated in necrotic cell death. Protein docking models predict that granulin 3 binds to and occludes the protease active site. Supporting this, ASP-3 activity is significantly reduced in granulin expressing animals. Furthermore, the expression of granulin 3 in a deg-1(d) background delays the cell death of mechanosensory neurons and prolongs response to gentle touch. In addition, human patients carrying progranulin mutations produce more granulin fragments in degenerative brain regions than age-matched controls, and this is accompanied by a significant reduction in CTSD activity. Our results identify granulins as a novel class of endogenous peptide aspartyl protease inhibitor, which in analogy to serpins and cystatins we call aspartins. Given that age and stress can increase granulin production, we propose that the regulated production of granulin peptides can impair protein homeostasis and contribute to neurodegenerative disease pathogenesis. These results also suggest that dysregulation of lysosomal protease activity may more generally contribute to aging and age-related diseases.

Fiannaca A et al. (2016) BMC Bioinformatics "MiRNATIP: a SOM-based miRNA-target interactions predictor."

Fiannaca A, Rizzo R, La Paglia L, La Rosa M, Urso A

[BMC Bioinformatics, 2016]

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNA sequences with regulatory functions to post-transcriptional level for several biological processes, such as cell disease progression and metastasis. MiRNAs interact with target messenger RNA (mRNA) genes by base pairing. Experimental identification of miRNA target is one of the major challenges in cancer biology because miRNAs can act as tumour suppressors or oncogenes by targeting different type of targets. The use of machine learning methods for the prediction of the target genes is considered a valid support to investigate miRNA functions and to guide related wet-lab experiments. In this paper we propose the miRNA Target Interaction Predictor (miRNATIP) algorithm, a Self-Organizing Map (SOM) based method for the miRNA target prediction. SOM is trained with the seed region of the miRNA sequences and then the mRNA sequences are projected into the SOM lattice in order to find putative interactions with miRNAs. These interactions will be filtered considering the remaining part of the miRNA sequences and estimating the free-energy necessary for duplex stability. RESULTS: We tested the proposed method by predicting the miRNA target interactions of both the Homo sapiens and the Caenorhbditis elegans species; then, taking into account validated target (positive) and non-target (negative) interactions, we compared our results with other target predictors, namely miRanda, PITA, PicTar, mirSOM, TargetScan and DIANA-microT, in terms of the most used statistical measures. We demonstrate that our method produces the greatest number of predictions with respect to the other ones, exhibiting good results for both species, reaching the for example the highest percentage of sensitivity of 31 and 30.5 %, respectively for Homo sapiens and for C. elegans. All the predicted interaction are freely available at the following url: http://tblab.pa.icar.cnr.it/public/miRNATIP/ . CONCLUSIONS: Results state miRNATIP outperforms or is comparable to the other six state-of-the-art methods, in terms of validated target and non-target interactions, respectively.