Gutwein, Michelle et al. (2013) International Worm Meeting "3' End Profiling of Gametogenesis in C. elegans Using RNA-Seq."

Gutwein, Michelle, Mecenas, Desirea, Piano, Fabio, Scheid, Paul, Ahmed, Rina, Gunsalus, Kris

[International Worm Meeting, 2013]

Post-transcriptional regulation of gene expression is largely mediated through sequence elements in the 3'UTR of protein-coding genes. One of our major interests is to understand post-transcriptional regulatory mechanisms during development. The C. elegans germline provides an excellent model to study post-transcriptional regulation because it is the primary determinant of gene expression in this organ (Merritt et al., Curr Biol 2008). We would like to characterize differences in 3'UTR isoform usage throughout gametogenesis by profiling 3'UTR ends in mitotic and meiotic regions of the gonad as well as in oocytes as a first step toward analyzing the contribution of putative regulatory elements in different regions of 3'UTRs. Next-generation sequencing has been useful to provide a deep sampling of transcriptional landscapes. However, transcript 3' termini are under-represented using standard RNA-seq library preparation protocols, rendering targeted analysis challenging. A number of specialized protocols to characterize the exact position of 3'end cleavage and polyadenylation have been developed, but they generally require larger sample sizes than are practical to extract from very specific tissues or cells in model organisms like C. elegans. Our aim is to develop new protocols that optimize 3'UTR endpoint analysis using small sample sizes for tissue-specific profiling with RNA-seq analysis. We have designed and tested a library preparation protocol for linear amplification and 3' end capture followed by deep sequencing on the Illumina HiSeq. We perform paired-end sequencing using a non-standard protocol designed specifically to avoid issues with sequencing the low-complexity polyA tail of transcripts. Here we present our progress in using this protocol with samples of RNA extracted from whole male and hermaphrodite N2 gonads, as well as dissected mitotic and meiotic regions and oocytes from hermaphrodite gonads in order to characterize 3'UTR diversity during germline development.

Mecenas, Desirea G. et al. (2015) International Worm Meeting "The Dynamics of Alternative Polyadenylation Usage in C. elegans Germ Cell Development."

Piano, Fabio, West, Sean M., Polanowska, Jola, Reboul, Jerome, Gutwein, Michelle, Gunsalus, Kristin C., Mecenas, Desirea G., Bian, Wenting

[International Worm Meeting, 2015]

Proper spatio-temporal control of gene activity is vital for C. elegans germline development and maintenance and is determined primarily by regulatory elements within 3'UTRs (Merritt et al., Curr Biol 2008). Because almost half of protein-coding genes in the genome are subject to alternative polyadenylation (Mangone et al., Science 2010; Jan et al., Nature 2011), we are investigating whether the regulatory potential of genes during germline development is controlled by alternative 3'UTR isoform expression. We have established a Low Input 3'-End Sequencing (LITE-Seq) method to simultaneously identify and quantify mRNA transcript abundance and 3'UTR isoforms from small RNA samples, and we have applied it to investigate differences in transcripts and 3'UTR isoforms expressed in oocyte- and sperm-producing germline and in three distinct developmental stages within the hermaphrodite germline (mitosis, early meiosis, and developing oocytes). We observe on a global level that 3'UTRs in sperm-producing germline tend to be shorter than those expressed in oocyte producing germline, and that 3'UTRs become progressively longer as germ cell nuclei proliferate, enter meiosis, and differentiate into oocytes. We have identified numerous transcripts whose abundance and/or 3'UTR isoforms differ in a sex- or developmental stage-dependent manner. We also detect examples of 3'UTR isoform switching between sexes or developmental stages, including for some genes whose total transcript abundance is similar. To test the idea that individual transcripts may be subject to differential post-transcriptional regulation by selective expression and/or degradation of alternative 3'UTR isoforms at different developmental times, we developed an in vivo assay that reports on the translational regulatory potential of alternative 3'UTR isoforms in the germline. The reporter construct enables the cloning of two distinct 3'UTR isoforms into a Gateway-compatible, two-color reporter system in which each fluorophore is subject to translational regulation by a single 3'UTR isoform. Using this reporter system, we found that protein expression for several genes identified above is altered in a 3'UTR isoform-dependent manner, and that protein levels vary in different developmental contexts. Future work to identify cis-regulatory elements within the variable regions of 3'UTRs will enable us to assay their relative contributions to specific spatio-temporal expression patterns of known developmental regulators in the germline and to ascertain their functional significance in different developmental processes.

Fradin, Helene et al. (2017) International Worm Meeting "Genome architecture and evolution of the unichromosomal asexual nematode, Diploscapter coronatus."

Piano, Fabio, Corcoran, David, Gunsalus, Kristin, Kovtun, Mikhail, Gutwein, Michelle, Zegar, Charles, Fradin, Helene, Baugh, Ryan, Lucas, Jessica, Fitch, David, Kiontke, Karin

[International Worm Meeting, 2017]

Long-lived clades of animals that reproduce exclusively asexually are rare, presumably because lack of variation in such species results in high extinction rates. Longevity of asexual clades appears to be correlated with the maintenance of heterozygosity across generations. To understand how successful asexual lineages evolve while maintaining heterozygosity, we investigated the reproductive biology and genome sequence of the nematode Diploscapter coronatus. D. coronatus is a species that belongs to the Protorhabditis group, the sister group to Caenorhabditis. We confirmed the existence of a long-lived (approx. 18 million years) asexual clade within Protorhabditis and resolved its phylogeny. We found that all asexual species in the clade have an unusual karyotype: a single pair of chromosomes. This karyotype evolved once from an ancestor with six chromosome pairs. This drastic drop in the number of chromosomes coincides with the transition from sexual to asexual reproduction. We determined the D. coronatus genome structure and sequence, and find evidence in the genome assembly that the single chromosome resulted from the fusion of ancestral chromosomes. This fusion is associated with extensive rearrangement among neighboring regions, which we used to infer a partial spatial order in which ancestral chromosomes fused. The genome can be organized into two divergent homologous haplotypes, confirming that heterozygosity is maintained in this species despite the asexual reproduction. Interestingly, two adjacent fused regions, corresponding to ancestral chromosomal domains I and X, show lower levels of heterozygosity than the other domains. These data are consistent with a scenario in which an initial X-I fusion became a neo-X chromosome in a sexual ancestor. This neo-X would have had a reduced effective population size and thus reduced heterozygosity and increased linkage disequilibrium relative to the autosomes. Consistent with chromosomal fusions, we find no evidence of typical nematode telomeres in the D. coronatus genome. Parthenogenesis likely evolved after chromosomal fusion. Cytological observations indicate that D. coronatus reproduces with a modified meiosis that skips Meiosis I, synapsis and recombination and results in a diploid embryo without fertilization. Consistent with this model, certain key conserved genes with roles in homologous pairing and recombination were not found in the D. coronatus genome. Also, oogenesis without Meiosis I is one way in which parthenogenetic organisms can maintain heterozygosity. As a prelude to functional studies, we also show that D. coronatus is amenable to experimental manipulation by RNAi.

Cipriani, Patricia Giselle et al. (2021) International Worm Meeting "LOTUS-domain containing proteins recruit C. elegans Vasa to germline granules and control the formation and size of the condensates"

Gan, Hin Hark, Fahs, Hala, Chung, George, Gunsalus, Kristin, Chen, Jiaxuan, Zinno, John, Duchaine, Thomas, Selbach, Matthias, Piano, Fabio, Mayya, Vinay, Gutwein, Michelle, Cipriani, Patricia Giselle, Bay, Olivia

[International Worm Meeting, 2021]

Limkain, Oskar and TUDOR 5 and 7 are LOTUS-domain containing proteins that have been described as important regulators of germline development and nucleators of ribonucleoprotein complexes in the germ cells of many metazoans. LOTUS-domain proteins have not been previously described in the C. elegans germ line. MIP-1 and MIP-2 are two previously uncharacterized paralogs that contain two LOTUS domains and extensive intrinsically disordered regions. Using co-immunoprecipitation, we found these proteins to be strong interactors of MEG-3, another intrinsically disordered protein that is a core component of embryonic P granules, and we named them MEG-3 Interacting Proteins 1 and 2. Fluorescently tagged and deletion alleles of the MIPs generated by CRIPSR-Cas9 show that the MIPs are constitutive components of germline granules in both embryos and the germ line. The simultaneous depletion of MIPs produces temperature sensitive sterility, a strong mortal germ line phenotype, and defects that affect all major developmental switches in germline development, including germline stem cell maintenance, the progression of cells through meiosis, and gametogenesis. The MIPs are jointly required for the condensation of other core P granule components, including MEG-3, the C. elegans Vasa homolog GLH-1, and PGL proteins. Interestingly, loss of function of either MIP individually affects the distribution and the size of remaining granules in the germ line in complementary ways. In oocytes, mip-1 deletion produces larger granules that are mostly cytoplasmic and mip-2 deletion produces smaller granules mostly attached to the nuclear membrane. Further biochemical and yeast-two hybrid analyses have shown that the MIPs physically interact with each other by forming homo- and heterodimers, and they directly bind GLH-1 through their N-terminal region, which contains the LOTUS domains. We propose that these proteins act as organizing centers in ribonucleoprotein networks and form a scaffold that helps recruit and balance essential RNA processing machinery within germline granules to regulate key developmental transitions in the germline.

Michelle S. Teng et al. (2006) European Worm Meeting "Expression of Mammalian GPCRs in C. elegans Generates Novel Behavioural Responses to Human Ligands"

John McCafferty, Martijn P.J. Dekkers, Andrew G. Fraser, Michelle S. Teng, Bee Ling Ng, Gert Jansen, Suzanne Rademakers

[European Worm Meeting, 2006]

Michelle S. Teng1, Martijn P.J. Dekkers2, Bee Ling Ng1, Suzanne Rademakers2, Gert Jansen2, Andrew G. Fraser1 & John McCafferty1. G protein coupled receptors (GPCRs) play a crucial role in many biological processes and represent a major class of drug targets. However purification of GPCRs for biochemical study is difficult and most methods of screening receptor-ligand interactions require cultured cells and endotoxin free compounds. In contrast, Caenorhabditis elegans is a soil dwelling nematode that feeds on bacteria and uses GPCRs expressed in chemosensory neurons to detect bacteria and environmental compounds. Here we report that expression of the mammalian somatostatin receptor (Sstr2) and chemokine receptor 5 (CCR5) in gustatory neurons allow C. elegans to specifically detect and respond to human somatostatin and MIP-1? respectively in a simple avoidance assay. The endogenous signalling components involved in this remarkable promiscuity of interaction, spanning 800 million years of evolution, are investigated. This system has practical utility in ligand screening. Using structure:function studies, we identified key amino acid residues involved in the interaction of somatostatin with its receptor. This in vivo system, which imparts novel avoidance behaviour on C. elegans, can therefore be used in screening impure GPCR ligands, including the identification of bacterial clones expressing agonists within recombinant libraries.

Woldemariam, Sarah et al. (2015) International Worm Meeting "Elucidating the role of cGMP dynamics in behavioral plasticity."

Schneider, Martin, Krzyzanowski, Michelle, Bethke, Mary, Nagpal, Jatin, Gottschalk, Alexander, Woldemariam, Sarah, Ferkey, Denise, L'Etoile, Noelle

[International Worm Meeting, 2015]

cGMP is a ubiquitous second messenger implicated in many important biological processes. In neurons, cGMP dynamics can regulate the function of ion channels and kinases, resulting in physiological changes. In the context of learning and memory, these changes result in short-term and long-term behavioral changes based on the organism's experience. We attempt to understand the molecular basis for long-term plasticity by studying the behavioral responses of the nematode C. elegans. Along with our collaborator Michelle Krzyzanowski from the Denise Ferkey lab in SUNY Buffalo, we are interested in how food might modulate behaviors. One food-modulated behavior is repulsion from quinine. This repulsion is mediated by the ASH neuron and it is down regulated by food withdrawal and the cGMP-dependent protein kinase EGL-4. This poses a conundrum since no guanylyl cyclases, which produce cGMP, are expressed in ASH. Genetic evidence suggests that guanylyl cyclases in other neurons are required for the food-modulated repulsion from quinine in ASH and that gap junctions are required for the transmission of cGMP from these neurons to ASH. In order to understand how cGMP dynamics in these neurons are modulated, we need a tool to visualize cGMP. To this end, we are using a cGMP sensor that will allow us to image cGMP dynamics in ASH and other neurons in the living behaving animal in the presence and absence of food.

Mangone, Marco et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "The landscape of C. elegans 3'UTRs."

Sugano, Sumio, Suzuki, Yukata, Salehi-Ashtiani, Kourosh, Gunsalus, Kris C, Rajewsky, Nikolaus, Harkins, Tim, Prasad Manoharan, Arun, Thierry-Mieg, Danielle, Thierry-Mieg, Jean, Mis, Emily, Mackowiak, Sebastian, Han, Ting, Khivansara, Vishal, Kim, John, Piano, Fabio, Zegar, Charles, Gutwein, Michelle, Mangone, Marco, Kohara, Yuji, Buffard, Pascal

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

Three-prime untranslated regions (3UTRs) of metazoan mRNAs contain numerous regulatory elements, yet the structure and developmental impact of 3UTRs remain largely uncharacterized. By integrating data from all Caenorhabditis elegans (C. elegans) developmental stages obtained by polyA capture, 3RACE, full-length cDNAs, and RNAseq, we define ~26,000 distinct mRNA 3 UTRs for ~85% of the 18,328 experimentally supported protein coding genes and refine the annotation of ~30% of gene models. Alternative 3UTR isoforms display widespread differential expression during development. Surprisingly, no canonical or variant polyadenylation signal (PAS) sequence is detec ted for 13% of polyA sites, most frequently among shorter alternative isoforms. Comparing trans-spliced and non trans-spliced genes reveals a strong correlation between the processing of transcript 5 and 3 ends: trans-spliced mRNAs possess longer 3UTRs and a higher frequency of no PAS or variant PAS motifs. We also predict conserved isoform-specific microRNA (miRNA) binding sites and identify additional evolutionarily constrained sequence blocks that may mediate 3UTR regulation. Thus, our data reveal a rich complexity of 3UTRs genome-wide and throughout development in C. elegans.

Martijn Dekkers et al. (2006) European Worm Meeting "Dissecting Gustatory Plasticity in C. elegans Using Mammalian Receptors"

Martijn Dekkers, Gert Jansen, John McCafferty, Michelle Teng

[European Worm Meeting, 2006]

Martijn Dekkers1, Michelle Teng2, John McCafferty, Gert Jansen1. We use C. elegans to study the molecular and cellular mechanisms of salt perception, using behavioural assays and calcium imaging. We discriminate three distinct responses to NaCl: First, attraction to NaCl concentrations ranging from 0.1 to 200 mM. Second, avoidance of higher concentrations. Third, avoidance of an otherwise attractive NaCl concentration after prolonged exposure. We call this latter behaviour gustatory plasticity. Previous studies have shown that chemo attraction to NaCl is mediated primarily by ASE, and to a lesser extent by ASI, ADF and ASG, and avoidance of high concentrations of NaCl is mediated by ASH (Bargmann & Horvitz, 1991). In our lab we have identified 85 proteins and five pairs of gustatory neurons that mediate gustatory plasticity. Based on our results we propose a model in which prolonged exposure to 100 mM of NaCl, elicits a signal from the ASE neurons, leading to sensitisation of the avoidance signalling ASI, ADF, ADL and ASH neurons. This results in avoidance of low concentrations of NaCl.. In an effort to identify the roles of the individual cells in gustatory plasticity we expressed either a TRP channel or a G-Protein Coupled Receptor (GPCR) in the neurons that have been implicated in gustatory plasticity. This allows us to specifically activate those cells. The TRP channel that we use is the mammalian capsaicin receptor VR-1. Normally C. elegans does not respond to capsaicin. Previously it has been shown that expression of VR-1 in the ASH neurons results in avoidance of capsaicin (Tobin et al 2002). We have generated animals that express the VR-1 receptor in the ASE, ASI, ADL and ADF neurons. We are currently testing their responses to capsaicin and the effects of preexposure to NaCl on this response, using behavioural assays.. The GPCRs that we have chosen are the mouse SSTR-2 somatostatin receptor and the human CCR-5 chemokine receptor. We have expressed these receptors in the ASH cells, and tested the responses in a novel avoidance assay. We found that the transgenic animals display specific avoidance behaviour to the ligands of the receptors, indicating that these GPCRs are integrated into the endogenous C. elegans signalling machinery, which is remarkable, given the evolutionary distance between the species. We are now making constructs to express these GPCRs in the other cells to assess their role in gustatory plasticity.