Beer, Madeline et al. (2021) International Worm Meeting "Investigating the Role of sRNA and Argonautes in Intercellular Communication"

Claycomb, Julie M., Beer, Madeline, Lao, Robert X., Seroussi, Uri

[International Worm Meeting, 2021]

The nematode Caenorhabditis elegans uses sRNA and Argonaute proteins (AGOs) to degrade, inhibit the translation of, or upregulate target transcripts in a process called RNA interference (RNAi). Remarkably, the worm is capable of taking up dsRNA from the environment via its intestine and transporting to distant tissues to elicit RNAi systemically. While some of the mechanisms of systemic RNAi are understood, one question that remains is: do AGOs themselves move throughout the animal? There is precedent for mobile AGOs in multiple species. For example, the pathogenic nematode Heligmosomoides bakeri secretes sRNAs and an AGO called exWAGO during infection to manipulate expression of mouse immunity genes. There are three homologs of exWAGO in C. elegans that localize to the apical membrane of the intestine (the intestinal Secondary AGOs, iSAGOs). Their apical intestinal localization places them at an interface with the environment. Our lab used IP/MS to identify protein interactors of iSAGOs and found interactors are involved in membrane and vesicular transport. I hypothesize that the localization of iSAGOs to the intestinal apical membrane allows them to take up dsRNA and sRNA from the environment, and transmit RNAi signals to other tissues in the worm. To test this, I will determine how iSAGOs are localized to the intestinal apical membrane, define the roles of iSAGOs in mediating host-pathogen interactions, and determine whether iSAGOs are necessary for systemic RNAi. This research will illuminate the mechanisms by which the iSAGOs are involved in intercellular communication.

Beer, Katharina et al. (2017) International Worm Meeting "Retromer-associated proteins and PAD-1 control TAT-5 flippase activity to inhibit extracellular vesicle budding."

Nance, Jeremy, Stigloher, Christian, Wehman, Ann, Fazeli, Gholamreza, Beer, Katharina

[International Worm Meeting, 2017]

Cells release extracellular vesicles (EV) that can mediate intercellular communication to influence development and disease (Beer & Wehman, Cell Adh Migr 2017). Despite their pleiotropic functions, the molecular details of EV release are poorly understood, especially for plasma membrane budding (ectocytosis). Previously, we showed that TAT-5 phospholipid flippase activity inhibits ectocytosis and maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the inner leaflet of the plasma membrane (Wehman et al., Curr Biol 2011). In a screen for additional proteins that inhibit EV budding, we identified new TAT-5 regulators related to the retromer recycling pathway (PI3Kinase VPS-34, Beclin1 homolog BEC-1, and RME-8) together with the Dopey domain protein PAD-1. PI3K, RME-8, and sorting nexins are required for the localization of TAT-5 to the plasma membrane, which is important to maintain PE asymmetry. PAD-1 also localizes to the plasma membrane, but is not required for TAT-5 localization. Rather, PAD-1 is required for the lipid flipping activity of TAT-5, further supporting the model that PE asymmetry regulates plasma membrane budding. Our study identifies new proteins that regulate extracellular vesicle release and pinpoints TAT-5 and phosphatidylethanolamine as key regulators of plasma membrane budding. Understanding the mechanisms of EV release will enable us to determine the in vivo roles of EVs during development and homeostasis.

Sinha, Amit et al. (2015) International Worm Meeting "Genome wide analysis of adaptation to acidic environments of the extremophile Turbatrix aceti."

Alkema, Mark, Sinha, Amit, Sarkies, Peter, Pirri, Jennifer

[International Worm Meeting, 2015]

The nematode Turbatrix aceti, commonly known as the vinegar eel, represents one of the rare examples of metazoans that are uniquely adapted to thrive in highly acidic environments. It can grow at a remarkable pH range from 2.5 to 9. To determine the molecular basis of mechanisms of adaptation to extreme environments we initiated a whole genome sequencing of T. aceti. Using Illumina sequencing platform, we have obtained about 11 Gigabases of data, from 55 million paired-end reads at 100 base pair length. We have assembled a first draft version of T. aceti genome to a total size of about 51 Megabases (Mb), indicating an average coverage of about 211-fold over the whole genome. This draft genome is comprised of around 83,000 contigs, with the largest contig at 78kb, and a N50 contig size of 2kb. Using the SNAP gene prediction software on this draft genome, we have identified about 25,000 protein coding genes. To improve the quality and contiguity of the genome assembly, we are currently using very long-read sequencing technology from Pacific Biosciences. T. aceti belongs to Clade IV of nematode phylogeny, which also harbors the beer mat nematode Panagrellus redivivus and many parasitic nematode species including, Strongyloides, and Globodera. We are analyzing the current set of gene predictions to search for novel gene families and protein domains specifically enriched in T. aceti as compared to other sequenced nematode genomes. Furthermore, to specifically identify genes potentially required for acid tolerance, we are using RNAseq to compare the gene expression profiles of T. aceti grown in acidic media versus in neutral media. We will present our analysis which should provide new insights to the physiological and genetic basis of adaptations to extreme environments.

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).