Sarkar, Debjani et al. (2013) International Worm Meeting "Evolution of Nematode Spliced Leader trans-splicing."

Muller, Berndt, Connolly, Bernadette, Sarkar, Debjani, Pettitt, Jonathan

[International Worm Meeting, 2013]

Spliced-leader (SL) trans-splicing is a pre-mRNA maturation event that has a pivotal role in processing polycistronic RNA transcripts from operons into mature monocistronic mRNAs. It is known to occur in multiple, widely distributed eukaryotic groups, including nematodes. Most of our knowledge on SL trans-splicing in nematodes has come from investigations in Caenorhabditis elegans and other nematodes from the Chromadorean clades. The identification of SL trans-splicing in nematodes that lie outside of the Chromadorea has led us to conclude that SL trans-splicing is likely to be a phylum-wide process and thus a trait found in the last common ancestor of the nematodes. In this project we investigate the nature of SL trans-splicing in nematodes outside of the Chromadorean clades. Previous studies have shown that the Dorylaimid Trichinella spiralis uses a range of highly polymorphic SL sequences that have only limited similarity to C. elegans SL1 and SL2 (Pettitt et al, 2008). In contrast, initial searches for SLs in Prionchulus punctatus have shown that it possesses clear SL2-like sequences (Harrison et al, 2010). In this study we carried out searches for putative SL sequences in Trichuris muris to address differences between SL sequences in T. spiralis and P. punctatus. Searches indicate that SLs in T. muris are similar to the SLs found in P. punctatus and C. elegans, which is unexpected given that T. muris shares a "recent" common ancestor with T. spiralis. This in turn highlights that the lineage leading to T. spiralis is derived in relation to SL trans-splicing. Our results provide us with a valuable insight into the likely nature of SL trans-splicing in the ancestral nematode and how it has evolved within the nematode phylum.

Brivio, Sarah et al. (2021) International Worm Meeting "Transgenic hph::gfp gene fusion allows streamlined screening for C. elegans gene knockouts"

Muller, Berndt, Fasimoye, Rotimi, Brivio, Sarah, Connolly, Bernadette, Pettitt, Jonathan

[International Worm Meeting, 2021]

We are interested in understanding the protein and RNA components of the spliced leader trans-splicing machinery. Previous work has implicated a set of novel non-coding RNA components, the SmY RNAs, in spliced leader trans-splicing. These RNAs are encoded by 12 distinct genes and we are in the process of systematically deleting them to understand the precise mechanistic roles of their products. To facilitate this approach we have developed a novel hph::gfp reporter transgene that allows tagging and knockout of any gene of interest in a single injection via CRISPR/Cas9 induced homology directed repair (HDR). This method generates a loss of function allele and a fluorescent protein fusion reporter, while the hph hygromycin resistance gene facilitates the selection process. The fusion protein is functional, successfully generating both broad cytoplasmic fluorescence and hygromycin resistance in the assayed worms. This hph::gfp repair template provides a simple and flexible approach - it can be modified by oligo cloning to be flanked by homology arms corresponding to the region upstream and downstream of any gene of interest. This approach significantly reduces the time and labour required to achieve each knockout, facilitating our goal of complete smy gene complement knockout. Our results to-date show that loss of ten of the twelve smy genes impair spliced leader trans-splicing, confirming the role of this enigmatic set of RNAs in this process.

Pettitt, Jonathan et al. (2013) International Worm Meeting "The evolution of nematode operons."

Goth, Henrike, Phillippe, Lucas, Sarkar, Debjani, Pettitt, Jonathan, Connolly, Bernadette, Muller, Berndt

[International Worm Meeting, 2013]

Operons are a means of organising multiple independent coding regions such that they are transcribed into a single, polycistronic RNA. The presence of operons in an organism's genome is strongly correlated with the ability to carry out spliced leader (SL) trans-splicing, consistent with the hypothesis that the processing of polycistronic RNAs in eukaryotes is dependent upon SL trans-splicing. Operons have been found in C. elegans and other nematodes that fall within the Chromodoria, one of the three major nematode clades. We have previously shown that the nematode, Trichinella spiralis, which lies in one of the other two main clades, the Dorylaimia, engages in SL trans-splicing, suggesting that it is a nematode-wide trait. An important question is whether the same is true of operons. We reasoned that if there is an intimate relationship between SL trans-splicing and polycistronic RNA processing, then we should also expect to be able to identify genes organised into operons in these nematodes. We have previously identified a set of T. spiralis genes whose mRNAs undergo SL trans-splicing, and using this dataset, we have demonstrated the existence of operons in this nematode. We have confirmed that they produce polycistronic RNAs and that they are present in the closely related Trichuris muris. At least two of the operons are conserved between nematodes in the Dorylaimia and the Chromodoria clades, suggesting that these represent operons likely present in the ancestor of the nematode phylum. We find that mRNAs derived from downstream genes in operons are SL trans-spliced, just as is found for other nematode operons, but there is no equivalent to the specialised SL2 found in C. elegans. We are currently expanding upon our limited set of data to build a more comprehensive picture of SL trans-splicing and operon organisation in the Dorylaimia, and thereby gain a better understanding of the influence these processes have had on the evolutionary dynamics of the nematode genome.

Wenzel, Marius et al. (2021) International Worm Meeting "SLIDR and SLOPPR: Computational pipelines for the discovery and characterisation of spliced leader trans-splicing and polycistronic RNA processing reveal the evolutionary dynamics of SL2 trans-splicing across the Nematoda"

Pettitt, Jonathan, Connolly, Bernadette, Wenzel, Marius, Muller, Berndt, Piccicacchi, Lucrezia, Marin, Andreea

[International Worm Meeting, 2021]

The organisation of genes into operons - clusters of genes which are transcribed as polycistronic RNAs - is a feature of all known nematode genomes. Spliced leader trans-splicing is essential for the expression of downstream operonic genes because the spliced leader provides the 5' cap to their otherwise uncapped transcripts. In C. elegans, a specialised spliced leader, SL2, is specific for transcripts derived from downstream operonic genes via a process that is mechanistically distinct from the more generic SL1 trans-splicing. Studies of nematodes outside of Clade V failed to detect SL2 trans-splicing, with downstream operonic gene transcripts being trans-spliced to SL1. This led to the hypothesis that SL2 trans-splicing is recent innovation and that SL1 trans-splicing is the ancestral mechanism for resolving nematode polycistronic RNAs. However, a rigorous investigation of this hypothesis requires the comprehensive genome-wide characterisation of both operons and spliced leader trans-splicing. This has, until recently, been challenging, and their identification has historically relied upon sequence similarity with C. elegans, which may bias the results.To systematically investigate spliced leader trans-splicing and operon organisation, we have developed two fully automated discovery and annotation pipelines, SLIDR and SLOPPR (https://doi.org/10.1186/s12859-021-04009-7), that enable the comprehensive characterisation of spliced leader trans-splicing and operon organisation in any organism using standard RNA-Seq datasets. Using these tools, we showed that SL2 trans-splicing is more broadly distributed than previous studies suggested; it is found in all Clade I nematodes that we investigated (http://www.rnajournal.org/cgi/doi/10.1261/rna.076414.120). However, we were unable to detect SL2 trans-splicing in any Clade III nematode, consistent with previous studies, and could only detect it in a small sub-set of Clade IV nematodes. These distributions can be explained either by loss of an ancestral SL2 trans-splicing mechanism in multiple lineages, with SL1 acquiring the role in processing polycistronic RNA; or by the convergent evolution of SL2 trans-splicing in selected lineages. I will present data that favours the former explanation provides a possible scenario to explain how SL1 might replace SL2 trans-splicing despite the latter's broad conservation and therefore functional importance.

Spencer, Rosie et al. (2021) International Worm Meeting "SNA-3: an essential, nematode-specific protein is a novel key component of the spliced leader trans-splicing machinery"

Pettitt, Jonathan, Soto-Martin, Eva, Eiljers, Peter, Elmassoudi, Haitem, Wenzel, Marius, Muller, Berndt, Connolly, Bernadette, Fasimoye, Rotimi, Spencer, Rosie

[International Worm Meeting, 2021]

We are investigating spliced leader (SL) trans-splicing and its key RNA and protein components as potential anthelmintic targets, using Caenorhabditis elegans as a model system. SL trans-splicing is an essential process in nematode gene expression that facilitates translation by replacement of the 5' untranslated region of most mRNAs with the spliced leader 1 (SL1). The splicing reaction involves an interaction between the SL1 snRNP, the nascent pre-mRNA and the spliceosome. Although SL trans-splicing was discovered more than 30 years ago, we know little about the molecular mechanism(s) by which this is achieved. To address this, we have carried out a comprehensive molecular characterisation of the SL1 snRNP. This work expands and refines our understanding of the proteins involved in SL1 trans-splicing: we have analysed factors co-immunoprecipitating with the SL1-specific protein SNA-1, giving us insight into the interaction of the SL1 snRNP with the spliceosome. Proteins critical for SL1 trans-splicing were identified using established RNAi-based qPCR and gfp-reporter gene assays (https://doi.org/10.1093/nar/gkx500). This led to the identification of a novel, essential trans-splicing factor termed SNA-3. SNA-3 is a highly conserved, nematode specific protein containing NADAR domains, which have been linked to NAD/ADP-ribose metabolism and may have N-glycosidase activity. SNA-3 interacts with several highly-conserved proteins associated with RNA processing including the CBC-ARS2 complex components NCBP-1 and SRRT/ARS2 involved in co-transcriptional determination of transcript fate. Together, these observations implicate SNA-3 in key steps linking SL1 trans-splicing to the transcriptional control of gene expression. The identification of another essential, nematode-specific protein involved in SL1trans-splicing strengthens the hypothesis that the acquisition of SL trans-splicing requires the evolution of novel machinery required to modify the activity of the spliceosome. The novelty of these proteins makes them ideal targets for the development of new anthelmintics.

Gray, Jeremy C et al. (2013) International Worm Meeting "Cross Species NILs - A Resource for Speciation, Evolution and Development."

Cutter, Asher D, Gray, Jeremy C, Bundus, Joanna

[International Worm Meeting, 2013]

Caenorhabditis sp 9 and Caenorhabditis briggsae are capable of forming hybrids at low rates, despite being separate species. Reproductive isolation is of long standing interest in evolutionary biology and the presence of interbreeding species in this genus presents a unique opportunity to probe the genetics of speciation in a well understood background. Here we detail the construction and genotyping of 200 near isogenic lines, consisting of small introgressions of one genome into the opposite species in both mitochondrial backgrounds. Introgressions were achieved by random mating and subsequent backcrosses. Next generation sequencing will be undertaken in order to determine which regions are introgressed, and regions which are under or overrepresented. Strains have been phenotyped for reproductive isolation, cytonuclear incompatibility and general developmental defects. The main thrust of the project is to identify and analyse Bateson-Dobzhansky-Muller incompatibilities within the genus, as well as to identify the genetic basis of the mating system differentiation in the two species. It is anticipated these strain will be made available to the community at large along with genotype information to allow further studies into the divergence of these two species.

DePellegrin Connelly, Tracey et al. (2009) International Worm Meeting "GENETICS, the peer-edited journal of the Genetics Society of America, reports on updates and special projects, including article links to WormBase."

DePellegrin Connelly, Tracey, Schedl, Tim

[International Worm Meeting, 2009]

GENETICS, the peer-edited journal of the Genetics Society of America has partnered with WormBase (Arun Rangarajan, Hans-Michael Muller and Paul Sternberg) to produce interactive journal articles (in full text/HTML, XML and PDF outputs). A reader who clicks on a gene or protein name, allele, transgene (or potentially any object found in the database) is taken directly to the corresponding page in WormBase. This innovative project integrates two major modes of communication used in the biological sciences: journal articles and databases. The project offers several benefits to readers, including fast access to relevant information associated with a genetic object in the text. This information can be general, providing an overview (e.g. gene summary), or highly specific, providing an important experimental detail (e.g. the molecular lesion of an allele). Also, the project promotes standardization of individual object nomenclature (e.g. gene names) and simplifies connections when there is a nomenclature change. Finally, the objects remain connected but evolve with advancesin knowledge. The benefits to WormBase include increased use of and interest in the database, more efficient and extensive corrections of information in the database by the community, facile incorporation of new information, reverse integration of the database with the primary data in the literature, all with minimal ongoing cost. We will show examples of the article links to WormBase, and discuss a number of other initiatives being undertaken by the journal GENETICS.

Susoy, Vladislav et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "EVOLUTIONARY DIVERSIFICATION THROUGH THE GAIN AND LOSS OF DEVELOPMENTAL PLASTICITY"

Sommer, Ralf J., Susoy, Vladislav, Ragsdale, Erik J.

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Developmental plasticity is common among plants and animals, yet its role in mediating evolutionary processes is debated. Several species of Rhabditina, including Pristionchus pacificus, can execute two alternative mouth phenotypes, one of which is associated with predatory feeding. Studies in P. pacificus have revealed that environmental factors act through a conserved genetic pathway that enables a rapid developmental response to the environment (Bento et al., 2010). In addition, a developmental switch that controls the expression of the alternative mouth phenotypes was recently identified (Ragsdale et al., 2013). In contrast, little is known about the macroevolutionary potential of the mouth plasticity. We studied the relationship between plasticity and morphological change by a macroevolutionary analysis of nematode mouthparts when accompanied by a dimorphism. To test whether plasticity facilitates or hinders morphological change, we analyzed variation in form and complexity in 90 nematode species with or without a mouth dimorphism. Our analyses revealed a two-step process of morphological diversification associated with the gain and loss of plasticity. First, acquisition of a dimorphism was accompanied by an increase in complexity, including structural innovations such as moveable teeth. Second, the fixation of a single mouth-phenotype in several nematode lineages was associated with a decrease in mouth complexity but a sharp increase in evolutionary rates when measured as change of shape and size. Thus, plasticity facilitates phenotypic diversification by fostering evolutionary novelties, whereas subsequent loss of the dimorphism enables acceleration of evolution by releasing novel morphologies from developmental constraints. 1. G. Bento, A. Ogawa, R. J. Sommer. Nature 466, 494-497 (2010). 2. E. J. Ragsdale, M. R. Muller, C. Rodelsperger, R. J. Sommer, Cell 155, 922-933 (2013).

Ashe A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Small RNAs With Characteristics Of 22Gs Are Present Multiple Generations After dsRNA Induced Silencing"

Miska E A, Goldstein L D, Ashe A, Lehrbach N J

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Since the discovery of RNAi in C. elegans in 1998, our knowledge of the complexity of small RNA pathways in this organism has grown rapidly. We now know that there are many exogenous and endogenous small RNA pathways interacting in complex ways within the organism. Much of this knowledge has come about through advances in sequencing technology that allow us to see in great detail the numerous small RNAs present in cells. Despite this abundance of data, there are still many aspects of small RNA biology that remain unclear. From the first reports of gene silencing induced by exogenous dsRNA, it has been observed that the silencing can be heritable (1). For RNAi of most genes, silencing of the target gene is limited to the F1 generation, with subsequent generations reverting back to normal levels of gene expression. However, there are a small number of reported cases where silencing of a specific gene can be transmitted across multiple generations (2, 3, 4). The mechanism of inheritance is unknown, but studies have suggested the involvement of a diffusible element (4), or chromatin modifiers (3). Here we show that silencing of a single-copy GFP transgene can be inherited for multiple generations, and that this silencing correlates with the presence of small RNAs targeted to the transgene. 1. H. Tabara, A. Grishok, C. Mello, Science 282, 430-431 (1998) 2. A. Grishok, H. Tabara, C. Mello, Science 287, 2494-2497 (2000) 3. N. Vastenhouw, K. Brunschwig, K. Okihara, F. Muller, M. Tijsterman, R. Plasterk, Nature, 442, 882 (2006) 4. R. Alcazar, R. Lin, A. Fire, Genetics, 180, 1275-1288 (2008)

Attila L Kovcs et al. (2005) International Worm Meeting "Electron microscopy of autophagy in Caenorhabditis elegans"

Judit Fehr, Tibor Vellai, Krisztina Takcs-Vellai, Zsolt Plfia, Jnos Kovcs, Attila L Kovcs

[International Worm Meeting, 2005]

Cellular autophagy is a process for the degradation of cytoplasmic constituents in eukaryotic cells. Since its discovery in 1957 in the epithelial cells of kidney of the newborn mice (1) electron microscopy has been and still remains an indispensable method for studying autophagy. One of the main reasons of the late start of autophagy research in C. elegans is the relative difficulty of performing transmission electron microscopy with worm samples. Recently we have developed a technique by which autophagic processes of the worm become accessible for systematic morphological and, in three major tissue types, for morphometric analysis by transmission electron microscopy (2). Our poster introduces the method, presents the criteria for the identification and morphological analysis of various types of autophagic vacuoles in all major cell types, and shows the latest morphometric data on autophagy in hypodermal, gut epithelial and body wall muscle cells during postembryonic development including all four larval, as well as the predauer, dauer and postdauer stages. Our results indicate that the cells of continuously feeding worms are practically devoid of autophagic vacuoles. Significant increase in the quantity of autophagic vacuoles can be observed at the end of each larval stage when the lethargus is reactivated. Systematic measurements on Daf-c mutants in the predauer period show that preparation for the dauer stage does not involve constitutive autophagic activity. (1) Clark S.L. (1957) Cellular differentiation in the kidneys of newborn mice studied with the electron microscope, J. Biophysic. Biochem. Cytol. 3, 349 (2) Kovacs AL, Vellai T, Muller F (2004) Autophagy in Caenorhabditis elegans. In: "Autophagy" Ed. Daniel J. Klionsky, Landes Bioscience 2004, Chapter 17, pp 216-223