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.

Peg MacMorris et al. (2005) International Worm Meeting "Specialization of the machinery for C. elegans polycistronic pre-mRNA processing: a small SL2 snRNP protein interacts with the 3 end formation protein CstF"

Peg MacMorris, Tom Blumenthal, Madhur Kumar

[International Worm Meeting, 2005]

In C. elegans, ~ 15 % of genes are transcribed in polycistronic transcription units, or operons. Generally, the 3 ends of upstream genes in operons are only ~100 bp from the 5 end of the next gene, which always begins with a trans-splice site that is spliced to the leader SL2. We have previously shown the SL2 snRNP can be found in a complex with CstF; we are currently analyzing its protein components. As a starting point we used the Nilsen lab observation that the SL1 snRNP of Ascaris, a distantly related nematode, has two unique proteins, essential for splicing. We find that the larger of these proteins is present in C. elegans (SL-75p) and is a component of both SL2 and SL1 snRNPs (used primarily for trans-splicing near the 5 ends of mRNAs). However, C. elegans has two homologs of the smaller protein. We have found that one of these, SL-21p, is a component of the SL1 snRNP, whereas the other, SL-26p, appears to be an SL2 snRNP component. Satisfyingly, we find that antibody to SL-26p, but not SL-21p, immunoprecipitates CstF, and it does so even when the SL2 RNA has been removed by RNase digestion. Thus it seems likely that SL-26p has evolved a novel binding site that allows it to interact with CstF at the site of 3 end formation. This interaction would allow the SL2 snRNP to be brought to the vicinity of its target ~50 nt downstream.Examination of the sequences of the small SL proteins from Ascaris and various Caenorhabditis species demonstrates that the N-terminal region is highly conserved; it contains two copies of a novel repeat. Since all of these proteins interact with SL-75p, this region is likely to be a binding site for the SL-75 protein. In contrast, the C-termini of SL-21p and SL-26p are unrelated to each other, even though both C-termini are highly conserved within the Caenorhabditis genus. We hypothesize that the C-terminus of each protein associates with a different partner to mediate SL1 and SL2-specific functions.

Philippe, L. et al. (2013) International Worm Meeting "A global genetic screen for the identification of factors involved in C. elegans spliced leader trans-splicing."

Connolly, B., Muller, B., Pettitt, J., Philippe, L.

[International Worm Meeting, 2013]

Spliced-leader (SL) trans-splicing is the precise joining of a short exonic sequence onto the 5' end of pre-mRNAs. In C. elegans, this splice reaction modifies mRNAs from ~70% of protein coding genes. C. elegans has two types of SL RNAs, SL1 and SL2. SL2 plays a crucial role in the resolution of operon transcripts. These polycistronic pre-mRNAs are processed into monocistronic RNAs through a mechanism involving SL2, allowing RNAs to be separated and capped. In contrast, SL1 is added to mRNAs encoded by monocistronic genes and also to the 5' end of operon transcripts. It is known from work in Ascaris and C. elegans that SL trans-splicing involves components of the splicing machinery as well as trans-splicing-specific factors. But despite the fact that the basic steps in spliced leader trans-splicing have been described and are similar to cis-splicing, how these steps are achieved at the molecular level is poorly understood. To identify new genes involved in SL trans-splicing, we have recently developed a novel and sensitive screening strategy. This strategy allows us, for the first time in any experimental system, to visualize loss of SL trans-splicing in vivo, based on a GFP reporter. We have validated our assay by showing that it is able to detect sub-lethal defects in this process, and have used it to show the involvement of three proteins previously implicated in in vitro experiments. We are now carrying out genetic screens to identify genes involved in SL trans-splicing. To date we have identified two mutant strains that display defects in SL trans-splicing and are currently charactering the molecular lesions that cause reduced SL trans-splicing in these strains.

Daigle SN et al. (1996) East Coast Worm Meeting "Transcription and Protein Localization of C. elegans Annexin Homologs nex-1, nex-2, and nex-3"

Snyder SL, Daigle SN, Redick J, Creutz CE

[East Coast Worm Meeting, 1996]

Annexins are a family of ubiquitous calcium-dependent phospholipid-binding proteins. The in vitro biochemical activities and localization of annexin proteins within mammalian systems suggest that annexins function in membrane trafficking, collagen deposition, and extracellular matrix formation. We have been developing the nematode Caenorhabditis elegans for use as a system for genetic analysis to investigate the in vivo function of annexins. We first isolated a 32 kDa protein from the soluble extract of a C. elegans culture by virtue of its ability to bind to phospholipid membranes in the presence of calcium. Peptide sequencing identified the 32 kDa protein as an annexin. Using an anti-nex-1 antiserum screen the annexin gene was cloned from a gt11 expression library. We sequenced the gene and named it nex-1. All of the peptides that were sequenced from the isolated 32 kDa protein originated from nex-1 protein. Two other annexin genes (nex-2 and nex-3) were identified by the nematode genomic sequencing project. However, protein from the nex-2 or nex-3 genes was never isolated from a mixed culture of nematodes. We use RT-PCR to detect mRNA from each of nex-1, nex-2, and nex-3, verifying that each of the known C. elegans annexin genes is transcribed. We also use RT-PCR to demonstrate that nex-1 mRNA and nex-3 mRNA are trans-spliced to splice leader sequence SL-1, while nex-2 mRNA is not trans-spliced. Previously, we had employed immunofluorescence and electron microscopy using immunogold labeling to localize nex-1 protein within adult N2 C. elegans. The nex-1 protein localized to the secretory gland cells in the pharynx, the uterine wall, the membranous folds of the spermathecal valve, and the yolk granules in maturing oocytes and is found in association with the grinder in the pharynx. To further investigate the localization of nex-1 protein and to begin examining the localization of the nex-3 protein, we have prepared constructs that will express the Green Fluorescent Protein under control of the nex-1 or nex-3 promoter, respectively. To begin examining the in vivo function of nex-1, we have developed a construct that will express nex-1 anti-sense RNA that, when expressed in C. elegans, will inhibit or attenuate nex-1 protein translation.

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.

Huang X-Y et al. (1991) International C. elegans Meeting "BIOCHEMICAL CHARACTERIZATION OF TWO PROTEINS SPECIFICALLY ASSOCIATED WITH THE TRANS-SPLICED LEADER"

Hirsh DI, Huang X-Y

[International C. elegans Meeting, 1991]

RNA trans-splicing in nematodes involves the transfer of an evolutionarily conserved 22-nucleotide spliced-leader (SL) and a trimethylguanosine (TMG) cap to the 5'-end of the recipient mRNA. The physiological role of trans-splicing, of the SL and the biological effect of TMG cap at the 5' ends of mRNAs are not yet understood. The possibility has been raised that SL itself has a catalytic role in splicing. The TMG cap has recently been shown to be an essential nuclear targeting signal. We hope to learn the functions of SL or transsplicing by identifying proteins specifically bound to SL and elucidating their functions. Electrophoretic mobility shift assays combined with competition analysis showed two proteins SLBP1 and SLPB2, bind specifically to SL1. The binding can be stimulated by a cap structure at the 5' end of SL. Although SLBP2 can bind SL1 RNA, SLBP1 seems unable to bind SL1 RNA. This may be due to the highly structured SL1 RNA interfering SLBP1 binding. We are presently testing the binding of SLBP1 and SLBP2 to SL2. UV cross-linking and SDS-PAGE revealed that SLBP1 has a molecular mass of 57kD and SLBP2 of 30kD. Nitrocellulose filter retention assay showed that the binding between SLBP1 and SL1, SLBP2 and SL1 conforms to a simple biomolecular reaction. SLBP1 has been purified to homogeneity as judged by silver staining on SDS-PAGE. Gel filtration analysis indicates that SLBP1 exists as a monomer and a dimer in solution. However we do not know whether dimerization is required for RNA binding. We intend to isolate their genes and generate antibodies against SLBP1 and SLBP2 to further characterize their biochemical properties and biological functions.

Cheng, X. et al. (2019) International Worm Meeting "Sphingolipidomic Analysis of C. elegans Reveals Development- and Environment-dependent Metabolic Features."

Cheng, X., Jiang, X., Zhang, H.

[International Worm Meeting, 2019]

Sphingolipids (SLs) serve as structural and signaling molecules in regulating various cellular events and growth. Given that SLs contain various bioactive species possessing distinct roles, quantitative analysis of sphingolipidome is essential for elucidating their differential requirement during development. Herein we developed a comprehensive sphingolipidomic profiling approach using liquid chromatography-mass spectrometry coupled with multiple reaction monitoring mode (LC-MS-MRM). SL profiling of C. elegans revealed organism-specific, development-dependent and environment-driven metabolic features. We showed for the first time a predominance of ceramide-1-phosphate (C1P) and the presence of a series of sphingoid bases in C. elegans sphingolipidome. Moreover, we successfully resolved growth-, temperature- and nutrition-dependent SL profiles at both individual metabolite-level and network-level. Sphingolipidomic analysis uncovered significant SL composition changes throughout development, with GluCers/C1Ps ratios increasing from L1- to L2-stage followed by a gradual decrease thereafter whereas total sphingolipid levels exhibiting opposing trends. We also identified a temperature-dependent alteration in C1Ps/(GluCers+SMs), suggesting an organism-specific strategy for environmental adaptation. Finally, we found serine-biased GluCer increases between serine- versus alanine-supplemented worms. Our study builds a "reference" resource for future SL analysis in the worm, provides insights into natural variability and plasticity of eukaryotic multicellular sphingolipidome and is highly valuable for investigating their functional significance. Acknowledgements: Work in our lab is supported by the Science and Technology Development Fund, Macao S.A.R. (FDCT) project 060/2015/A2, 018/2017/AMJ and 050/2018/A2, Multi-Year Research Grant, University of Macau (MYRG) projects 2016-00066-FHS and 2017-00082-FHS.

D Evans et al. (1999) International C. elegans Meeting "The sequence of the SL2 RNA spliced leader is not required for transcription or trans-splicing"

D Evans, T Blumenthal

[International C. elegans Meeting, 1999]

C. elegans utilizes a specialized spliced leader RNA, SL2 RNA, to process the products of downstream genes in operons. By comparative phylogenetic analysis of the 22 known SL2 RNA genes, we have identified conserved sequences: the SL 5' end, the trans -splice site, the Sm-binding site, and the top of the third stem-loop. To study the importance of these regions on expression and trans -splicing specificity, we expressed SL2 RNA variants in transgenic worms and measured trans -splicing to gpd-3 mRNA. Unlike SL1 RNA, SL2 RNA has no promoter element located within the SL sequence. Rather, expression is dependent on a proximal sequence element, similar to those of the snRNA genes. It had been hypothesized that base-pairing within the SL2 RNA, between the SL and the trans -splice site and bases just downstream, mimics that found between the 5' splice site and U1 snRNA, which plays no role in trans -splicing. To test this idea in SL2 trans -splicing, we mutated the 20 nt at the 5' end of the 22 nt SL. Since the predicted secondary structure is conserved among the natural variants of SL2 RNA, we expected that this large mutation would prevent trans -splicing due to an RNA structural change. Therefore, we also made mutations with compensatory changes that would allow formation of this first stem. Interestingly, the first mutation, which mutates nearly the entire SL, was nevertheless trans -spliced. Even more striking was the trans -splicing of mutant SL2 RNAs with changes in most of the first stem and loop, demonstrating that large mutations to the 'intron' sequences of SL2 RNA are also tolerated. In contrast, mutations to the conserved top of the third stem-loop do prevent trans -splicing, even though the mutant SL2 RNA is expressed at a high level, demonstrating the importance of this conserved sequence to SL2 RNA function. The top of the third stem-loop of SL1 RNA is not conserved, suggesting that SL2 RNA may have evolved a key function at this location.

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.

Pranali P Pathare et al. (2007) International Worm Meeting "Coordinate regulation of sphingolipid metabolism by a novel nuclear receptor partnership."

Marc Van Gilst, Pranali P Pathare

[International Worm Meeting, 2007]

The C. elegans NHR-49 is a lipid sensing nuclear receptor that has been shown to orchestrate multiple aspects of fat homeostasis, including mitochondrial <font face=symbol>b</font>-oxidation and fatty acid desaturation. To more comprehensively define the targets of NHR-49, we exploited global gene expression profiling. These experiments revealed a previously uncharacterized role for NHR-49 in the regulation of genes involved in membrane lipid metabolism, particularly glycosphingolipid processing. Indeed, deletion of nhr-49 resulted in dramatically increased expression of several sphingolipid (SL) metabolism genes, as well as other factors likely to influence phospholipid processing. Sphingolipids are important structural components of cell membranes as well as potent signaling molecules implicated in apoptosis, cell division and differentiation. However, little is known about the transcription factors that control SL gene expression. Consistent with NHR-49s impact on SL metabolism genes, biochemical analysis revealed a significantly altered sphingolipid profile in nhr-49 knockout mutants. Therefore, our findings reveal a new role for NHR-49 in the control of sphingolipid composition. Two hybrid studies have found that NHR-49 can bind to many different C. elegans NHRs1. Since nuclear receptors often regulate gene expression by forming heterodimers, we examined these potential binding partners to determine if they impacted NHR-49 gene regulation. Notably, we found that knockout of nhr-66 affected only a subset of NHR-49 targets. In particular, nhr-66 was exclusively required for the repression of NHR-49s sphingolipid and phospholipid metabolism targets. In contrast, deletion of nhr-66 did not impact NHR-49 activation of <font face=symbol>b</font>-oxidation and fatty acid desaturation genes. Thus, our findings support a model whereby NHR-49 and NHR-66 heterodimerize to mediate the repression of genes involved in membrane lipid composition. Therefore, NHR-49 independently regulates multiple lipid metabolism pathways, and the distinct lipid metabolism networks targeted by NHR-49 are determined by dimerization with different NHR partners. (1)Taubert S, Van Gilst M, Hansen M, and Yamamoto KR. (2006) Genes Dev 20, 1137-1149.