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[
International C. elegans Meeting,
1997]
The physical map of the 100 Mb C. elegans genome consists of 17,600 mapped cosmids and 3,000 mapped YACs, which together contain in excess of 95% of the genome, and more than 99% of the genes. The central gene-rich portions of the chromosomes are well represented in cosmids, and comprise about 60% of the genome. In contrast, the gene-poor chromosomal arms are not well represented in cosmids, and comprise about 40% of the genome. Approximately half of the chromosomal arm regions is represented by cosmids and the other half (or approximately 20% of the genome) is covered only by YAC clones. As the systematic large scale sequencing effort progresses (65% of the genome sequence is finished and another 20% of the genome is in active sequencing), we increasingly have focused our attention on strategies to provide sequence-ready clones for the remainder of the C. elegans genome (see abstract by Alan Coulson). To examine the extent to which these cosmid-lacking regions of the C. elegans genome can be recovered in single copy vectors, we have constructed a genomic library in the fosmid vector pFOS1. Approximately 18,000 C. elegans fosmids have been picked and gridded onto high density filter arrays (courtesy of Genome Systems, Inc., St. Louis, MO). The analysis of radioactive fingerprint data from random C. elegans fosmids and from those identified by direct probing has indicated that we may expect to recover half of the cosmid-lacking regions in fosmid clones. Furthermore, the deletion rate observed in random C. elegans fosmids is much lower than for random cosmids, and we are investigating the stability of selected C. elegans fosmids specific for regions known to delete when cloned in cosmids. Recently, we have generated an in silico fingerprint database derived from finished genomic sequence data and are incorporating agarose gel-based restriction digest data from random C. elegans fosmid clones. This method (see abstract by Marco Marra) could enable rapid identification of potential gap closing fosmid clones using a non-radioactive, high-throughput approach. The current progress of the use of fosmid clones to complement the existing YAC and cosmid coverage of the C. elegans genome will be presented.
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[
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.
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[
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.
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[
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.
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[
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.
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Goth, Henrike, Connolly, Bernadette, Phillippe, Lucas, Muller, Berndt, Pettitt, Jonathan, Sarkar, Debjani
[
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.
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[
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.
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[
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.
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[
International C. elegans Meeting,
1995]
Twelve contigs of cosmids and yeast artificial chromosomes (YACs) span more than 95Mb of the 100Mb C.elegans genome. 650 markers link the physical and genetic maps.Hybridisation of tag-sequenced cDNA clones to a map-representative set of YACs indicates that the map incorporates in excess of 99.8% of genes. The map is accessible in ACeDB. We (S.C.) are investigating the representation by bacterial artificial chromosomes (BACs) of regions of the genome not represented by cosmids. Two grids of YACs, of 958 clones ('Poly2') and 223 clones ('Suppoly') are available on request. The latter represents regions of the genome that have been characterised or better defined since the selection of clones for the former. Cosmid clones and YAC grids are available from the Sanger Centre (requests to alan@sanger.ac.uk; FAX 01223 494919). YAC clones and 'cm' series cDNA clones are available from the Sanger Centre or Washington University (rw@nematode. wustl.edu; FAX 314 362 2985).
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Connolly, Bernadette, Fasimoye, Rotimi, Wenzel, Marius, Eiljers, Peter, Soto-Martin, Eva, Pettitt, Jonathan, Muller, Berndt, Elmassoudi, Haitem, 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.