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[
Worm Breeder's Gazette,
1992]
Background. The extracellular matrix (ECM) plays an important role in maintaining the structural integrity and cellular functions of a multicellular organism. ECM components, including collagens, fibronectin, laminin, vitronectin, tenascin, entactin, and proteoglycans, have been identified and characterized in mammalian tissues. However, the structure and organization of these components in the intact matrix remain unclear. The inability to isolate mutants that are defective in a single ECM component in higher organisms impedes this type of analysis in vivo. With the advantage of the well-characterized genetics in C. elegans, we initiated a project to study the organization and structure of ECM using C. elegans as a model system. Our approach was to generate and characterize a panel of monoclonal antibodies against C. elegans ECM to use as markers for structural studies including immunofluorescent microscopy. In the future, we plan to use these monoclonal antibodies in affinity chromatography for the purification of ECM components and to use as probes for isolating cDNA. Experimental Approach. The ECM components were extracted from a mixed population of adult and juvenile C. elegans using the procedure shown in Figure 1. [See Figure 1] BALB/c mice were immunized intraperitoneally with 100 g of ECM extract homogenized in complete Freund's adjuvant followed by three bi-weekly injections of 50 g of ECM extract in incomplete Freund's adjuvant. Spleen cells from immunized mice were fused with myeloma cells and plated at a concentration of 2.5x10 +E5cells/well. Results. Of the 1,200 wells screened.approximately 32% of the hybridoma supernatants tested positive in ELISA against C. elegans ECM extracts. Supernatants from positive wells were tested by immunoblotting against C. elegans ECM extracts and by immunofluorescent microscopy on whole or fragmented C. elegans Protein species ranging from 20,000-180,000 daltons were detected by the hybridoma supernatants in immunoblotting, and supernatant recognition ranged from complex patterns of multiple bands to a single band. Immunofluorescent studies also revealed diverse staining patterns, which included staining between muscle and hypodermis, around muscle bundles, in layers surrounding intestines and gonads, and around the cuticle. Seven of the hybridomas were purified by limiting dilution cloning and injected into mice for ascites tumor production. We would like to make these monoclonal antibodies available to interested investigators in the near future. (This project was part of a course, Biotechnology Laboratory: Molecular Recognition (BSC 352), which was composed of 70% graduate students and 30% undergraduate students, and was supported by the Department of Biological Sciences and the College of Arts and Sciences at Illinois State University. We also would like to thank Robert Barstead for providing worm fragments and helpful discussion.)
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[
Mol Cell Biol,
1989]
The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a approximately 110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.
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[
Nature,
1988]
Maturation of some messenger RNAs in the nematode Caenorhabditis elegans involves the acquisition of a 22-base leader at their 5' ends. This 22-base leader, called the spliced leader (SL), is derived from the 5' end of a precursor RNA of 90-100 bases, called spliced leader RNA (SL RNA). SL RNA is transcribed from a 1-kilobase DNA repeat which also encodes the 5S ribosomal RNA. A subset of mRNAs in C. elegans acquire SL from SL RNA by a trans-splicing mechanism. SL behaves as a 5' exon in the trans-splicing reaction. Using antisera against the Sm antigen that is associated with small nuclear ribonucleoprotein particles (snRNPs), we precipitated SL RNA from extracts of C. elegans, indicating that it is bound by the Sm antigen in vivo. SL RNA also possesses the unique trimethylguanosine (
m32,2,7G) cap characteristic of most small nuclear RNAs. Therefore, SL RNA is a chimaeric molecule, made up of an snRNA attached to a 5' exon and is a constituent of a snRNP.
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[
RNA,
1996]
The 5'' exon donor in nematode trans-splicing, the SL RNA, is a small (approximately 100 nt) RNA that resembles cis-spliceosomal U snRNAs. Extensive analyses of the RNA sequence requirements for SL RNA function have revealed four essential elements, the core Sm binding site, three nucleotides immediately downstream of this site, a region of Stem-loop II, and a 5'' splice site. Although these elements are necessary and sufficient for SL RNA function in vitro, their respective roles in promoting SL RNA activity have not been elucidated. Furthermore, although it has been shown that assembly of the SL RNA into an Sm RNP is a prerequisite for function, the protein composition of the SL RNP has not been determined. Here, we have used oligoribonucleotide affinity to purify the SL RNP and find that it contains core Sm proteins as well as four specific proteins (175, 40, 30, and 28 kDa). Using in vitro assembly assays; we show that association of the 175- and 30-kDa SL-specific proteins correlates with SL RNP function in trans-splicing. Binding of these proteins depends upon the sequence of the core Sm binding site; SL RNAs containing the U1 snRNA Sm binding site assemble into Sm RNPs that contain core, but not SL-specific proteins. Furthermore, mutational and thiophosphate interference approaches reveal that both the primary nucleotide sequence and a specific phosphate oxygen within a segment of Stemloop II of the SL RNA are required for function. Finally, mutational activation of an unusual cryptic 5'' splice site within the SL sequence itself suggests that U5 snRNA may play a primary role in selecting and specifying the 5'' splice site in SL addition trans-splicing.
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[
Sci Rep,
2019]
Spliced leader trans-splicing (SLTS) plays a part in the maturation of pre-mRNAs in select species across multiple phyla but is particularly prevalent in Nematoda. The role of spliced leaders (SL) within the cell is unclear and an accurate assessment of SL occurrence within an organism is possible only after extensive sequencing data are available, which is not currently the case for many nematode species. SL discovery is further complicated by an absence of SL sequences from high-throughput sequencing results due to incomplete sequencing of the 5'-ends of transcripts during RNA-seq library preparation, known as 5'-bias. Existing datasets and novel methodology were used to identify both conserved SLs and unique hypervariable SLs within Heterodera glycines, the soybean cyst nematode. In H. glycines, twenty-one distinct SL sequences were found on 2,532 unique H. glycines transcripts. The SL sequences identified on the H. glycines transcripts demonstrated a high level of promiscuity, meaning that some transcripts produced as many as nine different individual SL-transcript combinations. Most uniquely, transcriptome analysis revealed that H. glycines is the first nematode to demonstrate a higher SL trans-splicing rate using a species-specific SL over well-conserved Caenorhabditis elegans SL-like sequences.
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[
Gigascience,
2018]
Background: The spliceosomal transfer of a short spliced leader (SL) RNA to an independent pre-mRNA molecule is called SL trans-splicing and is widespread in the nematode C. elegans. While RNA-seq data contain information on such events, properly documented methods to extract them are lacking. Findings: To address this, we developed SL-quant, a fast and flexible pipeline that adapts to paired-end and single-end RNA-seq data and accurately quantifies SL trans-splicing events. It is designed to work downstream of read mapping and uses the reads left unmapped as primary input. Briefly, the SL-sequences are identified with high specificity and are trimmed from the input reads, which are then re-mapped on the reference genome and quantified at the nucleotide position level (SL trans-splice sites) or at the gene level. Conclusions: SL-quant completes within 10 minutes on a basic desktop computer for typical C.elegans RNA-seq datasets, and can be applied to other species as well. Validating the method, the SL trans-splice sites identified display the expected consensus sequence and the results of the gene-level quantification are predictive of the gene position within operons. We also compared SL-quant to a recently published SL-containing read identification strategy which revealed being more sensitive, but less specific than SL-quant. Both methods are implemented as a bash script available under the MIT licence at https://github.com/cyaguesa/SL-quant. Full instructions for its installation, usage, and adaptation to other organisms are provided.
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[
Nature,
2002]
Pre-messenger-RNA maturation in nematodes and in several other lower eukaryotic phyla involves spliced leader (SL) addition trans-splicing. In this unusual RNA processing reaction, a short common 5'' exon, the SL, is affixed to the 5''-most exon of multiple pre-mRNAs. The nematode SL is derived from a trans-splicing-specific approximately 100-nucleotide RNA (SL RNA) that bears striking similarities to the cis-spliceosomal U small nuclear RNAs U1, U2, U4 and U5 (refs 3, 4); for example, the SL RNA functions only if it is assembled into an Sm small nuclear ribonucleoprotein (snRNP). Here we have purified and characterized the SL RNP and show that it contains two proteins (relative molecular masses 175,000 and 30,000 (M(r) 175K and 30K)) in addition to core Sm proteins. Immunodepletion and reconstitution with recombinant proteins demonstrates that both proteins are essential for SL trans-splicing; however, neither protein is required either for conventional cis-splicing or for bimolecular (trans-) splicing of fragmented cis constructs. The M(r) 175K and 30K SL RNP proteins are the first factors identified that are involved uniquely in SL trans-splicing. Several lines of evidence indicate that the SL RNP proteins function by participating in a trans-splicing specific network of protein protein interactions analogous to the U1 snRNP SF1/BBP U2AF complex that comprises the cross-intron bridge in cis-splicing.
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[
Nucleic Acids Res,
1988]
A 22 nt spliced leader (SL) is added to some C. elegans mRNAs by a process of discontinuous RNA synthesis. The SL is synthesized as part of a larger precursor RNA, the leader RNA (LR) which is about 100 nt long.
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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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[
Mol Cell Biol,
2010]
Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.