Understanding the mechanism of a nematode molecular Achilles' heel.
Lead Research Organisation:
University of Aberdeen
Department Name: Sch of Medicine, Medical Sci & Nutrition
Abstract
Parasitic nematodes are major challenges to human health and global food security. The impact of these animals is exacerbated by drug resistance, withdrawal of existing treatments due to environmental concerns, and expansion of parasites into new environments through climate change and human migration. There is thus an urgent need to develop new control methods. Ideal targets for the development of new therapeutics would be molecules and processes that are found only in nematodes and are absent from the animals and plants that they infect. Ideally these targets would be found in all nematodes, enabling the development of a drug active against a broad range of nematode infections. One such process is spliced leader trans-splicing, which is an essential step by which many nematode genes become active.
We have shown that two 'molecular machines' are required for spliced leader trans-splicing. Nematodes in which these machines are non-functional stop developing and are infertile. However, we don't have the detailed knowledge of these machines needed to understand how they function, a necessary precondition to properly develop drugs that inhibit them. In particular, we don't know all the components involved, and we don't understand those that we do know about. To address these challenges, we are systematically studying the components of these machines in the experimental nematode Caenorhabditis elegans. Our work so far has led to the identification of a new component which has properties that make it an ideal potential drug target.
The proposed research will extend our initial studies, to give a comprehensive picture of the molecular machinery that underpins this essential step in nematode gene expression. The process of spliced leader trans-splicing has evolved multiple times in different groups of organisms, many of which are also important parasites. Thus, as well as allowing the development of potential new drugs to treat nematode infections, our findings will serve as a starting point to investigate the similarities and differences between other 'versions' of trans-splicing.
We have shown that two 'molecular machines' are required for spliced leader trans-splicing. Nematodes in which these machines are non-functional stop developing and are infertile. However, we don't have the detailed knowledge of these machines needed to understand how they function, a necessary precondition to properly develop drugs that inhibit them. In particular, we don't know all the components involved, and we don't understand those that we do know about. To address these challenges, we are systematically studying the components of these machines in the experimental nematode Caenorhabditis elegans. Our work so far has led to the identification of a new component which has properties that make it an ideal potential drug target.
The proposed research will extend our initial studies, to give a comprehensive picture of the molecular machinery that underpins this essential step in nematode gene expression. The process of spliced leader trans-splicing has evolved multiple times in different groups of organisms, many of which are also important parasites. Thus, as well as allowing the development of potential new drugs to treat nematode infections, our findings will serve as a starting point to investigate the similarities and differences between other 'versions' of trans-splicing.
Technical Summary
Spliced leader trans-splicing is an essential gene expression step in many eukaryotes. It occurs by modification of the spliceosome to facilitate the intermolecular reaction between short, non-coding 'spliced leader' RNAs and their target pre-mRNAs. This results in addition of the spliced leader onto the 5' end of the mature mRNA. In nematodes, the majority of trans-splicing events depend on the SL1 and SmY ribonucleoproteins. However, we do not know how these complexes work, nor do we have complete knowledge of their compositions. To address these knowledge gaps, the proposed research will:
1. Determine the molecular composition of the two ribonucleoproteins critical for nematode SL1-type trans-splicing.
2. Define the activity, molecular determinants and structure of SNA-2, the essential protein component of the two ribonucleoproteins.
3. Understand the function of SNA-3, a newly identified essential component of the trans-splicing machinery.
Our research programme combines advantages of the model nematode C. elegans with advanced analytical techniques, including protein identification by mass spectrometry, next generation sequencing and structure analysis by X-ray crystallography and NMR spectroscopy. Validating a key approach - identification of ribonucleoprotein components by immunoprecipitation followed by mass spectrometry - we have identified SNA-3, a new protein involved in trans-splicing. We are in an excellent position for the successful completion of this programme that will provide detailed information about the molecular composition of ribonucleoproteins involved in SL1 trans-splicing, and the molecular function of the key components.
Based on our recent work, which showed trans-splicing is conserved throughout the nematode phylum, this research will have broad impacts on our understanding of nematode biology and mRNA splicing, and inform our understanding of gene expression in other organisms that employ this process.
1. Determine the molecular composition of the two ribonucleoproteins critical for nematode SL1-type trans-splicing.
2. Define the activity, molecular determinants and structure of SNA-2, the essential protein component of the two ribonucleoproteins.
3. Understand the function of SNA-3, a newly identified essential component of the trans-splicing machinery.
Our research programme combines advantages of the model nematode C. elegans with advanced analytical techniques, including protein identification by mass spectrometry, next generation sequencing and structure analysis by X-ray crystallography and NMR spectroscopy. Validating a key approach - identification of ribonucleoprotein components by immunoprecipitation followed by mass spectrometry - we have identified SNA-3, a new protein involved in trans-splicing. We are in an excellent position for the successful completion of this programme that will provide detailed information about the molecular composition of ribonucleoproteins involved in SL1 trans-splicing, and the molecular function of the key components.
Based on our recent work, which showed trans-splicing is conserved throughout the nematode phylum, this research will have broad impacts on our understanding of nematode biology and mRNA splicing, and inform our understanding of gene expression in other organisms that employ this process.
Planned Impact
Findings from this work will impact on the work of researchers that study gene expression in nematodes, and of researchers studying gene expression in a wide range of other eukaryotic organisms. It will inform the work of researchers studying mRNA splicing in other organisms and may impact on researchers working in the areas of synthetic biology and gene therapy.
Our work will impact upon the UK science skills base, and its concomitant effect on the country's economic competitiveness. The project helps address skill deficits in RNA splicing and fundamental biochemistry identified in the "BBSRC and MRC review of vulnerable skills and capabilities". The research workers employed on the project will benefit from training in the research specific skills involved. These skills will be further disseminated throughout the researchers' subsequent careers, ultimately benefitting the wider research base.
Since nematodes are major parasites of humans, livestock and economically important plants, some of the main beneficiaries will be commercial and non-commercial enterprises with an interest in the development of drugs to treat parasitic infections (anthelmintics). Our research is focussed on understanding an essential, conserved nematode gene expression process. Understanding this process will provide novel, nematode-specific targets for the development of new anthelmintics. The need for these drugs in the treatment of livestock infections is acute due to the global incidence of anthelmintic resistance, even to the most recently developed drug, monopantel. However, plant parasitic nematodes represent some of the greatest challenges to global crop production: almost every cultivated crop, for instance, is prone to infection by root knot nematodes. Previous treatments to control plant parasitic nematodes have been withdrawn due to their environmental impacts, so there is increased need to identify new compounds to treat these nematodes.
Impact will be achieved through publication of research findings, deposition of data in relevant archives, and plasmids and C. elegans strains that will be made through appropriate resource centres.
Finally, we plan to increase the societal impact of our research through a series of public engagement activities. Dr Pettitt has extensive, prize-winning expertise that covers a broad range of engagement events. We will exploit the skills and connections that he has previously established to raise awareness of the ecological importance of nematodes, and of their importance for animal welfare and food security.
Our work will impact upon the UK science skills base, and its concomitant effect on the country's economic competitiveness. The project helps address skill deficits in RNA splicing and fundamental biochemistry identified in the "BBSRC and MRC review of vulnerable skills and capabilities". The research workers employed on the project will benefit from training in the research specific skills involved. These skills will be further disseminated throughout the researchers' subsequent careers, ultimately benefitting the wider research base.
Since nematodes are major parasites of humans, livestock and economically important plants, some of the main beneficiaries will be commercial and non-commercial enterprises with an interest in the development of drugs to treat parasitic infections (anthelmintics). Our research is focussed on understanding an essential, conserved nematode gene expression process. Understanding this process will provide novel, nematode-specific targets for the development of new anthelmintics. The need for these drugs in the treatment of livestock infections is acute due to the global incidence of anthelmintic resistance, even to the most recently developed drug, monopantel. However, plant parasitic nematodes represent some of the greatest challenges to global crop production: almost every cultivated crop, for instance, is prone to infection by root knot nematodes. Previous treatments to control plant parasitic nematodes have been withdrawn due to their environmental impacts, so there is increased need to identify new compounds to treat these nematodes.
Impact will be achieved through publication of research findings, deposition of data in relevant archives, and plasmids and C. elegans strains that will be made through appropriate resource centres.
Finally, we plan to increase the societal impact of our research through a series of public engagement activities. Dr Pettitt has extensive, prize-winning expertise that covers a broad range of engagement events. We will exploit the skills and connections that he has previously established to raise awareness of the ecological importance of nematodes, and of their importance for animal welfare and food security.
Publications
Dix TC
(2022)
CMTr mediated 2'-O-ribose methylation status of cap-adjacent nucleotides across animals.
in RNA (New York, N.Y.)
Eijlers P
(2024)
A nematode-specific ribonucleoprotein complex mediates interactions between the major nematode spliced leader snRNP and its target pre-mRNAs
in Nucleic Acids Research
Fasimoye RY
(2022)
A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex.
in Nucleic acids research
Wenzel M
(2020)
Resolution of polycistronic RNA by SL2 trans-splicing is a widely conserved nematode trait.
in RNA (New York, N.Y.)
Wenzel MA
(2021)
SLIDR and SLOPPR: flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data.
in BMC bioinformatics
Description | We have identified key factors that interact with the C. elegans SNA-3 protein involved in spliced leader trans-splicing. These factors provide a first insight into the function of this protein. |
Exploitation Route | Understanding the function of SNA-3 protein will ultimately contribute to the understanding of the molecular machinery executing spliced leader trans-splicing, and may facilitate the development of new anthelminthic drugs. |
Sectors | Agriculture Food and Drink Healthcare Pharmaceuticals and Medical Biotechnology |
Description | International Partnership funding within URKI BBSRC Remit |
Amount | £38,530 (GBP) |
Organisation | United Kingdom Research and Innovation |
Sector | Public |
Country | United Kingdom |
Start | 01/2023 |
End | 03/2023 |
Title | Identification of RNAs associated with the Caenorhabditis elegans SL1 snRNP SNA-1 protein by RIP-Seq |
Description | RIP-Seq data available through ArrayExpress Accession E-MTAB-10289. This study aimed to identify RNAs bound to the C. elegans SL1 snRNP SNA-1 protein through use of RNA immunoprecipitation sequencing. Three IP sample replicates and three control sample replicates were sequenced. |
Type Of Material | Database/Collection of data |
Year Produced | 2022 |
Provided To Others? | Yes |
Impact | Impact has been realised in our group through publication of the data. |
URL | https://www.ebi.ac.uk/biostudies/arrayexpress/studies/E-MTAB-10289?query=%20E-MTAB-10289 |
Title | Molecular characterisation of the SL1 snRNP and other factors involved in spliced leader trans-splicing in the nematode Caenorhabditis elegans using immunoprecipitation and LC-MS/MS. |
Description | Spliced leader trans-splicing is an essential RNA processing step that is required for the formation of mRNA in many eukaryotes, including C. elegans. However, the factors involved in this reaction are not well known. Here we perform a molecular analysis of key components in this reaction by immunoprecipitation of GFP-tagged SNA-1 and SNA-3 proteins from C. elegans embryonic extracts treated with/without RNAseA/T1 followed by the identification of associated proteins using LC-MS/MS and label-free quantification. As control, embryonic extract from wild type N2 animals were also subjected to the same treatment. Note file names (e.g. PE906_SNA1_RNase_IP_anti-GFP_beads) indicate the name of the C. elegans line (PE906_), the name of the protein tagged with GFP (SNA1) and whether samples were treated with RNases A and T1 (RNase), and for raw files whether immunoprecipitation was done with anti-GFP nanobody coupled agarose beads (IP_anti_GFP_beads), or control agarose beads (IP_control_beads), respectively. Note that SNA-3 protein is only identified by its Uniprot identifier Q9GYR5. The data is available at the PRIDE archive. |
Type Of Material | Database/Collection of data |
Year Produced | 2022 |
Provided To Others? | Yes |
Impact | Impact was realised in our group by the publication of the data. |
URL | https://www.ebi.ac.uk/pride/archive?keyword=PXD024763 |
Title | SLIDR and SLOPPR: A suite of two pipelines for flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data |
Description | SLIDR and SLOPPR identify spliced leaders (SLs) from 5'-tails of RNA-Seq reads that are soft-clipped after read alignment to a reference genome or transcriptome. SLIDR (Spliced leader identification from RNA-Seq data) assembles these read tails into full-length SLs and functional SL RNA genes. SLOPPR (Spliced leader-informed operon prediction from RNA-Seq data) searches read tails for a set of known SLs, quantifies SL-containing reads against all genes in the genome and uses SL usage patterns across genes to predict operons. SLOPPR can incorporate known SL specialisation for resolving downstream operonic genes (e.g., SL1/SL2-type SLs in nematodes), infer such specialisation de novo, or handle scenarios without SL specialisation |
Type Of Material | Data analysis technique |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | Impact was realised in our group by publication. |
URL | https://github.com/wenzelm/slidr-sloppr |
Title | SLIDR and SLOPPR: Flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data |
Description | SLIDR and SLOPPR identify spliced leaders (SLs) from 5'-tails of RNA-Seq reads that are soft-clipped after read alignment to a reference genome or transcriptome. SLIDR (Spliced leader identification from RNA-Seq data) assembles these read tails into full-length SLs and functional SL RNA genes. SLOPPR (Spliced leader-informed operon prediction from RNA-Seq data) searches read tails for a set of known SLs, quantifies SL-containing reads against all genes in the genome and uses SL usage patterns across genes to predict operons. SLOPPR can incorporate known SL specialisation for resolving downstream operonic genes (e.g., SL1/SL2-type SLs in nematodes), infer such specialisation de novo, or handle scenarios without SL specialisation. |
Type Of Technology | Software |
Year Produced | 2020 |
Open Source License? | Yes |
Impact | This new software facilitates the identification of operons in eukaryotic genomes, and the analysis of their expression. We have already used this software to investigate nematode genome organisation and expression. |
Description | Genetic Society 2022 Communicating Your Science Workshop |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Professional Practitioners |
Results and Impact | Jonathan Pettitt- - organiser of Genetic Society residential workshop in science communication |
Year(s) Of Engagement Activity | 2022 |