| 1. |
- Aspegren, Anders, et al.
(författare)
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Novel non-coding RNAs in Dictyostelium discoideum and their expression during development
- 2004
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 32:15, s. 4646-4656
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Tidskriftsartikel (refereegranskat)abstract
- The quest for non-coding RNAs (ncRNAs) in the last few years has revealed a surprisingly large number of small RNAs belonging to previously known as well as entirely novel classes. Computational and experimental approaches have uncovered new ncRNAs in all kingdoms of life. In this work, we used a shotgun cloning approach to construct full-length cDNA libraries of small RNAs from the eukaryotic model organism Dictyostelium discoideum. Interestingly, two entirely novel classes of RNAs were identified of which one is developmentally regulated. The RNAs within each class share conserved 5'- and 3'-termini that can potentially form stem structures. RNAs of both classes show predominantly cytoplasmic localization. In addition, based on conserved structure and/or sequence motifs, several of the identified ncRNAs could be divided into classes known from other organisms, e.g. 18 small nucleolar RNA candidates (17 box C/D, of which a few are developmentally regulated, and one box H/ACA). Two ncRNAs showed a high degree of similarity to the small nuclear U2 RNA and signal recognition particle RNA (SRP RNA), respectively. Furthermore, the majority of the regions upstream of the sequences encoding the isolated RNAs share conserved motifs that may constitute new promoter elements.
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| 2. |
- Crona, Mikael, et al.
(författare)
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A Rare Combination of Ribonucleotide Reductases in the Social Amoeba Dictyostelium discoideum
- 2013
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 288:12, s. 8198-8208
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) catalyze the only pathway for de novo synthesis of deoxyribonucleotides needed for DNA replication and repair. The vast majority of eukaryotes encodes only a class I RNR, but interestingly some eukaryotes, including the social amoeba Dictyostelium discoideum, encode both a class I and a class II RNR. The amino acid sequence of the D. discoideum class I RNR is similar to other eukaryotic RNRs, whereas that of its class II RNR is most similar to the monomeric class II RNRs found in Lactobacillus spp. and a few other bacteria. Here we report the first study of RNRs in a eukaryotic organism that encodes class I and class II RNRs. Both classes of RNR genes were expressed in D. discoideum cells, although the class I transcripts were more abundant and strongly enriched during mid-development compared with the class II transcript. The quaternary structure, allosteric regulation, and properties of the diiron-oxo/radical cofactor of D. discoideum class I RNR are similar to those of the mammalian RNRs. Inhibition of D. discoideum class I RNR by hydroxyurea resulted in a 90% reduction in spore formation and decreased the germination viability of the surviving spores by 75%. Class II RNR could not compensate for class I inhibition during development, and an excess of vitamin B12 coenzyme, which is essential for class II activity, did not improve spore formation. We suggest that class I is the principal RNR during D. discoideum development and growth and is important for spore formation, possibly by providing dNTPs for mitochondrial replication.
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| 3. |
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| 5. |
- Dierker, Tabea, et al.
(författare)
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Nematodes join the family of chondroitin sulfate-synthesizing organisms : Identification of an active chondroitin sulfotransferase in Caenorhabditis elegans
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- Proteoglycans are proteins that carry sulfated glycosaminoglycans (GAGs). They help form and maintain morphogen gradients, guiding cell migration and differentiation during animal development. While no sulfated GAGs have been found in marine sponges, chondroitin sulfate (CS) and heparan sulfate (HS) have been identified in Cnidarians, Lophotrocozoans and Ecdysozoans. The general view that nematodes such as Caenorhabditis elegans, which belong to Ecdysozoa, produce HS but only chondroitin without sulfation has therefore been puzzling. We have analyzed GAGs in C. elegans using reversed-phase ion-pairing HPLC, mass spectrometry and immunohistochemistry. Our analyses included wild type C. elegans but also a mutant lacking two HS sulfotransferases (hst-6 hst-2), as we suspected that the altered HS structure could boost CS sulfation. We could indeed detect sulfated CS in both wild type and mutant nematodes. While 4-O-sulfation of galactosamine dominated, we also detected 6-O-sulfated galactosamine residues. Finally, we identified the product of the gene C41C4.1 as a C. elegans CS-sulfotransferase and renamed it chst-1 (CarboHydrate SulfoTransferase) based on loss of CS-4-O-sulfation in a C41C4.1 mutant and in vitro sulfotransferase activity of recombinant C41C4.1 protein. We conclude that C. elegans indeed manufactures CS, making this widely used nematode an interesting model for developmental studies involving CS.
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| 6. |
- Dube, Faruk, et al.
(författare)
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Gene co-expression network analysis reveal core responsive genes in Parascaris univalens tissues following ivermectin exposure
- 2024
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 19:2
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Tidskriftsartikel (refereegranskat)abstract
- Anthelmintic resistance in equine parasite Parascaris univalens, compromises ivermectin (IVM) effectiveness and necessitates an in-depth understanding of its resistance mechanisms. Most research, primarily focused on holistic gene expression analyses, may overlook vital tissue-specific responses and often limit the scope of novel genes. This study leveraged gene co-expression network analysis to elucidate tissue-specific transcriptional responses and to identify core genes implicated in the IVM response in P. univalens. Adult worms (n = 28) were exposed to 10−11 M and 10−9 M IVM in vitro for 24 hours. RNA-sequencing examined transcriptional changes in the anterior end and intestine. Differential expression analysis revealed pronounced tissue differences, with the intestine exhibiting substantially more IVM-induced transcriptional activity. Gene co-expression network analysis identified seven modules significantly associated with the response to IVM. Within these, 219 core genes were detected, largely expressed in the intestinal tissue and spanning diverse biological processes with unspecific patterns. After 10−11 M IVM, intestinal tissue core genes showed transcriptional suppression, cell cycle inhibition, and ribosomal alterations. Interestingly, genes PgR028_g047 (sorb-1), PgB01_g200 (gmap-1) and PgR046_g017 (col-37 & col-102) switched from downregulation at 10−11 M to upregulation at 10−9 M IVM. The 10−9 M concentration induced expression of cuticle and membrane integrity core genes in the intestinal tissue. No clear core gene patterns were visible in the anterior end after 10−11 M IVM. However, after 10−9 M IVM, the anterior end mostly displayed downregulation, indicating disrupted transcriptional regulation. One interesting finding was the non-modular calcium-signaling gene, PgR047_g066 (gegf-1), which uniquely connected 71 genes across four modules. These genes were enriched for transmembrane signaling activity, suggesting that PgR047_g066 (gegf-1) could have a key signaling role. By unveiling tissue-specific expression patterns and highlighting biological processes through unbiased core gene detection, this study reveals intricate IVM responses in P. univalens. These findings suggest alternative drug uptake of IVM and can guide functional validations to further IVM resistance mechanism understanding.
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| 7. |
- Dube, Faruk, et al.
(författare)
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Ivermectin-induced gene expression changes in adult Parascaris univalens and Caenorhabditis elegans : a comparative approach to study anthelminthic metabolism and resistance in vitro
- 2022
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Ingår i: Parasites & Vectors. - : Springer Nature. - 1756-3305. ; 15:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: The nematode Parascaris univalens is one of the most prevalent parasitic pathogens infecting horses but anthelmintic resistance undermines treatment approaches. The molecular mechanisms underlying drug activity and resistance remain poorly understood in this parasite since experimental in vitro models are lacking. The aim of this study was to evaluate the use of Caenorhabditis elegans as a model for P. univalens drug metabolism/resistance studies by a comparative gene expression approach after in vitro exposure to the anthelmintic drug ivermectin (IVM).Methods: Twelve adult P. univalens worms in groups of three were exposed to ivermectin (IVM, 10(-13) M, 10(-11) M, 10(-9 )M) or left unexposed for 24 h at 37 degrees C, and total RNA, extracted from the anterior end of the worms, was sequenced using Illumina NovaSeq. Differentially expressed genes (DEGs) involved in metabolism, transportation, or gene expression with annotated Caemorhabditis elegans orthologues were identified as candidate genes to be involved in IVM metabolism/resistance. Similarly, groups of 300 adult C. elegans worms were exposed to IVM (10(-9) M, 10(-8) M and 10(-7) M) or left unexposed for 4 h at 20 degrees C. Quantitative RT-PCR of RNA extracted from the C. elegans worm pools was used to compare against the expression of selected P. univalens candidate genes after drug treatment.Results: After IVM exposure, 1085 DEGs were found in adult P. univalens worms but the relative gene expression changes were small and large variabilities were found between different worms. Fifteen of the DEGs were chosen for further characterization in C. elegans after comparative bioinformatics analyses. Candidate genes, including the putative drug target Igc-37, responded to IVM in P. univalens, but marginal to no responses were observed in C. elegans despite dose-dependent behavioral effects observed in C. elegans after IVM exposure. Thus, the overlap in IVM-induced gene expression in this small set of genes was minor in adult worms of the two nematode species.Conclusion: This is the first time to our knowledge that a comparative gene expression approach has evaluated C. elegans as a model to understand IVM metabolism/resistance in P. univalens. Genes in P. univalens adults that responded to IVM treatment were identified. However, identifying conserved genes in P. univalens and C. elegans involved in IVM metabolism/resistance by comparing gene expression of candidate genes proved challenging. The approach appears promising but was limited by the number of genes studied (n = 15). Future studies comparing a larger number of genes between the two species may result in identification of additional candidate genes involved in drug metabolism and/or resistance.
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| 8. |
- Dube, Faruk, et al.
(författare)
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Transcriptomics of ivermectin response in Caenorhabditis elegans : Integrating abamectin quantitative trait loci and comparison to the Ivermectin-exposed DA1316 strain
- 2023
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 18:5
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Tidskriftsartikel (refereegranskat)abstract
- Parasitic nematodes pose a significant threat to human and animal health, as well as cause economic losses in the agricultural sector. The use of anthelmintic drugs, such as Ivermectin (IVM), to control these parasites has led to widespread drug resistance. Identifying genetic markers of resistance in parasitic nematodes can be challenging, but the free-living nematode Caenorhabditis elegans provides a suitable model. In this study, we aimed to analyze the transcriptomes of adult C. elegans worms of the N2 strain exposed to the anthelmintic drug Ivermectin (IVM), and compare them to those of the resistant strain DA1316 and the recently identified Abamectin Quantitative Trait Loci (QTL) on chromosome V. We exposed pools of 300 adult N2 worms to IVM (10(-7) and 10(-8) M) for 4 hours at 20 degrees C, extracted total RNA and sequenced it on the Illumina NovaSeq6000 platform. Differentially expressed genes (DEGs) were determined using an in-house pipeline. The DEGs were compared to genes from a previous microarray study on IVM-resistant C. elegans and Abamectin-QTL. Our results revealed 615 DEGs (183 up-regulated and 432 down-regulated genes) from diverse gene families in the N2 C. elegans strain. Of these DEGs, 31 overlapped with genes from IVM-exposed adult worms of the DA1316 strain. We identified 19 genes, including the folate transporter (folt-2) and the transmembrane transporter (T22F3.11), which exhibited an opposite expression in N2 and the DA1316 strain and were deemed potential candidates. Additionally, we compiled a list of potential candidates for further research including T-type calcium channel (cca-1), potassium chloride cotransporter (kcc-2), as well as other genes such as glutamate-gated channel (glc-1) that mapped to the Abamectin-QTL.
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| 9. |
- Hinas, Andrea, et al.
(författare)
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Identification of the Major Spliceosomal RNAs in Dictyostelium discoideum Reveals Developmentally Regulated U2 Variants and Polyadenylated snRNAs
- 2006
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Ingår i: Eukaryotic Cell. - 1535-9778 .- 1535-9786. ; 5:6, s. 924-934
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Tidskriftsartikel (refereegranskat)abstract
- Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5' cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5' end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.
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| 10. |
- Hinas, Andrea, et al.
(författare)
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SID-5 Is an Endosome-Associated Protein Required for Efficient Systemic RNAi in C. elegans
- 2012
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Ingår i: Current Biology. - : Elsevier BV. - 0960-9822 .- 1879-0445. ; 22:20, s. 1938-1943
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Tidskriftsartikel (refereegranskat)abstract
- In the nematode C. elegans, RNAi silencing signals are efficiently taken up from the environment and transported between cells and tissues [1-3]. Previous studies implicating endosomal proteins in systemic RNAi lack conclusive evidence [4, 5]. Here, we report the identification and characterization of SID-5, a C. elegans endosome-associated protein that is required for efficient systemic RNAi in response to both ingested and expressed double-stranded RNA (dsRNA). SID-5 is detected in cytoplasmic foci that partially colocalize with GFP fusions of late endosomal proteins RAB-7 and LMP-1. Furthermore, knockdown of various endosomal proteins similarly relocalizes both SID-5 and LMP-1::GFP. Consistent with a non-cell-autonomous function, intestine-specific SID-5 expression restored body wall muscle (bwm) target gene silencing in response to ingested dsRNA. Finally, we show that sid-5 is required for the previously described sid-/-independent transport of ingested RNAi triggers across the intestine [6]. Together, these data demonstrate that an endosome-associated protein, SID-5, promotes the transport of RNAi silencing signals between cells. Furthermore, SID-5 acts differently than the previously described SID-1, SID-2, and SID-3 proteins [3, 6-8], thus expanding the systemic RNAi pathway.
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