Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum.

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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Molecular cloning and sequencing of ama-1, the gene encoding the largest subunit of Caenorhabditis elegans RNA polymerase II

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4119-4130.1989 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4119-4130

Author(s):

D M Bird ◽

D L Riddle

Keyword(s):

Caenorhabditis Elegans ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Polymerase Iii ◽

Gene Encoding ◽

C Elegans ◽

A Subunit ◽

Rnap Ii ◽

Collagen Genes ◽

Splice Junctions

Two genomic sequences that share homology with Rp11215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster, have been isolated from the nematode Caenorhabditis elegans. One of these sequences was physically mapped on chromosome IV within a region deleted by the deficiency mDf4, 25 kilobases (kb) from the left deficiency breakpoint. This position corresponds to ama-1 (resistance to alpha-amanitin), a gene shown previously to encode a subunit of RNA polymerase II. Northern (RNA) blotting and DNA sequencing revealed that ama-1 spans 10 kb, is punctuated by 11 introns, and encodes a 5.9-kb mRNA. A cDNA clone was isolated and partially sequenced to confirm the 3' end and several splice junctions. Analysis of the inferred 1,859-residue ama-1 product showed considerable identity with the largest subunit of RNAP II from other organisms, including the presence of a zinc finger motif near the amino terminus, and a carboxyl-terminal domain of 42 tandemly reiterated heptamers with the consensus Tyr Ser Pro Thr Ser Pro Ser. The latter domain was found to be encoded by four exons. In addition, the sequence oriented ama-1 transcription with respect to the genetic map. The second C. elegans sequence detected with the Drosophila probe, named rpc-1, was found to encode a 4.8-kb transcript and hybridized strongly to the gene encoding the largest subunit of RNA polymerase III from yeast, implicating rpc-1 as encoding the analogous peptide in the nematode. By contrast with ama-1, rpc-1 was not deleted by mDf4 or larger deficiencies examined, indicating that these genes are no closer than 150 kb. Genes flanking ama-1, including two collagen genes, also have been identified.

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RNA polymerase II CTD S2P is dispensable for embryogenesis but mediates exit from developmental diapause in C. elegans

Science Advances ◽

10.1126/sciadv.abc1450 ◽

2020 ◽

Vol 6 (50) ◽

pp. eabc1450

Author(s):

C. Cassart ◽

C. Yague-Sanz ◽

F. Bauer ◽

P. Ponsard ◽

F. X. Stubbe ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Full Length ◽

C Elegans ◽

Developmental Program ◽

Genome Wide ◽

Trans Splicing ◽

Stimulatory Factor ◽

Rna Polymerase Ii Ctd

Serine 2 phosphorylation (S2P) within the CTD of RNA polymerase II is considered a Cdk9/Cdk12-dependent mark required for 3′-end processing. However, the relevance of CTD S2P in metazoan development is unknown. We show that cdk-12 lesions or a full-length CTD S2A substitution results in an identical phenotype in Caenorhabditis elegans. Embryogenesis occurs in the complete absence of S2P, but the hatched larvae arrest development, mimicking the diapause induced when hatching occurs in the absence of food. Genome-wide analyses indicate that when CTD S2P is inhibited, only a subset of growth-related genes is not properly expressed. These genes correspond to SL2 trans-spliced mRNAs located in position 2 and over within operons. We show that CDK-12 is required for maximal occupancy of cleavage stimulatory factor necessary for SL2 trans-splicing. We propose that CTD S2P functions as a gene-specific signaling mark ensuring the nutritional control of the C. elegans developmental program.

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Mutant Caenorhabditis elegans RNA polymerase II with a 20,000-fold reduced sensitivity to alpha-amanitin.

Genetics ◽

10.1093/genetics/126.4.889 ◽

1990 ◽

Vol 126 (4) ◽

pp. 889-898

Author(s):

T M Rogalski ◽

M Golomb ◽

D L Riddle

Keyword(s):

Caenorhabditis Elegans ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Temperature Sensitive ◽

Wild Type ◽

Cell Nuclei ◽

Spliced Leader ◽

Triton X ◽

Alpha Amanitin

Abstract A doubly mutant ama-1(m118m526) gene results in an RNA polymerase (Rpo) II that is unusually resistant to alpha-amanitin. Rpo II activity in isolated Caenorhabditis elegans cell nuclei is inhibited 50% by alpha-amanitin at a concentration of 150 micrograms/ml, making this enzyme 150 times more resistant to the toxin than Rpo II from the singly mutant allele, ama-1(m118), 20,000 times more resistant than the wild-type Rpo II, and about six times more resistant to amanitin than is Rpo III. It was determined that the SL1 spliced leader precursor is transcribed by Rpo II, and this transcript was used to measure Rpo II activity. The Rpo II activity is unstable in vitro, and the mutant strain has a temperature-sensitive sterile phenotype. The highly resistant double mutant was selected among four million progeny of the mutagenized ama-1(m118) parent by its ability to grow and reproduce in 200 micrograms/ml amanitin in the presence of a permeabilizing agent, Triton X-100.

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The RNA Polymerase II core subunit RPB-9 directs transcriptional elongation at piRNA loci in Caenorhabditis elegans

10.1101/2020.05.01.070433 ◽

2020 ◽

Author(s):

Ahmet C. Berkyurek ◽

Giulia Furlan ◽

Lisa Lampersberger ◽

Toni Beltran ◽

Eva-Maria Weick ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rapid Evolution ◽

Transcriptional Elongation ◽

Rna Pol Ii ◽

C Elegans ◽

Argonaute Proteins ◽

Endogenous Genes ◽

Pirna Pathway

ABSTRACTPIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA Polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. rpb-9 mutants fail to initiate heritable piRNA-mediated gene silencing. Furthermore, we show that RPB-9 is required to repress two DNA transposon families and a subset of somatic genes in the C. elegans germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis. We demonstrate that RPB-9 acts to promote transcriptional elongation/termination at endogenous piRNA loci. We conclude that as a part of its rapid evolution the piRNA pathway has co-opted another ancient machinery, this time for high-fidelity transcription.

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Post-transcriptional regulation of RNA polymerase II levels in Caenorhabditis elegans.

Genetics ◽

10.1093/genetics/133.2.237 ◽

1993 ◽

Vol 133 (2) ◽

pp. 237-245 ◽

Cited By ~ 1

Author(s):

B K Dalley ◽

T M Rogalski ◽

G E Tullis ◽

D L Riddle ◽

M Golomb

Keyword(s):

Enzyme Activity ◽

Steady State ◽

Caenorhabditis Elegans ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Gene Dosage ◽

C Elegans ◽

Steady State Levels ◽

Solubilized Enzyme ◽

Post Transcriptional Regulation

Abstract To investigate the regulation of RNA polymerase II levels in Caenorhabditis elegans, we have constructed nematode strains having one, two, or three copies of ama-1, the gene for the largest subunit of RNA polymerase II. Steady-state levels of RNA polymerase II polypeptides and solubilized enzyme activity are invariant with gene dosage, indicating regulatory compensation. However, steady-state levels of ama-1 mRNA are directly proportional to gene dosage. These results imply that RNA polymerase II levels in C. elegans are regulated post-transcriptionally.

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Molecular cloning and sequencing of ama-1, the gene encoding the largest subunit of Caenorhabditis elegans RNA polymerase II.

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4119 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4119-4130 ◽

Cited By ~ 50

Author(s):

D M Bird ◽

D L Riddle

Keyword(s):

Caenorhabditis Elegans ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Rna Polymerase Iii ◽

Gene Encoding ◽

C Elegans ◽

A Subunit ◽

Rnap Ii ◽

Collagen Genes ◽

Splice Junctions

Two genomic sequences that share homology with Rp11215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster, have been isolated from the nematode Caenorhabditis elegans. One of these sequences was physically mapped on chromosome IV within a region deleted by the deficiency mDf4, 25 kilobases (kb) from the left deficiency breakpoint. This position corresponds to ama-1 (resistance to alpha-amanitin), a gene shown previously to encode a subunit of RNA polymerase II. Northern (RNA) blotting and DNA sequencing revealed that ama-1 spans 10 kb, is punctuated by 11 introns, and encodes a 5.9-kb mRNA. A cDNA clone was isolated and partially sequenced to confirm the 3' end and several splice junctions. Analysis of the inferred 1,859-residue ama-1 product showed considerable identity with the largest subunit of RNAP II from other organisms, including the presence of a zinc finger motif near the amino terminus, and a carboxyl-terminal domain of 42 tandemly reiterated heptamers with the consensus Tyr Ser Pro Thr Ser Pro Ser. The latter domain was found to be encoded by four exons. In addition, the sequence oriented ama-1 transcription with respect to the genetic map. The second C. elegans sequence detected with the Drosophila probe, named rpc-1, was found to encode a 4.8-kb transcript and hybridized strongly to the gene encoding the largest subunit of RNA polymerase III from yeast, implicating rpc-1 as encoding the analogous peptide in the nematode. By contrast with ama-1, rpc-1 was not deleted by mDf4 or larger deficiencies examined, indicating that these genes are no closer than 150 kb. Genes flanking ama-1, including two collagen genes, also have been identified.

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A survey of the kinome pharmacopeia reveals multiple scaffolds and targets for the development of novel anthelmintics

Scientific Reports ◽

10.1038/s41598-021-88150-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Jessica Knox ◽

Nicolas Joly ◽

Edmond M. Linossi ◽

José A. Carmona-Negrón ◽

Natalia Jura ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Developmental Delay ◽

Small Molecule ◽

Parasitic Nematode ◽

Kinase Inhibitor ◽

Binding Pocket ◽

Drug Binding ◽

Nematode Infection ◽

Selective Inhibitors ◽

C Elegans

AbstractOver one billion people are currently infected with a parasitic nematode. Symptoms can include anemia, malnutrition, developmental delay, and in severe cases, death. Resistance is emerging to the anthelmintics currently used to treat nematode infection, prompting the need to develop new anthelmintics. Towards this end, we identified a set of kinases that may be targeted in a nematode-selective manner. We first screened 2040 inhibitors of vertebrate kinases for those that impair the model nematode Caenorhabditis elegans. By determining whether the terminal phenotype induced by each kinase inhibitor matched that of the predicted target mutant in C. elegans, we identified 17 druggable nematode kinase targets. Of these, we found that nematode EGFR, MEK1, and PLK1 kinases have diverged from vertebrates within their drug-binding pocket. For each of these targets, we identified small molecule scaffolds that may be further modified to develop nematode-selective inhibitors. Nematode EGFR, MEK1, and PLK1 therefore represent key targets for the development of new anthelmintic medicines.

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The Caenorhabditis elegans DEG-3/DES-2 Channel Is a Betaine-Gated Receptor Insensitive to Monepantel

Molecules ◽

10.3390/molecules27010312 ◽

2022 ◽

Vol 27 (1) ◽

pp. 312

Author(s):

Tina V. A. Hansen ◽

Heinz Sager ◽

Céline E. Toutain ◽

Elise Courtot ◽

Cédric Neveu ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Nicotinic Acetylcholine Receptors ◽

Acetylcholine Receptors ◽

Parasitic Nematode ◽

Nematode Species ◽

Xenopus Laevis Oocytes ◽

Allosteric Modulators ◽

C Elegans ◽

Electrode Voltage ◽

Insight Into

Natural plant compounds, such as betaine, are described to have nematocidal properties. Betaine also acts as a neurotransmitter in the free-living model nematode Caenorhabditis elegans, where it is required for normal motility. Worm motility is mediated by nicotinic acetylcholine receptors (nAChRs), including subunits from the nematode-specific DEG-3 group. Not all types of nAChRs in this group are associated with motility, and one of these is the DEG-3/DES-2 channel from C. elegans, which is involved in nociception and possibly chemotaxis. Interestingly, the activity of DEG-3/DES-2 channel from the parasitic nematode of ruminants, Haemonchus contortus, is modulated by monepantel and its sulfone metabolite, which belong to the amino-acetonitrile derivative anthelmintic drug class. Here, our aim was to advance the pharmacological knowledge of the DEG-3/DES-2 channel from C. elegans by functionally expressing the DEG-3/DES-2 channel in Xenopus laevis oocytes and using two-electrode voltage-clamp electrophysiology. We found that the DEG-3/DES-2 channel was more sensitive to betaine than ACh and choline, but insensitive to monepantel and monepantel sulfone when used as direct agonists and as allosteric modulators in co-application with betaine. These findings provide important insight into the pharmacology of DEG-3/DES-2 from C. elegans and highlight the pharmacological differences between non-parasitic and parasitic nematode species.

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Fine-structure genetics of ama-1, an essential gene encoding the amanitin-binding subunit of RNA polymerase II in Caenorhabditis elegans.

Genetics ◽

10.1093/genetics/120.2.423 ◽

1988 ◽

Vol 120 (2) ◽

pp. 423-434

Author(s):

A M Bullerjahn ◽

D L Riddle

Keyword(s):

Fine Structure ◽

Caenorhabditis Elegans ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Essential Gene ◽

Resistance Mutation ◽

Lethal Mutation ◽

Intragenic Recombination ◽

Lethal Mutations ◽

Binding Subunit

Abstract A fine-structure genetic map has been constructed for ama-1 IV, an essential gene in Caenorhabditis elegans encoding the amanitin-binding subunit of RNA polymerase II. Sixteen EMS-induced recessive-lethal mutations have been positioned in the gene by determining their intragenic recombination frequencies with m118, a mutation that confers dominant resistance to alpha-amanitin. The 16 mutants, all isolated in the ama-1(m118) background, include 13 that are early larval lethals, and three that are mid-larval lethals, at 25 degrees. Six of the mutants exhibit temperature-dependence in the severity of their phenotype. Intragenic recombination between the lethal site and the parental resistance mutation was detected by means of resistance to amanitin. Recombinants were detected at frequencies as low as 2 X 10(-6). The segregation of the closely linked flanking markers, unc-17 and unc-5, revealed whether the lethal mutation was to the left or the right of m118. By adding the distances between the extreme left and right mutations, the ama-1 gene is estimated to be 0.011 map unit long, with m118 positioned 0.004 map unit from the left-most lethal mutation. To order the lethal mutations with respect to each other, viable heteroallelic strains were constructed using the free duplication, mDp1[unc-17(e113) dpy-13(+) ama-1(+)]. The heteroallelic strains were sensitive to amanitin, and recombination events between the lethal mutations were specifically selected by means of the dominant amanitin resistance encoded on the recombinant chromosome. The segregation of outside markers revealed the left-right order of the lethal mutations. The position of mutations within the gene is nonrandom. Functional domains of the ama-1 gene indicated by the various lethal phenotypes are discussed.

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