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

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3543-3547.1989 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3543-3547

Author(s):

T W Nilsen ◽

J Shambaugh ◽

J Denker ◽

G Chubb ◽

C Faser ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Secondary Structure ◽

Rna Polymerase ◽

Binding Site ◽

Rna Polymerase Ii ◽

Ascaris Lumbricoides ◽

Parasitic Nematode ◽

Secondary Structures ◽

Spliced Leader ◽

C Elegans

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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RNA Polymerase II Localizes in Tetrahymena thermophila Meiotic Micronuclei When Micronuclear Transcription Associated with Genome Rearrangement Occurs

Eukaryotic Cell ◽

10.1128/ec.3.5.1233-1240.2004 ◽

2004 ◽

Vol 3 (5) ◽

pp. 1233-1240 ◽

Cited By ~ 34

Author(s):

Kazufumi Mochizuki ◽

Martin A. Gorovsky

Keyword(s):

Life Cycle ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Genome Rearrangement ◽

Tetrahymena Thermophila ◽

Germ Line ◽

Essential Gene ◽

Gene Encoding ◽

Sexual Process ◽

Rnap Ii

ABSTRACT The germ line micronucleus in Tetrahymena thermophila is transcriptionally silent in vegetatively growing cells. However, micronuclear transcription has been observed in the early (“crescent”) stages of the sexual process, conjugation. This transcription is proposed to play a central role in identifying sites for subsequent genome rearrangements that accompany development of the somatic macronucleus from the micronucleus. RPB3 (cnjC), a gene encoding a protein homologous to the third largest subunit of RNA polymerase II (RNAP II), was previously reported to be expressed specifically during conjugation, suggesting a role in micronucleus-specific transcription. Rpb3p localized in the micronucleus only during the meiotic prophase, when micronuclear transcription occurs, and its intranuclear distribution is strikingly similar to that for previously described sites of micronuclear RNA synthesis. By contrast, Rpc5p, the homologous subunit shared by RNAPs I and III, was not detectable in the micronucleus at any stage of the life cycle. However, Rpb3p is not specific to the transcribing micronucleus. Like Rpc5p, it also localizes to macronuclei in all stages of the life cycle. Rpb3p is encoded by a unique, essential gene in Tetrahymena. Thus, RNAP II is associated with both somatic transcription and crescent transcription and probably has an important role in genome rearrangement.

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Dimethylation of Histone H3 at Lysine 36 DemarcatesRegulatory and Nonregulatory ChromatinGenome-Wide

Molecular and Cellular Biology ◽

10.1128/mcb.25.21.9447-9459.2005 ◽

2005 ◽

Vol 25 (21) ◽

pp. 9447-9459 ◽

Cited By ~ 122

Author(s):

Bhargavi Rao ◽

Yoichiro Shibata ◽

Brian D. Strahl ◽

Jason D. Lieb

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nucleosome Occupancy ◽

Rna Polymerase Iii ◽

Histone H3 ◽

Open Reading Frames ◽

Gene Transcript ◽

Genome Wide ◽

Rnap Ii ◽

Reading Frames

ABSTRACT Set2p, which mediates histone H3 lysine 36 dimethylation (H3K36me2) in Saccharomyces cerevisiae, has been shown to associate with RNA polymerase II (RNAP II) at individual loci. Here, chromatin immunoprecipitation-microarray experiments normalized to general nucleosome occupancy reveal that nucleosomes within open reading frames (ORFs) and downstream noncoding chromatin were highly dimethylated at H3K36 and that Set2p activity begins at a stereotypic distance from the initiation of transcription genome-wide. H3K36me2 is scarce in regions upstream of divergently transcribed genes, telomeres, silenced mating loci, and regions transcribed by RNA polymerase III, providing evidence that the enzymatic activity of Set2p is restricted to its association with RNAP II. The presence of H3K36me2 within ORFs correlated with the “on” or“ off” state of transcription, but the degree of H3K36 dimethylation within ORFs did not correlate with transcription frequency. This provides evidence that H3K36me2 is established during the initial instances of gene transcription, with subsequent transcription having at most a maintenance role. Accordingly, newly activated genes acquire H3K36me2 in a manner that does not correlate with gene transcript levels. Finally, nucleosomes dimethylated at H3K36 appear to be refractory to loss from highly transcribed chromatin. Thus, H3K36me2, which is highly conserved throughout eukaryotic evolution, provides a stable molecular mechanism for establishing chromatin context throughout the genome by distinguishing potential regulatory regions from transcribed chromatin.

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3543 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3543-3547 ◽

Cited By ~ 47

Author(s):

T W Nilsen ◽

J Shambaugh ◽

J Denker ◽

G Chubb ◽

C Faser ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Secondary Structure ◽

Rna Polymerase ◽

Binding Site ◽

Rna Polymerase Ii ◽

Ascaris Lumbricoides ◽

Parasitic Nematode ◽

Secondary Structures ◽

Spliced Leader ◽

C Elegans

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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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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Functional Interaction between Ssu72 and the Rpb2 Subunit of RNA Polymerase II in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.20.22.8343-8351.2000 ◽

2000 ◽

Vol 20 (22) ◽

pp. 8343-8351 ◽

Cited By ~ 40

Author(s):

Donald L. Pappas ◽

Michael Hampsey

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Essential Gene ◽

Functional Interaction ◽

Gene Encoding ◽

General Transcription Factor ◽

Physical Interactions ◽

Rnap Ii

ABSTRACT SSU72 is an essential gene encoding a phylogenetically conserved protein of unknown function that interacts with the general transcription factor TFIIB. A recessive ssu72-1 allele was identified as a synthetic enhancer of a TFIIB (sua7-1) defect, resulting in a heat-sensitive (Ts−) phenotype and a dramatic downstream shift in transcription start site selection. Here we describe a new allele, ssu72-2, that confers a Ts− phenotype in a SUA7 wild-type background. In an effort to further define Ssu72, we isolated suppressors of thessu72-2 mutation. One suppressor is allelic toRPB2, the gene encoding the second-largest subunit of RNA polymerase II (RNAP II). Sequence analysis of the rpb2-100suppressor defined a cysteine replacement of the phylogenetically invariant arginine residue at position 512 (R512C), located within homology block D of Rpb2. The ssu72-2 andrpb2-100 mutations adversely affected noninduced gene expression, with no apparent effects on activated transcription in vivo. Although isolated as a suppressor of the ssu72-2Ts− defect, rpb2-100 enhanced the transcriptional defects associated with ssu72-2. The Ssu72 protein interacts directly with purified RNAP II in a coimmunoprecipitation assay, suggesting that the genetic interactions between ssu72-2 and rpb2-100 are a consequence of physical interactions. These results define Ssu72 as a highly conserved factor that physically and functionally interacts with the RNAP II core machinery during transcription initiation.

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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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Regulating expression of mistranslating tRNAs by readthrough RNA polymerase II transcription

10.1101/2021.09.16.460584 ◽

2021 ◽

Author(s):

Matthew D. Berg ◽

Joshua R Isaacson ◽

Ecaterina Cozma ◽

Julie Genereaux ◽

Patrick Lajoie ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Expression System ◽

Rna Polymerase Iii ◽

Cell Types ◽

Eukaryotic Cell ◽

Yeast Cells ◽

Activator Proteins ◽

Rnap Ii ◽

Rna Polymerase Ii Transcription

Transfer RNA (tRNA) variants that alter the genetic code increase protein diversity and have many applications in synthetic biology. Since the tRNA variants can cause a loss of proteostasis, regulating their expression is necessary to achieve high levels of novel protein. Mechanisms to positively regulate transcription with exogenous activator proteins like those often used to regulate RNA polymerase II (RNAP II) transcribed genes are not applicable to tRNAs as their expression by RNA polymerase III requires elements internal to the tRNA. Here, we show that tRNA expression is repressed by overlapping transcription from an adjacent RNAP II promoter. Regulating the expression of the RNAP II promoter allows inverse regulation of the tRNA. Placing either Gal4 or TetR-VP16 activated promoters downstream of a mistranslating tRNASer variant that mis-incorporates serine at proline codons in Saccharomyces cerevisiae allows mistranslation at a level not otherwise possible because of the toxicity of the unregulated tRNA. Using this inducible tRNA system, we explore the proteotoxic effects of mistranslation on yeast cells. High levels of mistranslation cause cells to arrest in G1 phase. These cells are impermeable to propidium iodide, yet growth is not restored upon repressing tRNA expression. High levels of mistranslation increase cell size and alter cell morphology. This regulatable tRNA expression system can be applied to study how native tRNAs and tRNA variants affect the proteome and other biological processes. Variations of this inducible tRNA system should be applicable to other eukaryotic cell types.

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