Faculty Opinions recommendation of RNA pol II subunit RPB7 is required for RNA pol I-mediated transcription in Trypanosoma brucei.

ABSTRACT In eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to α-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. brucei cell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, αβ-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

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Trypanosoma brucei ribonuclease H2A is an essential R-loop processing enzyme whose loss causes DNA damage during transcription initiation and antigenic variation

Nucleic Acids Research ◽

10.1093/nar/gkz644 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9180-9197 ◽

Cited By ~ 6

Author(s):

Emma Briggs ◽

Kathryn Crouch ◽

Leandro Lemgruber ◽

Graham Hamilton ◽

Craig Lapsley ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Trypanosoma Brucei ◽

Transcription Initiation ◽

Critical Role ◽

Rnase H ◽

Rna Pol Ii ◽

Pol Ii ◽

Pol I ◽

Rnase H2

Abstract Ribonucleotides represent a threat to DNA genome stability and transmission. Two types of Ribonuclease H (RNase H) excise ribonucleotides when they form part of the DNA strand, or hydrolyse RNA when it base-pairs with DNA in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast survives the absence of both enzymes. RNase H1 loss is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. Differential gene expression analysis reveals limited overall changes in RNA levels for RNA Pol II genes after TbRH2A loss, but increased perturbation of nucleotide metabolic genes. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, also leading to altered gene expression. Thus, we demonstrate separation of function between two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion.

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Development, embryonic genome activity and mitochondrial characteristics of bovine–pig inter-family nuclear transfer embryos

Reproduction ◽

10.1530/rep-09-0578 ◽

2010 ◽

Vol 140 (2) ◽

pp. 273-285 ◽

Cited By ~ 25

Author(s):

Irina Lagutina ◽

Helena Fulka ◽

Tiziana A L Brevini ◽

Stefania Antonini ◽

Dario Brunetti ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Nuclear Transfer ◽

Blastocyst Development ◽

Cell Stage ◽

Rna Pol Ii ◽

Pol Ii ◽

Pol I ◽

Embryonic Genome ◽

Genome Activity

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine–pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency gene NANOG and alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos, NANOG was not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16–25-cell stage for pig–bovine iSCNT embryos and at the four-cell stage for bovine–pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus–cytoplasm compatibility between these animal species.

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Effect of the Disruption of RNA Pol II on the Transcription by RNA Pol l by Trypanosoma brucei

Journal of Eukaryotic Microbiology ◽

10.1111/j.1550-7408.2005.05202003_3_8.x ◽

2005 ◽

Vol 52 (2) ◽

pp. 28S-34S

Author(s):

S. DEVAUX ◽

L. LECORDIER ◽

L. VANHAMME ◽

E. PAYS

Keyword(s):

Trypanosoma Brucei ◽

Rna Pol Ii ◽

Pol Ii

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Disruption of largest subunit RNA polymerase II genes in Trypanosoma brucei.

Molecular and Cellular Biology ◽

10.1128/mcb.13.6.3734 ◽

1993 ◽

Vol 13 (6) ◽

pp. 3734-3743 ◽

Cited By ~ 17

Author(s):

H M Chung ◽

M G Lee ◽

P Dietrich ◽

J Huang ◽

L H Van der Ploeg

Keyword(s):

Amino Acid ◽

Rna Polymerase Ii ◽

Trypanosoma Brucei ◽

Promoter Region ◽

Gene Promoter ◽

Amino Acid Substitutions ◽

Pol Ii ◽

Pol I ◽

Alpha Amanitin ◽

Gene Promoter Region

Two types of largest subunit RNA polymerase II (pol II) genes (pol IIA and pol IIB), differing in 3 amino acid substitutions, are encoded in the Trypanosoma brucei (stock 427-60) genome. As a result, the alpha-amanitin-resistant transcription of the procyclic acidic repetitive protein (PARP) and variant surface glycoprotein (VSG) genes was proposed to involve a modified, alpha-amanitin-resistant form of the largest subunit of pol II. Alternatively, pol I could transcribe the PARP and VSG genes. To discriminate between these two models, we deleted the N-terminal domain (about one-third of the polypeptide), which encodes the amino acid substitutions which discriminated the pol IIA and pol IIB genes, at both pol IIB alleles. The pol IIB- trypanosomes still transcribe the PARP genes and the VSG gene promoter region in insect-form trypanosomes by alpha-amanitin-resistant RNA polymerases, while control housekeeping genes are transcribed in an alpha-amanitin-sensitive manner, presumably by pol IIA. We conclude that the alpha-amanitin-resistant transcription of protein coding genes in T. brucei is not mediated by a diverged form of the largest subunit of pol II and that the presence of both the pol IIA and pol IIB genes is not essential for trypanosome viability. This conclusion was further supported by the finding that individual trypanosome variants exhibited allelic heterogeneity for the previously identified amino acid substitutions and that various permutations of the polymorphic amino acids generate at least four different types of largest subunit pol II genes. The expression of the PARP genes and the VSG gene promoter region by alpha-amanitin-resistant RNA polymerases in the pol IIB- trypanosomes provides evidence for transcription of these genes by pol I.

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Functional Characterization of a Trypanosoma brucei TATA-Binding Protein-Related Factor Points to a Universal Regulator of Transcription in Trypanosomes

Molecular and Cellular Biology ◽

10.1128/mcb.24.21.9610-9618.2004 ◽

2004 ◽

Vol 24 (21) ◽

pp. 9610-9618 ◽

Cited By ~ 42

Author(s):

Jia-peng Ruan ◽

George K. Arhin ◽

Elisabetta Ullu ◽

Christian Tschudi

Keyword(s):

Trypanosoma Brucei ◽

Binding Protein ◽

Functional Characterization ◽

Tata Binding Protein ◽

Related Factor ◽

Protein Coding ◽

Pol Ii ◽

Protein Coding Genes ◽

Pol I ◽

Rna Genes

ABSTRACT Transcriptional mechanisms remain poorly understood in trypanosomatid protozoa. In particular, there is no knowledge about the function of basal transcription factors, and there is an apparent rarity of promoters for protein-coding genes transcribed by RNA polymerase (Pol) II. Here we describe a Trypanosoma brucei factor related to the TATA-binding protein (TBP). Although this TBP-related factor (TBP-related factor 4 [TRF4]) has about 31% identity to the TBP core domain, several key residues involved in TATA box binding are not conserved. Depletion of the T. brucei TRF4 (TbTRF4) by RNA interference revealed an essential role in RNA Pol I, II, and III transcription. Using chromatin immunoprecipitation, we further showed that TRF4 is recruited to the Pol I-transcribed procyclic acidic repetitive genes, Pol II-transcribed spliced leader RNA genes, and Pol III-transcribed U-snRNA and 7SL RNA genes, thus supporting a role for TbTRF4 in transcription performed by all three nuclear RNA polymerases. Finally, a search for TRF4 binding sites in the T. brucei genome led to the identification of such sites in the 3′ portion of certain protein-coding genes, indicating a unique aspect of Pol II transcription in these organisms.

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Regulation of RNA Polymerase I-Dependent Promoters by the Hepatitis B Virus X Protein via Activated Ras and TATA-Binding Protein

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7086 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7086-7094 ◽

Cited By ~ 62

Author(s):

Horng-Dar Wang ◽

Alpa Trivedi ◽

Deborah L. Johnson

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Promoter Activity ◽

Tata Binding Protein ◽

S2 Cells ◽

X Protein ◽

Rna Pol Ii ◽

Pol Ii ◽

Pol I ◽

B Virus

ABSTRACT The hepatitis B virus (HBV) X protein is essential for viral infectivity, and evidence indicates that it is a strong contributor to HBV-mediated oncogenesis. X has been shown to transactivate a wide variety of RNA polymerase (Pol) II-dependent, as well as RNA Pol III-dependent, promoters. In this study, we have investigated the possibility that X modulates RNA Pol I-dependent rRNA transcription. In both human hepatoma Huh7 and Drosophila Schneider S2 cell lines, X expression stimulated rRNA promoter activity. Extracts prepared from X-expressing cells stably transfected with anX gene also exhibited an increased ability to transcribe the rRNA promoter. The mechanism for X transactivation was examined by determining whether this regulatory event was dependent on Ras activation and increased TATA-binding protein (TBP) levels. Our previous studies have demonstrated that X, and the activation of Ras, produces an increase in the cellular levels of TBP (H.-D. Wang, A. Trivedi, and D. L. Johnson, Mol. Cell. Biol. 17:6838–6846, 1997). Expression of a dominant negative form of Ras blocked the X-mediated induction of the rRNA promoters, whereas expression of a constitutively activated form of Ras mimicked the enhancing effect of X on rRNA promoter activity. When TBP was overexpressed in either Huh7 or S2 cells, a dose-dependent increase in rRNA promoter activity was observed. To analyze whether the increase in TBP was modulating rRNA promoter activity indirectly, by increasing activity of RNA Pol II-dependent promoters, a Drosophila TBP cDNA was constructed with a mutation that eliminated its ability to stimulate RNA Pol II-dependent promoters. Transient expression of wild-type TBP in S2 cells increased the activities of specific RNA Pol I- and Pol II-dependent promoters. Expression of the mutant TBP protein failed to enhance the activity of the RNA Pol II-dependent promoters, yet the protein completely retained its ability to stimulate the rRNA promoter. Furthermore, the addition of recombinant TBP to S2 extracts stimulated rRNA promoter activity in vitro. Together, these results demonstrate that the HBV X protein up-regulates RNA Pol I-dependent promoters via a Ras-activated pathway in two distinct cell lines. The enhanced promoter activity can, at least in part, be attributed to the X- and Ras-mediated increase in cellular TBP, a limiting transcription component.

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c-Myc co-ordinates mRNA cap methylation and ribosomal RNA production

Biochemical Journal ◽

10.1042/bcj20160930 ◽

2017 ◽

Vol 474 (3) ◽

pp. 377-384 ◽

Cited By ~ 4

Author(s):

Sianadh Dunn ◽

Olivia Lombardi ◽

Victoria H. Cowling

Keyword(s):

Ribosomal Rna ◽

Translation Initiation ◽

Rna Processing ◽

Nuclear Export ◽

Constitutive Expression ◽

Mrna Processing ◽

Rrna Synthesis ◽

Rna Pol Ii ◽

Pol Ii ◽

Pol I

The mRNA cap is a structure added to RNA pol II transcripts in eukaryotes, which recruits factors involved in RNA processing, nuclear export and translation initiation. RNA guanine-7 methyltransferase (RNMT)–RNA-activating miniprotein (RAM), the mRNA cap methyltransferase complex, completes the basic functional mRNA cap structure, cap 0, by methylating the cap guanosine. Here, we report that RNMT–RAM co-ordinates mRNA processing with ribosome production. Suppression of RNMT–RAM reduces synthesis of the 45S ribosomal RNA (rRNA) precursor. RNMT–RAM is required for c-Myc expression, a major regulator of RNA pol I, which synthesises 45S rRNA. Constitutive expression of c-Myc restores rRNA synthesis when RNMT–RAM is suppressed, indicating that RNMT–RAM controls rRNA production predominantly by controlling c-Myc expression. We report that RNMT–RAM is recruited to the ribosomal DNA locus, which may contribute to rRNA synthesis in certain contexts.

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Trypanosoma brucei ribonuclease H2A is an essential enzyme that resolves R-loops associated with transcription initiation and antigenic variation

10.1101/541300 ◽

2019 ◽

Author(s):

Emma Briggs ◽

Kathryn Crouch ◽

Leandro Lemgruber ◽

Graham Hamilton ◽

Craig Lapsley ◽

...

Keyword(s):

Gene Expression ◽

Trypanosoma Brucei ◽

Transcription Initiation ◽

Antigenic Variation ◽

Critical Role ◽

Rnase H ◽

Surface Glycoprotein ◽

Pol Ii ◽

Pol I ◽

Rnase H2

In every cell ribonucleotides represent a threat to the stability and transmission of the DNA genome. Two types of Ribonuclease H (RNase H) tackle such ribonucleotides, either by excision when they form part of the DNA strand, or by hydrolysing RNA when it base-pairs with DNA, in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast can prosper in the absence of both enzymes. Removal of RNase H1 is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of the catalytic subunit of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. In addition, differential gene expression of both RNA Pol I and II transcribed genes occurs after TbRH2A loss, including patterns that may relate to cytosolic DNA accumulation in humans with autoimmune disease. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, leading to altered variant surface glycoprotein expression. Thus, we demonstrate a separation of function between the two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion.

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