Active RNA Polymerase I of Trypanosoma brucei Harbors a Novel Subunit Essential for Transcription

ABSTRACT A unique characteristic of the protistan parasite Trypanosoma brucei is a multifunctional RNA polymerase I which, in addition to synthesizing rRNA as in other eukaryotes, transcribes gene units encoding the major cell surface antigens variant surface glycoprotein and procyclin. Thus far, purification of this enzyme has revealed nine orthologues of known subunits but no active enzyme. Here, we have epitope tagged the specific subunit RPB6z and tandem affinity purified RNA polymerase I from crude extract. The purified enzyme was active in both a nonspecific and a promoter-dependent transcription assay and exhibited enriched protein bands with apparent sizes of 31, 29, and 27 kDa. p31 and its trypanosomatid orthologues were identified, but their amino acid sequences have no similarity to proteins of other eukaryotes, nor do they contain a conserved sequence motif. Nevertheless, p31 cosedimented with purified RNA polymerase I, and RNA interferance-mediated silencing of p31 was lethal, affecting the abundance of rRNA. Moreover, extract of p31-silenced cells exhibited a specific defect in transcription of class I templates, which was remedied by the addition of purified RNA polymerase I, and an anti-p31 serum completely blocked RNA polymerase I-mediated transcription. We therefore dubbed this novel functional component of T. brucei RNA polymerase I TbRPA31.

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Promoter occupancy of the basal class I transcription factor A differs strongly between active and silent VSG expression sites in Trypanosoma brucei

Nucleic Acids Research ◽

10.1093/nar/gkt1301 ◽

2013 ◽

Vol 42 (5) ◽

pp. 3164-3176 ◽

Cited By ~ 24

Author(s):

Tu N. Nguyen ◽

Laura S. M. Müller ◽

Sung Hee Park ◽

T. Nicolai Siegel ◽

Arthur Günzl

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Trypanosoma Brucei ◽

Transcription Initiation ◽

Rna Polymerase I ◽

Surface Glycoprotein ◽

Class I ◽

Monoallelic Expression ◽

Promoter Occupancy ◽

Polymerase I

Abstract Monoallelic expression within a gene family is found in pathogens exhibiting antigenic variation and in mammalian olfactory neurons. Trypanosoma brucei, a lethal parasite living in the human bloodstream, expresses variant surface glycoprotein (VSG) from 1 of 15 bloodstream expression sites (BESs) by virtue of a multifunctional RNA polymerase I. The active BES is transcribed in an extranucleolar compartment termed the expression site body (ESB), whereas silent BESs, located elsewhere within the nucleus, are repressed epigenetically. The regulatory mechanisms, however, are poorly understood. Here we show that two essential subunits of the basal class I transcription factor A (CITFA) predominantly occupied the promoter of the active BES relative to that of a silent BES, a phenotype that was maintained after switching BESs in situ. In these experiments, high promoter occupancy of CITFA was coupled to high levels of both promoter-proximal RNA abundance and RNA polymerase I occupancy. Accordingly, fluorescently tagged CITFA-7 was concentrated in the nucleolus and the ESB. Because a ChIP-seq analysis found that along the entire BES, CITFA-7 is specifically enriched only at the promoter, our data strongly indicate that monoallelic BES transcription is activated by a mechanism that functions at the level of transcription initiation.

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Trypanosoma brucei ATR Links DNA Damage Signaling during Antigenic Variation with Regulation of RNA Polymerase I-Transcribed Surface Antigens

Cell Reports ◽

10.1016/j.celrep.2019.12.049 ◽

2020 ◽

Vol 30 (3) ◽

pp. 836-851.e5 ◽

Cited By ~ 3

Author(s):

Jennifer Ann Black ◽

Kathryn Crouch ◽

Leandro Lemgruber ◽

Craig Lapsley ◽

Nicholas Dickens ◽

...

Keyword(s):

Dna Damage ◽

Rna Polymerase ◽

Trypanosoma Brucei ◽

Antigenic Variation ◽

Surface Antigens ◽

Rna Polymerase I ◽

Dna Damage Signaling ◽

Polymerase I

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Quantification of RNA Polymerase I transcriptional attenuation at the active VSG expression site in Trypanosoma brucei

10.1101/2021.06.21.449234 ◽

2021 ◽

Author(s):

Nadine Weisert ◽

Klara Thein ◽

Helena Reis ◽

Christian J Janzen

Keyword(s):

Rna Polymerase ◽

Trypanosoma Brucei ◽

Antigenic Variation ◽

Rna Polymerase I ◽

Surface Glycoprotein ◽

Transcriptional Elongation ◽

Pol I ◽

Expression Site ◽

Polymerase I ◽

Large Repertoire

The cell surface of the extracellular pathogen Trypanosoma brucei consists of a dense coat of variant surface glycoprotein (VSG), which enables the parasite to evade the immune system of the vertebrate host. Only one VSG gene from a large repertoire is expressed from a so-called bloodstream form expression site (BES) at a given timepoint. There are several BES in every parasite but only one is transcriptionally active. Other BES are silenced by transcriptional attenuation. Periodic activation of a previously-silenced BES results in differential VSG transcription and escape from the immune response. A process called antigenic variation. In contrast to gene transcription in other eukaryotes, the BES is transcribed by RNA polymerase I (Pol I). It was proposed that this highly-processive polymerase is needed to provide a sufficiently high transcription rate at the VSG gene. Surprisingly, we discovered a position-dependent Pol I activity and attenuation of transcriptional elongation also at the active BES. Transcription rates at the VSG gene appear to be comparable to Pol II-mediated transcription of house-keeping genes. Although these findings are in contradiction to the long-standing concept of continuously high transcription rates at the active BES in Trypanosoma brucei, they are complementary to recent groundbreaking findings about transcriptional regulation of VSG genes.

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Characterization of a Novel Class I Transcription Factor A (CITFA) Subunit That Is Indispensable for Transcription by the Multifunctional RNA Polymerase I of Trypanosoma brucei

Eukaryotic Cell ◽

10.1128/ec.00250-12 ◽

2012 ◽

Vol 11 (12) ◽

pp. 1573-1581 ◽

Cited By ~ 20

Author(s):

Tu N. Nguyen ◽

Bao N. Nguyen ◽

Ju Huck Lee ◽

Aswini K. Panigrahi ◽

Arthur Günzl

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Trypanosoma Brucei ◽

Rna Polymerase I ◽

Class I ◽

Mammalian Host ◽

Subunit Structure ◽

Content Type ◽

Pol I ◽

Polymerase I

ABSTRACT Trypanosoma brucei is the only organism known to have evolved a multifunctional RNA polymerase I (pol I) system that is used to express the parasite's ribosomal RNAs, as well as its major cell surface antigens, namely, the variant surface glycoprotein (VSG) and procyclin, which are vital for establishing successful infections in the mammalian host and the tsetse vector, respectively. Thus far, biochemical analyses of the T. brucei RNA pol I transcription machinery have elucidated the subunit structure of the enzyme and identified the class I transcription factor A (CITFA). CITFA binds to RNA pol I promoters, and its CITFA-2 subunit was shown to be absolutely essential for RNA pol I transcription in the parasite. Tandem affinity purification (TAP) of CITFA revealed the subunits CITFA-1 to -6, which are conserved only among kinetoplastid organisms, plus the dynein light chain DYNLL1. Here, by tagging CITFA-6 instead of CITFA-2, a complex was purified that contained all known CITFA subunits, as well as a novel proline-rich protein. Functional studies carried out in vivo and in vitro , as well as a colocalization study, unequivocally demonstrated that this protein is a bona fide CITFA subunit, essential for parasite viability and indispensable for RNA pol I transcription of ribosomal gene units and the active VSG expression site in the mammalian-infective life cycle stage of the parasite. Interestingly, CITFA-7 function appears to be species specific, because expression of an RNA interference (RNAi)-resistant CITFA-7 transgene from Trypanosoma cruzi could not rescue the lethal phenotype of silencing endogenous CITFA-7 .

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RNA polymerase I-mediated protein-coding gene expression in Trypanosoma brucei

Parasitology Today ◽

10.1016/0169-4758(92)90194-7 ◽

1992 ◽

Vol 8 (12) ◽

pp. 414-418 ◽

Cited By ~ 30

Author(s):

H-M. Chung ◽

M.G-S. Lee ◽

L.H.T. Van der Ploeg

Keyword(s):

Gene Expression ◽

Rna Polymerase ◽

Trypanosoma Brucei ◽

Rna Polymerase I ◽

Protein Coding ◽

Polymerase I

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A New Strategy of RNA Interference That Targets Heterologous Sequences Reveals CITFA1 as an Essential Component of Class I Transcription Factor A in Trypanosoma brucei

Eukaryotic Cell ◽

10.1128/ec.00014-14 ◽

2014 ◽

Vol 13 (6) ◽

pp. 785-795 ◽

Cited By ~ 5

Author(s):

Sung Hee Park ◽

Bao N. Nguyen ◽

Justin K. Kirkham ◽

Tu N. Nguyen ◽

Arthur Günzl

Keyword(s):

Transcription Factor ◽

Rna Interference ◽

Rna Polymerase ◽

Trypanosoma Brucei ◽

Transcription Initiation ◽

Single Copy ◽

Rna Polymerase I ◽

Class I ◽

Content Type ◽

Polymerase I

ABSTRACTConditional gene silencing by RNA interference inTrypanosoma bruceican be inconclusive if knockdowns are inefficient or have off-target effects. To enable efficient, specific silencing of single-copy genes in mammalian-infective, bloodstream form trypanosomes, we developed a system that targets the heterologous and functionalTrypanosoma cruziU2AF353′ untranslated region (UTR) (Tc3) or, alternatively, the sequence of the PTP tag, which can be fused to any mRNA of interest. Two cell lines were created, single-marker Tc3 (smTc3) and smPTP, which conditionally express Tc3 and PTP double-stranded RNA (dsRNA), respectively. The system depends on manipulating both alleles of the gene of interest so that cells exclusively express the target mRNA as a fusion to one of these heterologous sequences. We generated allele integration vectors in which the C-terminal part of a gene's coding sequence can be fused to either heterologous sequence in a single cloning step. We first tested this system withCITFA7, which encodes a well-characterized subunit of the class I transcription factor A (CITFA), an essential factor for transcription initiation by RNA polymerase I. Targeting either Tc3 or PTP fused to theCITFA7mRNA resulted in gene knockdowns that were as efficient and specific as targeting the endogenousCITFA7mRNA. Moreover, application of this system toCITFA1, which could not be silenced by established methods, demonstrated that the gene encodes an essential CITFA subunit that mediates binding of the transcription factor complex to RNA polymerase I promoters.

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