scholarly journals An assembly of nuclear bodies associates with the active VSG expression site in African trypanosomes

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
James Budzak ◽  
Robert Jones ◽  
Christian Tschudi ◽  
Nikolay G. Kolev ◽  
Gloria Rudenko
Keyword(s):  
Rna Polymerase I ◽  
Nuclear Bodies ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Cajal Bodies ◽  
African Trypanosomes ◽  
Spliced Leader ◽  
Pol I ◽  
Expression Site ◽  
Polymerase I

AbstractA Variant Surface Glycoprotein (VSG) coat protects bloodstream form Trypanosoma brucei. Prodigious amounts of VSG mRNA (~7-10% total) are generated from a single RNA polymerase I (Pol I) transcribed VSG expression site (ES), necessitating extremely high levels of localised splicing. We show that splicing is required for processive ES transcription, and describe novel ES-associated T. brucei nuclear bodies. In bloodstream form trypanosomes, the expression site body (ESB), spliced leader array body (SLAB), NUFIP body and Cajal bodies all frequently associate with the active ES. This assembly of nuclear bodies appears to facilitate the extraordinarily high levels of transcription and splicing at the active ES. In procyclic form trypanosomes, the NUFIP body and SLAB do not appear to interact with the Pol I transcribed procyclin locus. The congregation of a restricted number of nuclear bodies at a single active ES, provides an attractive mechanism for how monoallelic ES transcription is mediated.

scholarly journals VEX1 controls the allelic exclusion required for antigenic variation in trypanosomes

2016 ◽  
Vol 113 (26) ◽  
pp. 7225-7230 ◽  
Author(s):  
Lucy Glover ◽  
Sebastian Hutchinson ◽  
Sam Alsford ◽  
David Horn
Keyword(s):  
Antigenic Variation ◽  
Negative Regulation ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Allelic Exclusion ◽  
Positive Regulation ◽  
African Trypanosomes ◽  
Pol I ◽  
Winner Takes All ◽  
Polymerase I

Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for defects in telomere-exclusive expression. VEX1 was sequestered by the active VSG and silencing of other VSGs failed when VEX1 was either ectopically expressed or depleted, indicating positive and negative regulation, respectively. Positive regulation affected VSGs and nontelomeric pol-I–transcribed genes, whereas negative regulation primarily affected VSGs. Negative regulation by VEX1 also affected telomeric pol-I–transcribed reporter constructs, but only when they contained blocks of sequence sharing homology with a pol-I–transcribed locus. We conclude that restricted positive regulation due to VEX1 sequestration, combined with VEX1-dependent, possibly homology-dependent silencing, drives a “winner-takes-all” mechanism of allelic exclusion.

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

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.

scholarly journals Nucleosomes Are Depleted at the VSG Expression Site Transcribed by RNA Polymerase I in African Trypanosomes

2009 ◽  
Vol 9 (1) ◽  
pp. 148-154 ◽  
Author(s):  
Luisa M. Figueiredo ◽  
George A. M. Cross
Keyword(s):  
Rna Polymerase ◽  
Antigenic Variation ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Micrococcal Nuclease ◽  
Rrna Genes ◽  
Host Immune System ◽  
African Trypanosomes ◽  
Pol I ◽  
Polymerase I

ABSTRACT In most eukaryotes, RNA polymerase I (Pol I) exclusively transcribes long arrays of identical rRNA genes (ribosomal DNA [rDNA]). African trypanosomes have the unique property of using Pol I to also transcribe the variant surface glycoprotein VSG genes. VSGs are important virulence factors because their switching allows trypanosomes to escape the host immune system, a mechanism known as antigenic variation. Only one VSG is transcribed at a time from one of 15 bloodstream-form expression sites (BESs). Although it is clear that switching among BESs does not involve DNA rearrangements and that regulation is probably epigenetic, it remains unknown why BESs are transcribed by Pol I and what roles are played by chromatin structure and histone modifications. Using chromatin immunoprecipitation, micrococcal nuclease digestion, and chromatin fractionation, we observed that there are fewer nucleosomes at the active BES and that these are irregularly spaced compared to silent BESs. rDNA coding regions are also depleted of nucleosomes, relative to the rDNA spacer. In contrast, genes transcribed by Pol II are organized in a more compact, regularly spaced, nucleosomal structure. These observations provide new insight on antigenic variation by showing that chromatin remodeling is an intrinsic feature of BES regulation.

The PARP and rRNA promoters of Trypanosoma brucei are composed of dissimilar sequence elements that are functionally interchangeable

1994 ◽  
Vol 14 (9) ◽  
pp. 5804-5811
Author(s):  
L Janz ◽  
C Clayton
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
African Trypanosomes ◽  
Sequence Elements ◽  
Major Surface Proteins ◽  
Major Surface ◽  
Polymerase I ◽  
Repetitive Protein

The African trypanosomes express two major surface proteins, the variant surface glycoprotein (VSG) and the procyclic acidic repetitive protein (PARP). The RNA polymerase that transcribes the VSG and PARP genes shares many characteristics with RNA polymerase I. We show that although there is very little similarity in nucleotide sequence, the functional structure of a trypanosome rRNA promoter is almost identical to that of the PARP promoter. Further, domains from the PARP promoter can functionally substitute for the corresponding parts of the rRNA promoter, and vice versa.

scholarly journals Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes

2015 ◽  
Vol 112 (21) ◽  
pp. E2803-E2812 ◽  
Author(s):  
Igor Cestari ◽  
Ken Stuart
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Inositol Phosphate ◽  
Rna Polymerase I ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Activator Protein 1 ◽  
African Trypanosomes ◽  
Polymerase I

African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing.

scholarly journals Active VSG Expression Sites in Trypanosoma brucei Are Depleted of Nucleosomes

2009 ◽  
Vol 9 (1) ◽  
pp. 136-147 ◽  
Author(s):  
Tara M. Stanne ◽  
Gloria Rudenko
Keyword(s):  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Histone H3 ◽  
Bloodstream Form ◽  
Surface Glycoprotein ◽  
Micrococcal Nuclease ◽  
Pcr Analysis ◽  
Marker Genes ◽  
African Trypanosomes ◽  
Pol I

ABSTRACT African trypanosomes regulate transcription differently from other eukaryotes. Most of the trypanosome genome is constitutively transcribed by RNA polymerase II (Pol II) as large polycistronic transcription units while the genes encoding the major surface proteins are transcribed by RNA polymerase I (Pol I). In bloodstream form Trypanosoma brucei, the gene encoding the variant surface glycoprotein (VSG) coat is expressed in a monoallelic fashion from one of about 15 VSG bloodstream form expression sites (BESs). Little is known about the chromatin structure of the trypanosome genome, and the chromatin state of active versus silent VSG BESs remains controversial. Here, we determined histone H3 occupancy within the genome of T. brucei, focusing on active versus silent VSG BESs in the bloodstream form. We found that histone H3 was most enriched in the nontranscribed 50-bp and 177-bp repeats and relatively depleted in Pol I, II, and III transcription units, with particular depletion over promoter regions. Using two isogenic T. brucei lines containing marker genes in different VSG BESs, we determined that histone H3 is 11- to 40-fold depleted from active VSG BESs compared with silent VSG BESs. Quantitative PCR analysis of fractionated micrococcal nuclease-digested chromatin revealed that the active VSG BES is depleted of nucleosomes. Therefore, in contrast to earlier views, nucleosome positioning appears to be involved in the monoalleleic control of VSG BESs in T. brucei. This may provide a level of epigenetic regulation enabling bloodstream form trypanosomes to efficiently pass on the transcriptional state of active and silent BESs to daughter cells.

scholarly journals The PARP and rRNA promoters of Trypanosoma brucei are composed of dissimilar sequence elements that are functionally interchangeable.

1994 ◽  
Vol 14 (9) ◽  
pp. 5804-5811 ◽  
Author(s):  
L Janz ◽  
C Clayton
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Surface Proteins ◽  
Surface Glycoprotein ◽  
African Trypanosomes ◽  
Sequence Elements ◽  
Major Surface Proteins ◽  
Major Surface ◽  
Polymerase I ◽  
Repetitive Protein

The African trypanosomes express two major surface proteins, the variant surface glycoprotein (VSG) and the procyclic acidic repetitive protein (PARP). The RNA polymerase that transcribes the VSG and PARP genes shares many characteristics with RNA polymerase I. We show that although there is very little similarity in nucleotide sequence, the functional structure of a trypanosome rRNA promoter is almost identical to that of the PARP promoter. Further, domains from the PARP promoter can functionally substitute for the corresponding parts of the rRNA promoter, and vice versa.

scholarly journals Dynamic colocalization of 2 simultaneously active VSG expression sites within a single expression-site body in Trypanosoma brucei

2019 ◽  
Vol 116 (33) ◽  
pp. 16561-16570 ◽  
Author(s):  
James Budzak ◽  
Louise E. Kerry ◽  
Aris Aristodemou ◽  
Belinda S. Hall ◽  
Kathrin Witmer ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Chromatin Immunoprecipitation ◽  
Surface Glycoprotein ◽  
Monoallelic Expression ◽  
Drug Selection ◽  
Wild Type ◽  
Pol I ◽  
Dna Base ◽  
Expression Site ◽  
Polymerase I

Monoallelic exclusion ensures that the African trypanosome Trypanosoma brucei exclusively expresses only 1 of thousands of different variant surface glycoprotein (VSG) coat genes. The active VSG is transcribed from 1 of 15 polycistronic bloodstream-form VSG expression sites (ESs), which are controlled in a mutually exclusive fashion. Unusually, T. brucei uses RNA polymerase I (Pol I) to transcribe the active ES, which is unprecedented among eukaryotes. This active ES is located within a unique extranucleolar Pol I body called the expression-site body (ESB). A stringent restriction mechanism prevents T. brucei from expressing multiple ESs at the same time, although how this is mediated is unclear. By using drug-selection pressure, we generated VSG double-expresser T. brucei lines, which have disrupted monoallelic exclusion, and simultaneously express 2 ESs in a dynamic fashion. The 2 unstably active ESs appear epigenetically similar to fully active ESs as determined by using chromatin immunoprecipitation for multiple epigenetic marks (histones H3 and H1, TDP1, and DNA base J). We find that the double-expresser cells, similar to wild-type single-expresser cells, predominantly contain 1 subnuclear ESB, as determined using Pol I or the ESB marker VEX1. Strikingly, simultaneous transcription of the 2 dynamically transcribed ESs is normally observed only when the 2 ESs are both located within this single ESB. This colocalization is reversible in the absence of drug selection. This discovery that simultaneously active ESs dynamically share a single ESB demonstrates the importance of this unique subnuclear body in restricting the monoallelic expression of VSG.

scholarly journals The Trypanosomatid-Specific N Terminus of RPA2 Is Required for RNA Polymerase I Assembly, Localization, and Function

2012 ◽  
Vol 11 (5) ◽  
pp. 662-672 ◽  
Author(s):  
Jan-Peter Daniels ◽  
Keith Gull ◽  
Bill Wickstead
Keyword(s):  
Rna Polymerase ◽  
Nuclear Localization ◽  
Rna Polymerase I ◽  
Growth Defect ◽  
Protein Coding ◽  
African Trypanosomes ◽  
Protein Coding Genes ◽  
Pol I ◽  
N Terminus ◽  
Polymerase I

ABSTRACT African trypanosomes are the only organisms known to use RNA polymerase I (pol I) to transcribe protein-coding genes. These genes include VSG , which is essential for immune evasion and is transcribed from an extranucleolar expression site body (ESB). Several trypanosome pol I subunits vary compared to their homologues elsewhere, and the question arises as to how these variations relate to pol I function. A clear example is the N-terminal extension found on the second-largest subunit of pol I, RPA2. Here, we identify an essential role for this region. RPA2 truncation leads to nuclear exclusion and a growth defect which phenocopies single-allele knockout. The N terminus is not a general nuclear localization signal (NLS), however, and it fails to accumulate unrelated proteins in the nucleus. An ectopic NLS is sufficient to reinstate nuclear localization of truncated RPA2, but it does not restore function. Moreover, NLS-tagged, truncated RPA2 has a different subnuclear distribution to full-length protein and is unable to build stable pol I complexes. We conclude that the RPA2 N-terminal extension does not have a role exclusive to the expression of protein-coding genes, but it is essential for all pol I functions in trypanosomes because it directs trypanosomatid-specific interactions with RPA1.

Sign in / Sign up

Export Citation Format

Share Document