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MCM-BP is required for repression of life-cycle specific genes transcribed by RNA polymerase I in the mammalian infectious form of Trypanosoma brucei.

ABSTRACT: Trypanosoma brucei variant surface glycoprotein (VSG) expression is a classic example of allelic exclusion. While the genome of T. brucei contains >2,000 VSG genes and VSG pseudogenes, only one allele is expressed at the surface of each infectious trypanosome and the others are repressed. Along with recombinatorial VSG switching, allelic exclusion provides a major host evasion mechanism for trypanosomes, a phenomenon known as antigenic variation. To extend our understanding of how trypanosomes escape host immunity by differential expression of VSGs, we attempted to identify genes that contribute to VSG silencing, by performing a loss-of-silencing screen in T. brucei using a transposon-mediated random insertional mutagenesis. One identified gene, which we initially named LOS1, encodes a T. brucei MCM-Binding Protein (TbMCM-BP). Here we show that TbMCM-BP is essential for viability of infectious bloodstream-form (BF) trypanosome and is required for proper cell-cycle progression. Tandem affinity purification of TbMCM-BP followed by mass spectrometry identified four subunits (MCM4-MCM7) of the T. brucei MCM complex, a replicative helicase, and MCM8, a subunit that is uniquely co-purified with TbMCM-BP. TbMCM-BP is required not only for repression of subtelomeric VSGs but also for silencing of life-cycle specific, insect-stage genes, procyclin and procyclin-associated genes (PAGs), that are normally repressed in BF trypanosomes and are transcribed by RNA polymerase I. Our study uncovers a functional link between chromosome maintenance and RNA pol I-mediated gene silencing in T. brucei.

SUBMITTER: Kim HS

PROVIDER: S-EPMC3581582 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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MCM-BP is required for repression of life-cycle specific genes transcribed by RNA polymerase I in the mammalian infectious form of Trypanosoma brucei.

Kim Hee-Sook HS Park Sung Hee SH Günzl Arthur A Cross George A M GA

PloS one 20130225 2

Trypanosoma brucei variant surface glycoprotein (VSG) expression is a classic example of allelic exclusion. While the genome of T. brucei contains >2,000 VSG genes and VSG pseudogenes, only one allele is expressed at the surface of each infectious trypanosome and the others are repressed. Along with recombinatorial VSG switching, allelic exclusion provides a major host evasion mechanism for trypanosomes, a phenomenon known as antigenic variation. To extend our understanding of how trypanosomes e ...[more]

PMID: 23451133

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Project description:The infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vThe infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic Trapanosoma forms. We used an in vitro system based on expressing the RNA binding protein 6 (RBP6) to obtain infectious metacyclics and determined their protein and mRNA repertoires by mass-spectrometry (MS) based proteomics and mRNA sequencing (RNAseq) in comparison to non-infectious procyclic trypanosomes. This comparison showed that metacyclics are quiescent cells, and we propose this influences the choice of a monocistronic variant surface glycoprotein expression site. Metacyclics have a largely bloodstream-form type transcriptome, and thus are programmed to translate a bloodstream-form type proteome upon entry into the mammalian host and resumption of cell division. Genes encoding cell surface components showed the largest changes between procyclics and metacyclics, observed at both the transcript and protein levels. Genes encoding metabolic enzymes exhibited expression in metacyclics with features of both procyclic and bloodstream forms, suggesting that this intermediate-type metabolism is dictated by the availability of nutrients in the tsetse fly vector. ector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic Trapanosoma forms. We used an in vitro system based on expressing the RNA binding protein 6 (RBP6) to obtain infectious metacyclics and determined their protein and mRNA repertoires by mass-spectrometry (MS) based proteomics and mRNA sequencing (RNAseq) in comparison to non-infectious procyclic trypanosomes. This comparison showed that metacyclics are quiescent cells, and we propose this influences the choice of a monocistronic variant surface glycoprotein expression site. Metacyclics have a largely bloodstream-form type transcriptome, and thus are programmed to translate a bloodstream-form type proteome upon entry into the mammalian host and resumption of cell division. Genes encoding cell surface components showed the largest changes between procyclics and metacyclics, observed at both the transcript and protein levels. Genes encoding metabolic enzymes exhibited expression in metacyclics with features of both procyclic and bloodstream forms, suggesting that this intermediate-type metabolism is dictated by the availability of nutrients in the tsetse fly vector.

Dataset Information

MCM-BP is required for repression of life-cycle specific genes transcribed by RNA polymerase I in the mammalian infectious form of Trypanosoma brucei.

Publications

MCM-BP is required for repression of life-cycle specific genes transcribed by RNA polymerase I in the mammalian infectious form of Trypanosoma brucei.

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