Transcription by the multifunctional RNA polymerase I in Trypanosoma brucei functions independently of RPB7

Park, Sung Hee; Nguyen, Tu N.; Kirkham, Justin K.; Lee, Ju Huck; Günzl, Arthur

doi:10.1016/j.molbiopara.2011.06.008

Cited by 15 publications

(21 citation statements)

References 50 publications

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“…BFs were cultured in HMI-9 medium as described previously (22). For stable BF transfection, 3 ϫ 10 7 cells, grown at exponential phase, were washed once in 2 ml Cytomix (120 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 , 25 mM HEPES-KOH, pH 7.6, 2 mM EDTA, and 5 mM MgCl 2 ), resuspended in 0.4 ml Cytomix, and mixed with 10 g of linearized plasmid DNA or 5 g of PCR product.…”

Section: Methodsmentioning

confidence: 99%

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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

Nguyen

Lee

et al. 2012

Eukaryot Cell

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cTrypanosoma 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. In eukaryotes, RNA polymerase I (pol I) transcribes exclusively the large ribosomal gene units in the nucleolus. The unicellular parasite Trypanosoma brucei is exceptional in this regard because it is the only organism known to have evolved a multifunctional RNA pol I system that is used for rRNA synthesis and for the expression of proteins that are crucial for the parasite's successful interaction with its hosts. T. brucei is a tsetse-borne parasite in sub-Saharan Africa that causes lethal diseases in humans and livestock animals (2). It lives freely in the mammalian bloodstream by virtue of a dense coat of variant surface glycoprotein (VSG) which shields invariant membrane proteins from immune recognition (32) and whose antigenic variation enables the parasite to evade the host's immune system. There are ϳ10 million VSG copies on the surface of a bloodstream-form (BF) trypanosome, all of which are expressed from a single gene drawn from a repertoire of up to 2,000 VSG genes (16). To accommodate the dense coat, the active VSG gene, which resides in one of 15 telomeric expression sites (ESs) (11), needs to be transcribed at extremely high rates; it was estimated that VSG RNA synthesis from the active ES exceeds that of a single ␤-tubulin gene by ϳ50-fold (4). This high VSG expression is not only required for antigenic variation but essential to BF viability itself, since VSG silencing led to a rapid b...

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Section: Methodsmentioning

confidence: 99%

“…The notion that the missing ortholog of the essential yeast RPA43 subunit could be replaced in T. brucei by its RNA pol II paralog RPB7 (13) could not be substantiated (22). Instead, an essential RNA pol I subunit, termed T. brucei RPA31 (TbRPA31), which is either a novel subunit or an extremely divergent ortholog of yeast RPA43, was found (18).…”

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confidence: 99%

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

Nguyen

Lee

et al. 2012

Eukaryot Cell

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“…Cells. T. brucei cell culture, transfection, and the generation of stable cell lines by selection and limiting dilution were carried out as described previously (27,38). In RNAi experiments, double-stranded RNA (dsRNA) synthesis was induced with 2 g/ml of doxycycline.…”

Section: Dnasmentioning

confidence: 99%

Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1

Badjatia

Ambrósio

Lee

et al. 2013

Molecular and Cellular Biology

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, RPB1, mediates the enzyme's promoter escape and binding of RNA-processing factors, such as the m 7 G capping enzymes. The first critical step, Ser 5 phosphorylation, is carried out by cyclin-dependent kinase 7 (CDK7), a subunit of the basal transcription factor TFIIH. Many early-diverged protists, such as the lethal human parasite Trypanosoma brucei, however, lack the heptad repeats and, apparently, a CDK7 ortholog. Accordingly, characterization of trypanosome TFIIH did not identify a kinase component. The T. brucei CTD, however, is phosphorylated and essential for transcription. Here we show that silencing the expression of T. brucei cdc2-related kinase 9 (CRK9) leads to a loss of RPB1 phosphorylation. Surprisingly, this event did not impair RNA Pol II transcription or cotranscriptional m 7 G capping. Instead, we observed that CRK9 silencing led to a block of spliced leader (SL) trans splicing, an essential step in trypanosome mRNA maturation, that was caused by hypomethylation of the SL RNA's unique cap4.

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“…Quantitative RT-PCR of CITFA1 mRNA was carried out either with the oligonucleotide pair 5=-ATCGGATGTTGAGTCGCTGCGTTG G-3=/5=-AAAGTCATTC CATGCCACTGGAACC-3= (CITFA1 coding sequence) or the pair 5=-AA TACGCCAG GCAGATTGATGC-3=/5=-TTAAGCGTAGTCAGGTACG TCGTAAGG-3= (CITFA7 coding region/HS). BES and RRNA promoter consensus oligonucleotides and oligonucleotides specific to the TFIIB gene and the ␤-/␣-tubulin intergenic region, used in quantitative PCR (qPCR), were previously specified (21,27).…”

Section: Methodsmentioning

confidence: 99%

“…BFs were cultured in HMI-9 medium as specified previously (27). Transfections were done with 1 ϫ 10 7 to 2 ϫ 10 7 BF trypanosomes using the Amaxa Basic Parasite Nucleofector kit (Lonza).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2014

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Conditional gene silencing by RNA interference in Trypanosoma brucei can 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 functional Trypanosoma cruzi U2AF35 3= 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 with CITFA7, 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 the CITFA7 mRNA resulted in gene knockdowns that were as efficient and specific as targeting the endogenous CITFA7 mRNA. Moreover, application of this system to CITFA1, 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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Transcription by the multifunctional RNA polymerase I in Trypanosoma brucei functions independently of RPB7

Cited by 15 publications

References 50 publications

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

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

Trypanosome cdc2-Related Kinase 9 Controls Spliced Leader RNA cap4 Methylation and Phosphorylation of RNA Polymerase II Subunit RPB1

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

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