scholarly journals Correction for Kirkham et al., “Dynein Light Chain LC8 Is Required for RNA Polymerase I-Mediated Transcription in Trypanosoma brucei, Facilitating Assembly and Promoter Binding of Class I Transcription Factor A”

2017 ◽  
Vol 37 (12) ◽  
Author(s):  
Justin K. Kirkham ◽  
Sung Hee Park ◽  
Tu N. Nguyen ◽  
Ju Huck Lee ◽  
Arthur Günzl
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Light Chain ◽  
Rna Polymerase I ◽  
Class I ◽  
Dynein Light Chain ◽  
Polymerase I

scholarly journals 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

2012 ◽  
Vol 11 (12) ◽  
pp. 1573-1581 ◽  
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 .

scholarly journals 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

2014 ◽  
Vol 13 (6) ◽  
pp. 785-795 ◽  
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.

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

2013 ◽  
Vol 42 (5) ◽  
pp. 3164-3176 ◽  
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.

scholarly journals The dynein light chain LC8 is required for RNA polymerase I-mediated transcription inTrypanosoma brucei,facilitating assembly and promoter binding of class I transcription factor A

2015 ◽  
pp. MCB.00705-15 ◽  
Author(s):  
Justin K Kirkham ◽  
Sung Hee Park ◽  
Tu N Nguyen ◽  
Ju Huck Lee ◽  
Arthur Günzl
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Light Chain ◽  
Antigenic Variation ◽  
Surface Glycoprotein ◽  
Class I ◽  
Dynein Light Chain ◽  
Pol I ◽  
Independent Functions

Dynein light chain LC8 is highly conserved among eukaryotes and has both dynein-dependent and dynein-independent functions. Interestingly, LC8 was identified as a subunit of the class I transcription factor A (CITFA), which is essential for transcription by RNA polymerase (pol) I in the parasiteTrypanosoma brucei.Given that LC8 has never been identified with a basal transcription factor and thatT. bruceirelies on RNA pol I for expressing the variant surface glycoprotein (VSG), the key protein in antigenic variation, we investigated the CITFA-specific role of LC8. Depletion of LC8 from mammalian-infective bloodstream trypanosomes affected cell cycle progression, reduced the abundances of rRNA andVSGmRNA, and resulted in rapid cell death. Sedimentation analysis, co-immunoprecipitation of recombinant proteins, and bioinformatic analysis revealed an LC8 binding site near the N-terminus of the subunit CITFA2. Mutation of this site prevented the formation of a CITFA2-LC8 heterotetramer and,in vivo, was lethal, affecting assembly of a functional CITFA complex. Gel shift assays and UV-crosslinking experiments identified CITFA2 as a promoter-binding CITFA subunit. Accordingly, silencing ofLC8orCITFA2resulted in a loss of CITFA from RNA pol I promoters. Hence, we discovered an LC8 interaction that, unprecedentedly, has a basal function in transcription.

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

1992 ◽  
Vol 8 (12) ◽  
pp. 414-418 ◽  
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

scholarly journals The RNA Polymerase I Transcription Factor UBF Is the Product of a Primary Response Gene

1995 ◽  
Vol 270 (9) ◽  
pp. 4209-4212 ◽  
Author(s):  
Marija Glibetic ◽  
Laura Taylor ◽  
Dawn Larson ◽  
Ross Hannan ◽  
Bruce Sells ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Primary Response ◽  
Rna Polymerase I ◽  
Response Gene ◽  
Primary Response Gene ◽  
Polymerase I
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