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

Abstract

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...

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

“…To analyze specific functions of individual subunits, we attempted to silence the expression of each subunit gene. Our previous results demonstrated that CITFA2 and CITFA7 are essential for RNA pol I transcription and trypanosome viability (13,21). We also found that three of the eight subunits are required to maintain the integrity of the complex (T. N. Nguyen and A. Günzl, unpublished results).…”

“…S1 in the supplemental material) has two cassettes, a C-terminal tagging module and a selectable marker cassette. It is a direct derivative of pCITFA7-HA-BLA (21) and was obtained by replacing the T. brucei RPA1 3= gene flank with Tc3 using the vector's XhoI and ClaI restriction sites. Furthermore, our tagging vectors are derived from pBluescript II SK(ϩ), which has a T7 promoter that, after integration into an endogenous allele, could lead to overexpression of downstream genes in T7 RNA polymerase-expressing sm BF and 29-13 PF cells.…”

“…Data obtained thus far strongly indicate that CITFA is a promoter-binding transcription initiation factor. CITFA stably bound the BES promoter in gel shift assays and required both promoter elements for efficient binding, depletion or inhibition of CITFA resulted in loss of transcription within 121 to 146 bp of the transcription initiation site, CITFA7 silencing strongly reduced promoter-proximal RNA pol I occupancy at RRNA repeats, and a genome-wide chromatin immunoprecipitation-sequencing (ChIP-seq) analysis found CITFA7 occupancy within a BES to be restricted to the promoter region (13,21,22). To analyze specific functions of individual subunits, we attempted to silence the expression of each subunit gene.…”

“…The parasite uses RNA pol I to transcribe the RRNA array, as in all other eukaryotes, yet also employs it to express its major cell surface proteins, procyclins in PFs and variant surface glycoprotein (VSG) in BFs (13,21,22). The latter is expressed from a single VSG gene, drawn from a large repertoire, in one of 15 40-to 60-kb-long BF telomeric expression sites (BESs) (23).…”

The VEXing problem of monoallelic expression in the African trypanosome

“…If one imagines VEX1 as a gatekeeper for entry to an ESBassociated VSG transcription "center," then loss of VEX1 would allow a "free-for-all" entry of additional BESs within the ESBassociated center and access to the transcription factors housed within it [Pol I, class I transcription factor A (CITFA), or nucleoplasminlike protein (NLP) (12,20), and possibly others], resulting in increased transcription of VSGs at the newly associated BESs. Conversely, overabundance of VEX1 would establish additional gated centers for VSG expression (which might not necessarily coincide with the ESB).…”