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ABSTRACTIn trypanosomatid parasites, spliced leader (SL) transsplicing is an essential nuclear mRNA maturation step which caps mRNAs posttranscriptionally and, in conjunction with polyadenylation, resolves individual mRNAs from polycistronic precursors. While all trypanosomatid mRNAs are transspliced, intron removal by cissplicing is extremely rare and predicted to occur in only four pre-mRNAs. trans- and cis-splicing reactions are carried out by the spliceosome, which consists of U-rich small nuclear ribonucleoprotein particles (U snRNPs) and of non-snRNP factors. Mammalian and yeast spliceosome complexes are well characterized and found to be associated with up to 170 proteins. Despite the central importance of transsplicing in trypanosomatid gene expression, only the core RNP proteins and a few snRNP-specific proteins are known. To characterize the trypanosome spliceosomal protein repertoire, we conducted a proteomic analysis by tagging and tandem affinity-purifying the canonical core RNP protein SmD1 in Trypanosoma bruceiand by identifying copurified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes thus far and 21 proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified 10 divergent orthologues of known splicing factors, including the missing U1-specific protein U1A. In addition, a novel U5-specific, and, as we show, an essential splicing factor was identified that shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and was thus tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.

ABSTRACTTrypanosomatid parasites share a gene expression mode which differs greatly from that of their human and insect hosts. In these unicellular eukaryotes, protein-coding genes are transcribed polycistronically and individual mRNAs are processed from precursors by spliced leader (SL) transsplicing and polyadenylation. In transsplicing, the SL RNA is consumed through a transfer of its 5'-terminal part to the 5' end of mRNAs. Since all mRNAs are transspliced, the parasites depend on strong and continuous SL RNA synthesis mediated by RNA polymerase II. As essential factors for SL RNA gene transcription in Trypanosoma brucei, the general transcription factor (GTF) IIB and a complex, consisting of the TATA-binding protein-related protein 4, the small nuclear RNA-activating protein complex, and TFIIA, were recently identified. Although T. bruceiTFIIA and TFIIB are extremely divergent to their counterparts in other eukaryotes, their characterization suggested that trypanosomatids do form a class II transcription preinitiation complex at the SL RNA gene promoter and harbor orthologues of other known GTFs. TFIIH is a GTF which functions in transcription initiation, DNA repair, and cell cycle control. Here, we investigated whether a T. bruceiTFIIH is important for SL RNA gene transcription and found that silencing the expression of the highly conserved TFIIH subunit XPD in T. bruceiaffected SL RNA gene synthesis in vivo, and depletion of this protein from extract abolished SL RNA gene transcription in vitro. Since we also identified orthologues of the TFIIH subunits XPB, p52/TFB2, and p44/SSL1 copurifying with TbXPD, we concluded that the parasite harbors a TFIIH which is indispensable for SL RNA gene transcription.

ABSTRACTPlasmodium falciparumis the causative agent of severe human malaria, responsible for over 2 million deaths annually. Of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. A major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. One approach to assign function to these proteins is by identifying their physiological partners. Here we describe the use of tandem affinity purification (TAP) and mass spectrometry for identification of native protein interactions and purification of protein complexes in P. falciparum. Transgenic parasites were generated which express the translation elongation factor PfEF-1ß harboring a C-terminal PTP tag which consists of the protein C epitope, a tobacco etch virus protease cleavage site, and two protein A domains. Purification of PfEF-1ß-PTP from crude extracts followed by mass spectrometric analysis revealed, in addition to the tagged protein itself, the presence of the native PfEF-1ß, the G-protein PfEF-1a, and two new proteins that we named PfEF-1? and PfEF-1d based on their homology to other eukaryotic ? and d translation elongation factor subunits. These data, which constitute the first application of TAP for purification of a protein complex under native conditions in P. falciparum, revealed that the translation elongation complex in this organism contains at least two subunits of PfEF-1ß. The success of this approach will set the stage for a systematic analysis of protein interactions in this important human pathogen.

ABSTRACTCiliated protists are model organisms for a number of molecular phenomena including telomerase function, self-splicing introns, and an RNA interference-related mechanism in programmed DNA elimination. Despite this relevance, our knowledge about promoters and transcriptional regulation in these organisms is very limited. The macronuclear genome of stichotrichous ciliates consists of minichromosomes which typically encode a single gene. The 5' nontranscribed spacers are usually no longer than 400 bp and highly suitable for promoter characterizations. We used microinjection of two artificial and differently tagged a1 tubulin minichromosomes into the macronucleus of Stylonychia lemnaeas a means to characterize in detail the corresponding promoter. Clonal cell lines that stably maintained both minichromosomes were generated, enabling comparative expression analysis by primer extension assays. Deletion and block substitution mutations of one of the minichromosomes revealed a TATA-like element, a putative initiator element, and two distinct upstream sequence elements (USEs). Determination of transcription initiation sites and a sequence alignment indicated that both TATA-like and initiator elements are conserved components of S. lemnaeminichromosomes, whereas the USEs appear to be specific for the a1 tubulin minichromosome. The a2 tubulin minichromosome promoter is very short, comprising the two proximal elements but not the USEs. Despite the latter finding, up-regulation of a-tubulin expression in cells treated with concanavalin A activated the a2 but not the a1 tubulin promoter. These results therefore show that gene expression regulation in S. lemnaeoccurs at the level of transcription initiation on the basis of structurally different promoters.

ABSTRACTTandem affinity purification (TAP) allows for rapid and efficient purification of epitope-tagged protein complexes from crude extracts under native conditions. The method was established in yeast and has been successfully applied to other organisms, including mammals and trypanosomes. However, we found that the original method, which is based on the TAP tag, consisting of a duplicate protein A epitope, a tobacco etch virus protease cleavage site, and the calmodulin-binding peptide (CBP), did not yield enough recovery of transcription factor SNAPc (for small nuclear RNA-activating protein complex) from crude trypanosome extracts for protein identification. Specifically, the calmodulin affinity chromatography step proved to be inefficient. To overcome this problem, we replaced CBP by the protein C epitope (ProtC) and termed this new epitope combination PTP tag. ProtC binds with high affinity to the monoclonal antibody HPC4, which has the unique property of requiring calcium for antigen recognition. Thus, analogous to the calcium-dependent CBP-calmodulin interaction, ProtC-tagged proteins can be released from immobilized HPC4 by a chelator of divalent cations. While this property was retained, epitope substitution improved purification in our experiments by eliminating the inefficiency of calmodulin affinity chromatography and by providing an alternative way of elution using the ProtC peptide in cases where EGTA inactivated protein function. Furthermore, HPC4 allowed highly sensitive and specific detection of ProtC-tagged proteins after protease cleavage. Thus far, we have successfully purified and characterized the U1 small nuclear ribonucleoprotein particle, the transcription factor complex TATA-binding protein related factor 4 (TRF4)/SNAPc/transcription factor IIA (TFIIA), and RNA polymerase I of Trypanosoma brucei.

ABSTRACTIn eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to a-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. bruceicell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, aß-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. bruceiharbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

ABSTRACTThe trypanosome variant surface glycoprotein (VSG) is first expressed during differentiation to the infective, metacyclic population in tsetse fly salivary glands. Unlike the VSGgenes expressed by bloodstream form trypanosomes, metacyclic VSGs (MVSGs) have their own promoters. The scarcity of metacyclic cells has meant that only indirect approaches have been used to study these promoters, and not even their identities have been agreed on. Here, we isolated trypanosomes by dissection from salivary glands and used an approach involving 5' rapid amplification of cDNA ends to identify the transcription start site of three MVSGs. This shows that the authentic start site is that proposed for the MVATseries of MVSGs (K. S. Kim and J. E. Donelson, J. Biol. Chem. 272:24637-24645, 1997). In the more readily accessible procyclic trypanosome stage, where MVSGs are normally silent, we used reporter gene assays and linker scanning analysis to confirm that the 67 bp upstream of the start site is a promoter. This is confirmed further by accurate initiation in a homologous in vitro transcription system. We show also that MVSGpromoters become derepressed when tested outwith their endogenous, subtelomeric loci. The MVSGpromoters are only loosely conserved with bloodstream VSGpromoters, and our detailed analysis of the 1.63 MVSGpromoter reveals that its activity depends on the start site itself and sequences 26 to 49 bp and 56 to 60 bp upstream. These are longer than those necessary for the bloodstream promoter.

ABSTRACTIn Trypanosoma brucei, transcription resistant to the mushroom toxin a-amanitin is not restricted to the rRNA genes (rDNA), as in higher eukaryotes, but extends to genes encoding the major cell surface proteins variant surface glycoprotein (VSG) and procyclin or procyclic acidic repetitive protein (PARP). Here, we report the development of a homologous cell extract from procyclic T. bruceicells in which rDNA and PARP A and VSG gene promoters drive efficient, accurate, and a-amanitin-resistant transcription. A comparative analysis revealed that transcription from the three promoters generally required identical reaction conditions for maximal efficiency. Nevertheless, PARP promoter transcription proved to be exceptional by its high efficiency, its lag phase, a high template DNA concentration optimum, and its tolerance to increasing concentrations of Mn2+. Mutational analysis for both the PARP and rDNA promoters showed that the proximal and distal core elements were essential for efficient transcription in vitro. Deletion of the upstream control regions (UCRs), however, had a different effect. Whereas PARP UCR deletion reduced transcription efficiency almost 10-fold, deletion of the rDNA UCR had only a minor effect on transcription efficiency.