Arthur Günzl

Highly Efficient Tandem Affinity Purification of Trypanosome Protein Complexes Based on a Novel Epitope Combination

ABSTRACT Tandem 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.

RNA Polymerase I Transcribes Procyclin Genes and Variant Surface Glycoprotein Gene Expression Sites in Trypanosoma brucei

ABSTRACT In 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 α-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. brucei cell 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, αβ-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

Characterization of a multisubunit transcription factor complex essential for spliced-leader RNA gene transcription in Trypanosoma brucei.

ABSTRACT In the unicellular human parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., the spliced-leader (SL) RNA is a key molecule in gene expression donating its 5′-terminal region in SL addition trans splicing of nuclear pre-mRNA. While there is no evidence that this process exists in mammals, it is obligatory in mRNA maturation of trypanosomatid parasites. Hence, throughout their life cycle, these organisms crucially depend on high levels of SL RNA synthesis. As putative SL RNA gene transcription factors, a partially characterized small nuclear RNA-activating protein complex (SNAPc) and the TATA-binding protein related factor 4 (TRF4) have been identified thus far. Here, by tagging TRF4 with a novel epitope combination termed PTP, we tandem affinity purified from crude T. brucei extracts a stable and transcriptionally active complex of six proteins. Besides TRF4 these were identified as extremely divergent subunits of SNAPc and of transcription factor IIA (TFIIA). The latter finding was unexpected since genome databases of trypanosomatid parasites appeared to lack general class II transcription factors. As we demonstrate, the TRF4/SNAPc/TFIIA complex binds specifically to the SL RNA gene promoter upstream sequence element and is absolutely essential for SL RNA gene transcription in vitro.

The pre-mRNA splicing machinery of trypanosomes: complex or simplified?

ABSTRACT Trypanosomatids are early-diverged, protistan parasites of which Trypanosoma brucei, Trypanosoma cruzi, and several species of Leishmania cause severe, often lethal diseases in humans. To better combat these parasites, their molecular biology has been a research focus for more than 3 decades, and the discovery of spliced leader (SL) trans splicing in T. brucei established a key difference between parasites and hosts. In SL trans splicing, the capped 5′-terminal region of the small nuclear SL RNA is fused onto the 5′ end of each mRNA. This process, in conjunction with polyadenylation, generates individual mRNAs from polycistronic precursors and creates functional mRNA by providing the cap structure. The reaction is a two-step transesterification process analogous to intron removal by cis splicing which, in trypanosomatids, is confined to very few pre-mRNAs. Both types of pre-mRNA splicing are carried out by the spliceosome, consisting of five U-rich small nuclear RNAs (U snRNAs) and, in humans, up to ∼170 different proteins. While trypanosomatids possess a full set of spliceosomal U snRNAs, only a few splicing factors were identified by standard genome annotation because trypanosomatid amino acid sequences are among the most divergent in the eukaryotic kingdom. This review focuses on recent progress made in the characterization of the splicing factor repertoire in T. brucei, achieved by tandem affinity purification of splicing complexes, by systematic analysis of proteins containing RNA recognition motifs, and by mining the genome database. In addition, recent findings about functional differences between trypanosome and human pre-mRNA splicing factors are discussed.

Identification of a Novel Chromosomal Passenger Complex and Its Unique Localization during Cytokinesis in Trypanosoma brucei

Aurora B kinase is a key component of the chromosomal passenger complex (CPC), which regulates chromosome segregation and cytokinesis. An ortholog of Aurora B was characterized in Trypanosoma brucei (TbAUK1), but other conserved components of the complex have not been found. Here we identified four novel TbAUK1 associated proteins by tandem affinity purification and mass spectrometry. Among these four proteins, TbKIN-A and TbKIN-B are novel kinesin homologs, whereas TbCPC1 and TbCPC2 are hypothetical proteins without any sequence similarity to those known CPC components from yeasts and metazoans. RNAi-mediated silencing of each of the four genes led to loss of spindle assembly, chromosome segregation and cytokinesis. TbKIN-A localizes to the mitotic spindle and TbKIN-B to the spindle midzone during mitosis, whereas TbCPC1, TbCPC2 and TbAUK1 display the dynamic localization pattern of a CPC. After mitosis, the CPC disappears from the central spindle and re-localizes at a dorsal mid-point of the mother cell, where the anterior tip of the daughter cell is tethered, to start cell division toward the posterior end, indicating a most unusual CPC-initiated cytokinesis in a eukaryote.

Antimalarial activity of the anticancer and proteasome inhibitor bortezomib and its analog ZL3B

BackgroundThe high rate of mortality due to malaria and the worldwide distribution of parasite resistance to the commonly used antimalarial drugs chloroquine and pyrimethamine emphasize the urgent need for the development of new antimalarial drugs. An alternative approach to the long and uncertain process of designing and developing new compounds is to identify among the armamentarium of drugs already approved for clinical treatment of various human diseases those that may have strong antimalarial activity.MethodsProteasome inhibitor bortezomib (Velcade™: [(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl) amino]propyl]amino]butyl] boronic acid), which has been approved for treatment of patients with multiple myeloma, and a second boronate analog Z-Leu-Leu-Leu-B(OH)2 (ZL3B), were tested against four different strains of P. falciparum (3D7, HB3, W2 and Dd2) that are either sensitive or have different levels of resistance to the antimalarial drugs pyrimethamine and chloroquine.ResultsBortezomib and ZL3B are equally effective against drug-sensitive and -resistant parasites and block intraerythrocytic development prior to DNA synthesis, but have no effect on parasite egress or invasion.ConclusionThe identification of bortezomib and its analog as potent antimalarial drugs will set the stage for the advancement of this class of compounds, either alone or in combination therapy, for treatment of malaria, and emphasize the need for large-scale screens to identify new antimalarials within the library of clinically approved compounds.

Multifunctional class I transcription in Trypanosoma brucei depends on a novel protein complex

The vector‐borne, protistan parasite Trypanosoma brucei is the only known eukaryote with a multifunctional RNA polymerase I that, in addition to ribosomal genes, transcribes genes encoding the parasites major cell‐surface proteins—the variant surface glycoprotein (VSG) and procyclin. In the mammalian bloodstream, antigenic variation of the VSG coat is the parasites means to evade the immune response, while procyclin is necessary for effective establishment of trypanosome infection in the fly. Moreover, the exceptionally high efficiency of mono‐allelic VSG expression is essential to bloodstream trypanosomes since its silencing caused rapid cell‐cycle arrest in vitro and clearance of parasites from infected mice. Here we describe a novel protein complex that recognizes class I promoters and is indispensable for class I transcription; it consists of a dynein light chain and six polypeptides that are conserved only among trypanosomatid parasites. In accordance with an essential transcriptional function of the complex, silencing the expression of a key subunit was lethal to bloodstream trypanosomes and specifically affected the abundance of rRNA and VSG mRNA. The complex was dubbed class I transcription factor A.

Spliceosomal Proteomics in Trypanosoma brucei Reveal New RNA Splicing Factors

ABSTRACT In trypanosomatid parasites, spliced leader (SL) trans splicing 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 trans spliced, intron removal by cis splicing 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 trans splicing 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 brucei and 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.

Transcriptionally active TFIIH of the early-diverged eukaryote Trypanosoma brucei harbors two novel core subunits but not a cyclin-activating kinase complex

Trypanosoma brucei is a member of the early-diverged, protistan family Trypanosomatidae and a lethal parasite causing African Sleeping Sickness in humans. Recent studies revealed that T. brucei harbors extremely divergent orthologues of the general transcription factors TBP, TFIIA, TFIIB and TFIIH and showed that these factors are essential for initiating RNA polymerase II-mediated synthesis of spliced leader (SL) RNA, a trans splicing substrate and key molecule in trypanosome mRNA maturation. In yeast and metazoans, TFIIH is composed of a core of seven conserved subunits and the ternary cyclin-activating kinase (CAK) complex. Conversely, only four TFIIH subunits have been identified in T. brucei. Here, we characterize the first protistan TFIIH which was purified in its transcriptionally active form from T. brucei extracts. The complex consisted of all seven core subunits but lacked the CAK sub-complex; instead it contained two trypanosomatid-specific subunits, which were indispensable for parasite viability and SL RNA gene transcription. These findings were corroborated by comparing the molecular structures of trypanosome and human TFIIH. While the ring-shaped core domain was surprisingly congruent between the two structures, trypanosome TFIIH lacked the knob-like CAK moiety and exhibited extra densities on either side of the ring, presumably due to the specific subunits.

SMN-assisted assembly of snRNP-specific Sm cores in trypanosomes.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring (U1, U5, spliced leader snRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4 snRNPs). Searching for specificity factors, we identified SMN and Gemin2 proteins that are highly divergent from known orthologs. SMN is splicing-essential in trypanosomes and nuclear-localized, suggesting that Sm core assembly in trypanosomes is nuclear. We demonstrate in vitro that SMN is sufficient to confer specificity of canonical Sm core assembly and to discriminate against binding to nonspecific RNA and to U2 and U4 snRNAs. SMN interacts transiently with the SmD3B subcomplex, contacting specifically SmB. SMN remains associated throughout the assembly of the Sm heteroheptamer and dissociates only when a functional Sm site is incorporated. These data establish a novel role of SMN, mediating snRNP specificity in Sm core assembly, and yield new biochemical insight into the mechanism of SMN activity.