Single p197 molecules of the mitochondrial genome segregation system of Trypanosoma brucei determine the distance between basal body and outer membrane.

The tripartite attachment complex (TAC) couples the segregation of the single unit mitochondrial DNA of trypanosomes with the basal body (BB) of the flagellum. Here, we studied the architecture of the exclusion zone filament (EZF) of the TAC, the only known component of which is p197, that connects the BB with the mitochondrial outer membrane (OM). We show that p197 has three domains that are all essential for mitochondrial DNA inheritance. The C terminus of p197 interacts with the mature and probasal body (pro-BB), whereas its N terminus binds to the peripheral OM protein TAC65. The large central region of p197 has a high α-helical content and likely acts as a flexible spacer. Ultrastructure expansion microscopy (U-ExM) of cell lines exclusively expressing p197 versions of different lengths that contain both N- and C-terminal epitope tags demonstrates that full-length p197 alone can bridge the ∼270-nm distance between the BB and the cytosolic face of the OM. Thus U-ExM allows the localization of distinct domains within the same molecules and suggests that p197 is the TAC subunit most proximal to the BB. In addition, U-ExM revealed that p197 acts as a spacer molecule, as two shorter versions of p197, with the repeat domain either removed or replaced by the central domain of the Trypanosoma cruzi p197 ortholog reduced the distance between the BB and the OM in proportion to their predicted molecular weight.

p166 links membrane and intramitochondrial modules of the trypanosomal tripartite attachment complex.

The protist parasite Trypanosoma brucei has a single mitochondrion with a single unit genome termed kinetoplast DNA (kDNA). Faithfull segregation of replicated kDNA is ensured by a complicated structure termed tripartite attachment complex (TAC). The TAC physically links the basal body of the flagellum with the kDNA spanning the two mitochondrial membranes. Here, we characterized p166 as the only known TAC subunit that is anchored in the inner membrane. Its C-terminal transmembrane domain separates the protein into a large N-terminal region that interacts with the kDNA-localized TAC102 and a 34 aa C-tail that binds to the intermembrane space-exposed loop of the integral outer membrane protein TAC60. Whereas the outer membrane region requires four essential subunits for proper TAC function, the inner membrane integral p166, via its interaction with TAC60 and TAC102, would theoretically suffice to bridge the distance between the OM and the kDNA. Surprisingly, non-functional p166 lacking the C-terminal 34 aa still localizes to the TAC region. This suggests the existence of additional TAC-associated proteins which loosely bind to non-functional p166 lacking the C-terminal 34 aa and keep it at the TAC. However, binding of full length p166 to these TAC-associated proteins alone would not be sufficient to withstand the mechanical load imposed by the segregating basal bodies.

tRNA 3' shortening by LCCR4 as a response to stress in Trypanosoma brucei.

Sensing of environmental cues is crucial for cell survival. To adapt to changes in their surroundings cells need to tightly control the repertoire of genes expressed at any time. Regulation of translation is key, especially in organisms in which transcription is hardly controlled, like Trypanosoma brucei. In this study, we describe the shortening of the bulk of the cellular tRNAs during stress at the expense of the conserved 3' CCA-tail. This tRNA shortening is specific for nutritional stress and renders tRNAs unsuitable substrates for translation. We uncovered the nuclease LCCR4 (Tb927.4.2430), a homologue of the conserved deadenylase Ccr4, as being responsible for tRNA trimming. Once optimal growth conditions are restored tRNAs are rapidly repaired by the trypanosome tRNA nucleotidyltransferase thus rendering the recycled tRNAs amenable for translation. This mechanism represents a fast and efficient way to repress translation during stress, allowing quick reactivation with a low energy input.

The highly diverged trypanosomal MICOS complex is organized in a nonessential integral membrane and an essential peripheral module.

The mitochondrial contact site and cristae organization system (MICOS) mediates the formation of cristae, invaginations in the mitochondrial inner membrane. The highly diverged MICOS complex of the parasitic protist Trypanosoma brucei consists of nine subunits. Except for two Mic10-like and a Mic60-like protein, all subunits are specific for kinetoplastids. Here, we determined on a proteome-wide scale how ablation of individual MICOS subunits affects the levels of the other subunits. The results reveal co-regulation of TbMic10-1, TbMic10-2, TbMic16 and TbMic60, suggesting that these nonessential, integral inner membrane proteins form an interdependent network. Moreover, the ablation of TbMic34 and TbMic32 reveals another network consisting of the essential, intermembrane space-localized TbMic20, TbMic32, TbMic34 and TbMic40, all of which are peripherally associated with the inner membrane. The downregulation of TbMic20, TbMic32 and TbMic34 also interferes with mitochondrial protein import and reduces the size of the TbMic10-containing complexes. Thus, the diverged MICOS of trypanosomes contains two subcomplexes: a nonessential membrane-integrated one, organized around the conserved Mic10 and Mic60, that mediates cristae formation, and an essential membrane-peripheral one consisting of four kinetoplastid-specific subunits, that is required for import of intermembrane space proteins.

Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of Trypanosoma brucei.

Mitochondria cannot form de novo but require mechanisms that mediate their inheritance to daughter cells. The parasitic protozoan Trypanosoma brucei has a single mitochondrion with a single-unit genome that is physically connected across the two mitochondrial membranes with the basal body of the flagellum. This connection, termed the tripartite attachment complex (TAC), is essential for the segregation of the replicated mitochondrial genomes prior to cytokinesis. Here we identify a protein complex consisting of three integral mitochondrial outer membrane proteins-TAC60, TAC42 and TAC40-which are essential subunits of the TAC. TAC60 contains separable mitochondrial import and TAC-sorting signals and its biogenesis depends on the main outer membrane protein translocase. TAC40 is a member of the mitochondrial porin family, whereas TAC42 represents a novel class of mitochondrial outer membrane ß-barrel proteins. Consequently TAC40 and TAC42 contain C-terminal ß-signals. Thus in trypanosomes the highly conserved ß-barrel protein assembly machinery plays a major role in the biogenesis of its unique mitochondrial genome segregation system.

Correction: TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes.

[This corrects the article DOI: 10.1371/journal.ppat.1005586.].

TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes.

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization.

Functional characterization of the trypanosome translational repressor SCD6.

The storage of translationally inactive mRNAs in cytosolic granules enables cells to react flexibly to environmental changes. In eukaryotes, Scd6 (suppressor of clathrin deficiency 6)/Rap55 (RNA-associated protein 55), a member of the LSm14 (like-Sm14) family, is an important factor in the formation and activity of P-bodies, where mRNA decay factors accumulate, in stress granules that store mRNAs under adverse conditions and in granules that store developmentally regulated mRNAs. SCD6 from Trypanosoma brucei (TbSCD6) shares the same domain architecture as orthologous proteins in other organisms and is also present in cytosolic granules (equivalent to P-bodies). We show that TbSCD6 is a general repressor of translation and that its depletion by RNAi results in a global increase in protein synthesis. With few exceptions, the steady-state levels of proteins are unchanged. TbSCD6 is not required for the formation of starvation-induced granules in trypanosomes, and unlike Scd6 from yeast, Plasmodium and all multicellular organisms analysed to date, it does not form a complex with the helicase Dhh1 (DExD/H-box helicase 1). In common with Xenopus laevis RAP55, TbSCD6 co-purifies with two arginine methyltransferases; moreover, TbSCD6 itself is methylated on three arginine residues. Finally, a detailed analysis identified roles for the Lsm and N-rich domains in both protein localization and translational repression.

The nuclear mRNA export receptor Mex67-Mtr2 of Trypanosoma brucei contains a unique and essential zinc finger motif.

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis. Trypanosomes are early diverged protozoan parasites and show significant differences in their gene expression compared with higher eukaryotes. Due to a lack of individual gene promoters, large polycistronic transcripts are produced and individual mRNAs mature by trans-splicing and polyadenylation. In the absence of transcriptional control, regulation of gene expression occurs post-transcriptionally mainly by control of transcript stability and translation. Regulation of mRNA export from the nucleus to the cytoplasm might be an additional post-transcriptional event involved in gene regulation. However, our knowledge about mRNA export in trypanosomes is very limited. Although export factors of higher eukaryotes are reported to be conserved, only a few orthologues can be readily identified in the genome of T. brucei. Hence, biochemical approaches are needed to identify the export machinery of trypanosomes. Here, we report the functional characterization of the essential mRNA export factor TbMex67. TbMex67 contains a unique and essential N-terminal zinc finger motif. Furthermore, we could identify two interacting export factors namely TbMtr2 and the karyopherin TbIMP1. Our data show that the general heterodimeric export receptor Mex67-Mtr2 is conserved throughout the eukaryotic kingdom albeit exhibiting parasite-specific features.

Nucleolar proteins regulate stage-specific gene expression and ribosomal RNA maturation in Trypanosoma brucei.

Different life-cycle stages of Trypanosoma brucei are characterized by stage-specific glycoprotein coats. GPEET procyclin, the major surface protein of early procyclic (insect midgut) forms, is transcribed in the nucleolus by RNA polymerase I as part of a polycistronic precursor that is processed to monocistronic mRNAs. In culture, when differentiation to late procyclic forms is triggered by removal of glycerol, the precursor is still transcribed, but accumulation of GPEET mRNA is prevented by a glycerol-responsive element in the 3' UTR. A genome-wide RNAi screen for persistent expression of GPEET in glycerol-free medium identified a novel protein, NRG1 (Nucleolar Regulator of GPEET 1), as a negative regulator. NRG1 associates with GPEET mRNA and with several nucleolar proteins. These include two PUF proteins, TbPUF7 and TbPUF10, and BOP1, a protein required for rRNA processing in other organisms. RNAi against each of these components prolonged or even increased GPEET expression in the absence of glycerol as well as causing a significant reduction in 5.8S rRNA and its immediate precursor. These results indicate that components of a complex used for rRNA maturation can have an additional role in regulating mRNAs that originate in the nucleolus.

Fetal RHD Screening in RH1 Negative Pregnant Women: Experience in Switzerland.

RH1 incompatibility between mother and fetus can cause hemolytic disease of the fetus and newborn. In Switzerland, fetal RHD genotyping from maternal blood has been recommended from gestational age 18 onwards since the year 2020. This facilitates tailored administration of RH immunoglobulin (RHIG) only to RH1 negative women carrying a RH1 positive fetus. Data from 30 months of noninvasive fetal RHD screening is presented. Cell-free DNA was extracted from 7192 plasma samples using a commercial kit, followed by an in-house qPCR to detect RHD exons 5 and 7, in addition to an amplification control. Valid results were obtained from 7072 samples, with 4515 (64%) fetuses typed RHD positive and 2556 (36%) fetuses being RHD negative. A total of 120 samples led to inconclusive results due to the presence of maternal or fetal RHD variants (46%), followed by women being serologically RH1 positive (37%), and technical issues (17%). One sample was typed false positive, possibly due to contamination. No false negative results were observed. We show that unnecessary administration of RHIG can be avoided for more than one third of RH1 negative pregnant women in Switzerland. This reduces the risks of exposure to a blood-derived product and conserves this limited resource to women in actual need.

tRNASec is transcribed by RNA polymerase II in Trypanosoma brucei but not in humans.

Nuclear-encoded tRNAs are universally transcribed by RNA polymerase III (Pol-III) and contain intragenic promoters. Transcription of vertebrate tRNA(Sec) however requires extragenic promoters similar to Pol-III transcribed U6 snRNA. Here, we present a comparative analysis of tRNA(Sec) transcription in humans and the parasitic protozoa Trypanosoma brucei, two evolutionary highly diverged eukaryotes. RNAi-mediated ablation of Pol-II and Pol-III as well as oligo-dT induced transcription termination show that the human tRNA(Sec) is a Pol-III transcript. In T. brucei protein-coding genes are polycistronically transcribed by Pol-II and processed by trans-splicing and polyadenylation. tRNA genes are generally clustered in between polycistrons. However, the trypanosomal tRNA(Sec) genes are embedded within a polycistron. Their transcription is sensitive to α-amanitin and RNAi-mediated ablation of Pol-II, but not of Pol-III. Ectopic expression of the tRNA(Sec) outside but not inside a polycistron requires an added external promoter. These experiments demonstrate that trypanosomal tRNA(Sec), in contrast to its human counterpart, is transcribed by Pol-II. Synteny analysis shows that in trypanosomatids the tRNA(Sec) gene can be found in two different polycistrons, suggesting that it has evolved twice independently. Moreover, intron-encoded tRNAs are present in a number of eukaryotic genomes indicating that Pol-II transcription of tRNAs may not be restricted to trypanosomatids.

Single- and duplex TaqMan-quantitative PCR for determining the copy numbers of integrated selection markers during site-specific mutagenesis in Toxoplasma gondii by CRISPR-Cas9.

Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.

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 parasite's major cell-surface proteins-the variant surface glycoprotein (VSG) and procyclin. In the mammalian bloodstream, antigenic variation of the VSG coat is the parasite's 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.

Active RNA polymerase I of Trypanosoma brucei harbors a novel subunit essential for transcription.

A unique characteristic of the protistan parasite Trypanosoma brucei is a multifunctional RNA polymerase I which, in addition to synthesizing rRNA as in other eukaryotes, transcribes gene units encoding the major cell surface antigens variant surface glycoprotein and procyclin. Thus far, purification of this enzyme has revealed nine orthologues of known subunits but no active enzyme. Here, we have epitope tagged the specific subunit RPB6z and tandem affinity purified RNA polymerase I from crude extract. The purified enzyme was active in both a nonspecific and a promoter-dependent transcription assay and exhibited enriched protein bands with apparent sizes of 31, 29, and 27 kDa. p31 and its trypanosomatid orthologues were identified, but their amino acid sequences have no similarity to proteins of other eukaryotes, nor do they contain a conserved sequence motif. Nevertheless, p31 cosedimented with purified RNA polymerase I, and RNA interferance-mediated silencing of p31 was lethal, affecting the abundance of rRNA. Moreover, extract of p31-silenced cells exhibited a specific defect in transcription of class I templates, which was remedied by the addition of purified RNA polymerase I, and an anti-p31 serum completely blocked RNA polymerase I-mediated transcription. We therefore dubbed this novel functional component of T. brucei RNA polymerase I TbRPA31.

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

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 (SNAP(c)) 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 SNAP(c) 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/SNAP(c)/TFIIA complex binds specifically to the SL RNA gene promoter upstream sequence element and is absolutely essential for SL RNA gene transcription in vitro.

A TFIIB-like protein is indispensable for spliced leader RNA gene transcription in Trypanosoma brucei.

The lack of general class II transcription factors was a hallmark of the genomic sequences of the human parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. However, the recent identification of TFIIA as part of a protein complex essential for RNA polymerase II-mediated transcription of SLRNA genes, which encode the trans splicing-specific spliced leader RNA, suggests that trypanosomatids assemble a highly divergent set of these factors at the SLRNA promoter. Here we report the identification of a trypanosomatid TFIIB-like (TFIIB(like)) protein which has limited overall sequence homology to eukaryotic TFIIB and archaeal TFB but harbors conserved residues within the N-terminal zinc ribbon domain, the B finger and cyclin repeat I. In accordance with the function of TFIIB, T.brucei TFIIB(like) is encoded by an essential gene, localizes to the nucleus, specifically binds to the SLRNA promoter, interacts with RNA polymerase II, and is absolutely required for SLRNA transcription.

The Diverged Trypanosome MICOS Complex as a Hub for Mitochondrial Cristae Shaping and Protein Import.

The mitochondrial contact site and cristae organization system (MICOS) is a multiprotein complex responsible for cristae formation. Even though cristae are found in all mitochondria capable of oxidative phosphorylation, only Mic10 and Mic60 appear to be conserved throughout eukaryotes. The remaining 4 or 5 known MICOS subunits are specific to the supergroup Opisthokonta, which includes yeast and mammals that are the only organisms in which this complex has been analyzed experimentally. We have isolated the MICOS from Trypanosoma brucei, a member of the supergroup Excavata that is profoundly diverged from opisthokonts. We show that it is required for the maintenance of the unique discoidal cristae that typify excavates, such as euglenids and kinetoplastids, the latter of which include trypanosomes. The trypanosome MICOS consists of 9 subunits, most of which are essential for normal growth. Unlike in opisthokonts, it contains two distinct Mic10 orthologs and an unconventional putative Mic60 that lacks a mitofilin domain. Interestingly, one of the essential trypanosomatid-specific MICOS subunits called TbMic20 is a thioredoxin-like protein that appears to be involved in import of intermembrane space proteins, including respiratory chain complex assembly factors. This result points to trypanosome MICOS coordinating cristae shaping and population of its membrane with proteins involved in respiration, the latter via the catalytic activity of TbMic20. Thus, trypanosome MICOS allows us to define which of its features are conserved in all eukaryotes and decipher those that represent lineage-specific adaptations.

U1 small nuclear RNP from Trypanosoma brucei: a minimal U1 snRNA with unusual protein components.

Processing of primary transcripts in trypanosomes requires trans splicing and polyadenylation, and at least for the poly(A) polymerase gene, also internal cis splicing. The trypanosome U1 snRNA, which is most likely a cis-splicing specific component, is unusually short and has a relatively simple secondary structure. Here, we report the identification of three specific protein components of the Trypanosoma brucei U1 snRNP, based on mass spectrometry and confirmed by in vivo epitope tagging and in vitro RNA binding. Both T.brucei U1-70K and U1C are only distantly related to known counterparts from other eukaryotes. The T.brucei U1-70K protein represents a minimal version of 70K, recognizing the first loop sequence of U1 snRNA with the same specificity as the mammalian protein. The trypanosome U1C-like protein interacts with 70K directly and binds the 5' terminal sequence of U1 snRNA. Surprisingly, instead of U1A we have identified a novel U1 snRNP-specific protein, TbU1-24K. U1-24K lacks a known RNA-binding motif and integrates in the U1 snRNP via interaction with U1-70K. These data result in a model of the trypanosome U1 snRNP, which deviates substantially from our classical view of the U1 particle and may reflect the special requirements for splicing of a small set of cis-introns in trypanosomes.

Purification of an eight subunit RNA polymerase I complex in Trypanosoma brucei.

Trypanosoma brucei harbors a unique multifunctional RNA polymerase (pol) I which transcribes, in addition to ribosomal RNA genes, the gene units encoding the major cell surface antigens variant surface glycoprotein and procyclin. In consequence, this RNA pol I is recruited to three structurally different types of promoters and sequestered to two distinct nuclear locations, namely the nucleolus and the expression site body. This versatility may require parasite-specific protein-protein interactions, subunits or subunit domains. Thus far, data mining of trypanosomatid genomes have revealed 13 potential RNA pol I subunits which include two paralogous sets of RPB5, RPB6, and RPB10. Here, we analyzed a cDNA library prepared from procyclic insect form T. brucei and found that all 13 candidate subunits are co-expressed. Moreover, we PTP-tagged the largest subunit TbRPA1, tandem affinity-purified the enzyme complex to homogeneity, and determined its subunit composition. In addition to the already known subunits RPA1, RPA2, RPC40, 1RPB5, and RPA12, the complex contained RPC19, RPB8, and 1RPB10. Finally, to evaluate the absence of RPB6 in our purifications, we used a combination of epitope-tagging and reciprocal coimmunoprecipitation to demonstrate that 1RPB6 but not 2RPB6 binds to RNA pol I albeit in an unstable manner. Collectively, our data strongly suggest that T. brucei RNA pol I binds a distinct set of the RPB5, RPB6, and RPB10 paralogs.