Summary: 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.

Summary: 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 ( N ucleolar R egulator 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.

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.

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 inToxoplasma gondiiknockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the insertedmdhfr-tsand of the wild-type (WT)dhfr-ts(wtdhfr-ts) which is present as single copy gene inToxoplasmaand encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of thedhfr-tsfragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number ofdhfr-tscopies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bpdhfr-tsfragment and theT.gondii 529-bprepeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given bydhfr-tsamplification is equal to the number of tachyzoites determined by amplification of theToxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration ofmdhfr-ts, the calculated ratio is 2. We then applied both approaches to testT.gondiiRH mutants in which themajor surface antigen(SAG1) was disrupted through insertion ofmdhfr-tsusing CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integratedmdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic ofT.gondiiKOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions ofmdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers inToxoplasma. TaqMan qPCR tools may contribute to more frequent use of WTToxoplasmastrains during functional genomics.

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.