The genome of Trypanosoma brucei is unusual in being regulated almost entirely at the post-transcriptional level. In terms of regulation, the best-studied genes are procyclins, which encode a family of major surface GPI-anchored glycoproteins (EP1, EP2, EP3, GPEET) that show differential expression in the parasite's tsetse-fly vector. Although procyclin mRNA cis-regulatory sequences have provided the paradigm for post-transcriptional control in kinetoplastid parasites, trans-acting regulators of procyclin mRNAs are unidentified, despite intensive effort over 15 years. Here we identify the developmental regulator, TbZFP3, a CCCH-class predicted RNA binding protein, as an isoform-specific regulator of Procyclin surface coat expression in trypanosomes. We demonstrate (i) that endogenous TbZFP3 shows sequence-specific co-precipitation of EP1 and GPEET, but not EP2 and EP3, procyclin mRNA isoforms, (ii) that ectopic overexpression of TbZFP3 does not perturb the mRNA abundance of procyclin transcripts, but rather that (iii) their protein expression is regulated in an isoform-specific manner, as evidenced by mass spectrometric analysis of the Procyclin expression signature in the transgenic cell lines. The TbZFP3 mRNA–protein complex (TbZFP3mRNP) is identified as a trans-regulator of differential surface protein expression in trypanosomes. Moreover, its sequence-specific interactions with procyclin mRNAs are compatible with long-established predictions for Procyclin regulation. Combined with the known association of TbZFP3 with the translational apparatus, this study provides a long-sought missing link between surface protein cis-regulatory signals and the gene expression machinery in trypanosomes.

The post-transcriptional control of gene expression is becoming increasingly important in the understanding of regulated events in eukaryotic cells. The parasitic kinetoplastids have a unique reliance on such processes, because their genome is organized into polycistronic transcription units in which adjacent genes are not coordinately regulated. Indeed, the number of RNA-binding proteins predicted to be encoded in the genome of kinetoplastids is unusually large, invoking the presence of unique RNA regulators dedicated to gene expression in these evolutionarily ancient organisms. Here, we report that a small CCCH zinc finger protein, TbZFP3, enhances development between life-cycle stages in Trypanosoma brucei. Moreover, we demonstrate that this protein interacts both with the translational machinery and with other small CCCH proteins previously implicated in trypanosome developmental control. Antibodies to this protein also co-immunoprecipitate EP procyclin mRNA and encode the major surface antigen of insect forms of T. brucei. Strikingly, although TbZFP3 is constitutively expressed, it exhibits developmentally regulated association with polyribosomes, and mutational analysis demonstrates that this association is essential for the expression of phenotype.

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Leishmania spp. are protozoan parasites that are transmitted by sandfly vectors during blood sucking to vertebrate hosts and cause a spectrum of diseases called leishmaniases. It has been demonstrated that host cholesterol plays an important role during Leishmania infection. Nevertheless, little is known about the intracellular distribution of this lipid early after internalization of the parasite. Here, pulse‐chase experiments with radiolabeled cholesteryl esterified to fatty acids bound to low‐density lipoproteins indicated that retention of this source of cholesterol is increased in parasite‐containing subcellular fractions, while uptake is unaffected. This is correlated with a reduction or absence of detectable NPC1 (Niemann–Pick disease, type C1), a protein responsible for cholesterol efflux from endocytic compartments, in the Leishmania mexicana habitat and infected cells. Filipin staining revealed a halo around parasites within parasitophorous vacuoles (PV) likely representing free cholesterol accumulation. Labeling of host cell membranous cholesterol by fluorescent cholesterol species before infection revealed that this pool is also trafficked to the PV but becomes incorporated into the parasites’ membranes and seems not to contribute to the halo detected by filipin. This cholesterol sequestration happened early after infection and was functionally significant as it correlated with the upregulation of mRNA‐encoding proteins required for cholesterol biosynthesis. Thus, sequestration of cholesterol by Leishmania amastigotes early after infection provides a basis to understand perturbation of cholesterol‐dependent processes in macrophages that were shown previously by others to be necessary for their proper function in innate and adaptive immune responses.