Extracellular vesicles (EV) secreted by pathogens function in a variety of biological processes. Here, we demonstrate that in the protozoan parasite Trypanosoma brucei, exosome secretion is induced by stress that affects trans-splicing. Following perturbations in biogenesis of spliced leader RNA, which donates its spliced leader (SL) exon to all mRNAs, or after heat-shock, the SL RNA is exported to the cytoplasm and forms distinct granules, which are then secreted by exosomes. The exosomes are formed in multivesicular bodies (MVB) utilizing the endosomal sorting complexes required for transport (ESCRT), through a mechanism similar to microRNA secretion in mammalian cells. Silencing of the ESCRT factor, Vps36, compromised exosome secretion but not the secretion of vesicles derived from nanotubes. The exosomes enter recipient trypanosome cells. Time-lapse microscopy demonstrated that cells secreting exosomes or purified intact exosomes affect social motility (SoMo). This study demonstrates that exosomes are delivered to trypanosome cells and can change their migration. Exosomes are used to transmit stress signals for communication between parasites. Trypanosomes are the causative agent of major parasitic diseases such as African sleeping sickness, leishmaniosis and Chagas' disease that affect millions of people. These parasites cycle between an insect and a mammalian host. Communication between the parasites and the host must be essential for executing a productive infection and for cycling of the parasite between its hosts. Exosomes are 40-100nm vesicles of endocytic origin, and were shown to affect a variety of biological processes and human diseases. Exosomes were also shown to help pathogens evade the immune system. In this study, we demonstrate that exosomes are secreted from Trypanosoma brucei parasites when trans-splicing is inhibited. These exosomes contain, among many other constituents, a type of RNA known as spliced leader RNA (SL RNA), which is essential in these parasites for formation of all mature mRNA. These exosomes are able to enter neighboring trypanosomes, and only intact exosomes affect the social motility of these parasites. We propose that exosomes can potentially control parasite migration in the insect host by acting as a repellent that drives the fit parasites away from either damaged cells or an unfavorable environment. This mechanism could secure a productive infection.

The protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa, undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector. However, little is known about how the parasite performs most molecular functions in such different environments. Here, we provide evidence for the intriguing possibility that pseudouridylation of rRNA plays an important role in the capacity of the parasite to transit between the insect midgut and the mammalian bloodstream. Briefly, we mapped pseudouridines (Ψ) on rRNA by Ψ-seq in procyclic form (PCF) and bloodstream form (BSF) trypanosomes. We detected 68 Ψs on rRNA, which are guided by H/ACA small nucleolar RNAs (snoRNA). The small RNome of both life cycle stages was determined by HiSeq and 83 H/ACAs were identified. We observed an elevation of 21 Ψs modifications in BSF as a result of increased levels of the guiding snoRNAs. Overexpression of snoRNAs guiding modification on H69 provided a slight growth advantage to PCF parasites at 30 °C. Interestingly, these modifications are predicted to significantly alter the secondary structure of the large subunit (LSU) rRNA suggesting that hypermodified positions may contribute to the adaption of ribosome function during cycling between the two hosts.

The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an insect and a mammalian host. Here, we investigated the presence of pseudouridines (Ψs) on the spliceosomal small nuclear RNAs (snRNAs), which may enable growth at the very different temperatures characterizing the two hosts. To this end, we performed the first high-throughput mapping of spliceosomal snRNA Ψs by small RNA Ψ-seq. The analysis revealed 42 Ψs on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding Ψ on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA Ψs are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth at elevated temperatures. We further demonstrate that Ψ strengthens U4/U6 RNA–RNA and U2B"/U2A’ proteins-U2 snRNA interaction at elevated temperatures. The existence of single hairpin RNAs that modify both the spliceosome and ribosome RNAs is unique for these parasites, and may be related to their ability to cycle between their two hosts that differ in temperature.

In trypanosomes, mRNAs are processed by trans-splicing; in this process, a common exon, the spliced leader, is added to all mRNAs from a small RNA donor, the spliced leader RNA (SL RNA). However, l...

In trypanosomes, in contrast to most eukaryotes, the large subunit (LSU) ribosomal RNA is fragmented into two large and four small ribosomal RNAs (srRNAs) pieces, and this additional processing likely requires trypanosome-specific factors. Here, we examined the role of 10 abundant small nucleolar RNAs (snoRNAs) involved in rRNA processing. We show that each snoRNA involved in LSU processing associates with factors engaged in either early or late biogenesis steps. Five of these snoRNAs interact with the intervening sequences of rRNA precursor, whereas the others only guide rRNA modifications. The function of the snoRNAs was explored by silencing snoRNAs. The data suggest that the LSU rRNA processing events do not correspond to the order of rRNA transcription, and that srRNAs 2, 4 and 6 which are part of LSU are processed before srRNA1. Interestingly, the 6 snoRNAs that affect srRNA1 processing guide modifications on rRNA positions that span locations from the protein exit tunnel to the srRNA1, suggesting that these modifications may serve as check-points preceding the liberation of srRNA1. This study identifies the highest number of snoRNAs so far described that are involved in rRNA processing and/or rRNA folding and highlights their function in the unique trypanosome rRNA maturation events.