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.

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.

SUMMARYApproximately 20% of plant genes possess upstream open-reading frames (uORFs). The effect of uORFs on gene expression has mainly been studied at the translational level. Very little is known about the impact of plant uORFs on transcript content through the nonsense-mediated mRNA decay (NMD) pathway, which degrades transcripts bearing premature termination codons (PTCs). Here we examine the impact of the uORF of the Arabidopsis AtMHX gene on transcript accumulation. The suggestion that this uORF exposes transcripts containing it to NMD is supported by (i) the increase in transcript levels upon eliminating the uORF from constructs containing it, (ii) experiments with a modified uORF-peptide, which excluded peptide-specific degradation mechanisms, (iii) the increase in levels of the native AtMHX transcript upon treatment with cycloheximide, which inhibits translation and blocks NMD, and (iv) the sensitivity of transcripts containing the uORF of AtMHX to the presence of introns. We also showed that introns can increase NMD efficiency not only in transcripts having relatively short 3¢ untranslated regions (UTRs), but also in uORF-containing transcripts. AtMHX transcript levels were almost unaltered in mutants of the NMD factors UPF3 and UPF1. Possible reasons, including the existence of a NMD-compensatory mechanism, are discussed. Interestingly, the levels of UPF3 transcript were higher in upf1 mutants, suggesting a compensatory mechanism that links weak function of the NMD machinery to increased expression of UPF3. Our findings highlight that uORFs, which are abundant in plants, can not only inhibit translation but also strongly affect transcript accumulation.

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, little is known regarding how this process is regulated. In this study, we investigated the function of two serine-arginine-rich proteins, TsR1 and TsR1IP, implicated in trans-splicing in Trypanosoma brucei. Depletion of these factors by RNai suggested their role in both cisand trans-splicing. Microarray was used to examine the transcriptome of the silenced cells. The level of hundreds of mRNas was changed, suggesting that these proteins have a role in regulating only a subset of T. brucei mRNas. Massspectrometry analyses of complexes associated with these proteins suggest that these factors function in mRNa stability, translation, and rRNa processing. We further demonstrate changes in the stability of mRNa as a result of depletion of the two TsR proteins. In addition, rRNa defects were observed under the depletion of U2aF35, TsR1, and TsR1IP, but not sF1, suggesting involvement of sR proteins in rRNa processing. ©2014 Landes Bioscience. Do not distribute 716RNa Biology Volume 11 Issue 6 among the hundreds of proteins present in the RNA polymerase II transcription complex, and are often loaded co-transcriptionally and accompany the fully spliced mRNA to the cytoplasm. 19 Since splice site consensus sequences are not sufficient to direct assembly of the spliceosome, sequences present in exons or introns such as exonic and intronic splicing enhancers (ESE and ISE), or exonic and intronic splicing silencers (ESS or ISS), are used to bind factors that regulate spliceosome assembly. SR proteins stabilize interactions between the U1 snRNP at the 5′ splice site and U2AF65 at the 3′ splice site. SR proteins are also known to bind to ESE and antagonize the activity of hnRNP proteins recognizing ESS. 20 In metazoa such as C. elegans, several SR proteins are essential, but others are not. 21 In mouse, many SR proteins are essential for life. 22 SR proteins have other functions in addition to their role in splicing, such as nuclear export, non-sense-mediated decay, and translation. SR proteins affect translation directly and indirectly. SF2/ASF was shown to associate with polyribosomes and to enhance translation, probably via release of 4E-BP, a competitive inhibitor of cap-dependent translation. 17 Recent studies support the role of SR proteins not only as splicing regulators, but also implicate these proteins in genome stability, chromatin binding, transcription elongation, mRNA stability, mRNA export, and translation (see review 23 ).The function of SR proteins is regulated by phosphorylation and de-phosphorylation. The RS domain is extensively phosphorylated on serine residues and this modification controls the localization of the protein. Mammalian SR proteins become dephosphorylated during the course of pre-mRNA processing, and promote mRNP transit through the nuclear pore complex. 24 SR proteins associate with the exon-junct...

(2015) Genome-wide analysis of small nucleolar RNAs of Leishmaniamajor reveals a rich repertoire of RNAs involved in modification and processing of rRNA, RNA Biology, 12:11, 1222-1255, DOI: 10.1080/15476286.2015 Trypanosomatids are protozoan parasites and the causative agent of infamous infectious diseases. These organisms regulate their gene expression mainly at the post-transcriptional level and possess characteristic RNA processing mechanisms. In this study, we analyzed the complete repertoire of Leishmania major small nucleolar (snoRNA) RNAs by performing RNA-seq analysis on RNAs that were affinity-purified using the C/D snoRNA core protein, SNU13, and the H/ ACA core protein, NHP2. This study revealed a large collection of C/D and H/ACA snoRNAs, organized in gene clusters generally containing both snoRNA types. Abundant snoRNAs were identified and predicted to guide trypanosomespecific rRNA cleavages. The repertoire of snoRNAs was compared to that of the closely related Trypanosoma brucei, and 80% of both C/D and H/ACA molecules were found to have functional homologues. The comparative analyses elucidated how snoRNAs evolved to generate molecules with analogous functions in both species. Interestingly, H/ACA RNAs have great flexibility in their ability to guide modifications, and several of the RNA species can guide more than one modification, compensating for the presence of single hairpin H/ACA snoRNA in these organisms. Placing the predicted modifications on the rRNA secondary structure revealed hypermodification regions mostly in domains which are modified in other eukaryotes, in addition to trypanosome-specific modifications.