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 6snoRNAs 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.

The parasite Trypanosoma brucei is the causative agent of African sleeping sickness and is known for its unique RNA processing mechanisms that are common to all the kinetoplastidea including Leishmania and Trypanosoma cruzi. Trypanosomes possess two canonical RNA poly (A) polymerases (PAPs) termed PAP1 and PAP2. PAP1 is encoded by one of the only two genes harboring cis-spliced introns in this organism, and its function is currently unknown. In trypanosomes, all mRNAs, and non-coding RNAs such as small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs), undergo trans-splicing and polyadenylation. Here, we show that the function of PAP1, which is located in the nucleus, is to polyadenylate non-coding RNAs, which undergo trans-splicing and polyadenylation. Major substrates of PAP1 are the snoRNAs and lncRNAs. Under the silencing of either PAP1 or PAP2, the level of snoRNAs is reduced. The dual polyadenylation of snoRNA intermediates is carried out by both PAP2 and PAP1 and requires the factors essential for the polyadenylation of mRNAs. The dual polyadenylation of the precursor snoRNAs by PAPs may function to recruit the machinery essential for snoRNA processing.

The infectious metacyclic forms of Trypanosoma brucei result from a complex development in the tsetse fly vector. When they infect mammals, they cause African sleeping sickness in humans. Due to scarcity of biological material and difficulties of the tsetse fly as an experimental system, very limited information is available concerning the gene expression profile of metacyclic forms. We used an in vitro system based on expressing the RNA binding protein 6 to obtain infectious metacyclics and determined their protein and mRNA repertoires by mass-spectrometry (MS) based proteomics and mRNA sequencing (RNA-Seq) in comparison to non-infectious procyclic trypanosomes. We showed that metacyclics are quiescent cells, and propose this influences the choice of a monocistronic variant surface glycoprotein expression site. Metacyclics have a largely bloodstream-form type transcriptome, and thus are programmed to translate a bloodstream-form type proteome upon entry into the mammalian host and resumption of cell division. Genes encoding cell surface components showed the largest changes between procyclics and metacyclics, observed at both the transcript and protein levels. Genes encoding metabolic enzymes exhibited expression in metacyclics with features of both procyclic and bloodstream forms, suggesting that this intermediate-type metabolism is dictated by the availability of nutrients in the tsetse fly vector.

One distinctive feature of the Trypanosoma brucei life cycle is the presence of two discrete populations that are based on differential expression of variant surface glycoproteins (VSGs). Both are adapted to the environmental pressures they face and more importantly, both contribute directly to transmission. Metacyclics in the tsetse fly enable transmission to a new mammalian host, whereas bloodstream trypanosomes must avoid immune destruction to the extent that sufficient numbers are available for transmission, when the insect vector takes a blood meal. At present, there are few investigations on the molecular aspects of parasite biology in the tsetse vector and specifically about the activation of metacyclic VSG gene expression. Here we used an established in vitro differentiation system based on the overexpression of the RNA-binding protein 6 (RBP6), to monitor two metacyclic VSGs (VSG 397 and VSG 653) during development from procyclics to infectious metacyclic forms. We observed that activation of these two mVSGs was simultaneous both at the transcript and protein level, and manifested by the appearance of only one of the mVSGs in individual cells.

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 trypanosome-specific 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.

Background: Although technical advances in genomics and proteomics research have yielded a better understanding of the coding capacity of a genome, one major challenge remaining is the identification of all expressed proteins, especially those less than 100 amino acids in length. Such information can be particularly relevant to human pathogens, such as Trypanosoma brucei, the causative agent of African trypanosomiasis, since it will provide further insight into the parasite biology and life cycle.Results: Starting with 993 T. brucei transcripts, previously shown by RNA-Sequencing not to coincide with annotated coding sequences (CDS), homology searches revealed that 173 predicted short open reading frames in these transcripts are conserved across kinetoplastids with 13 also conserved in representative eukaryotes. Mining mass spectrometry data sets revealed 42 transcripts encoding at least one matching peptide. RNAi-induced down-regulation of these 42 transcripts revealed seven to be essential in insect-form trypanosomes with two also required for the bloodstream life cycle stage. To validate the specificity of the RNAi results, each lethal phenotype was rescued by co-expressing an RNAi-resistant construct of each corresponding CDS. These previously non-annotated essential small proteins localized to a variety of cell compartments, including the cell surface, mitochondria, nucleus and cytoplasm, inferring the diverse biological roles they are likely to play in T. brucei. We also provide evidence that one of these small proteins is required for replicating the kinetoplast (mitochondrial) DNA.Conclusions: Our studies highlight the presence and significance of small proteins in a protist and expose potential new targets to block the survival of trypanosomes in the insect vector and/or the mammalian host.

High-throughput RNA sequencing (RNA-Seq) has quickly occupied center stage in the repertoire of available tools for transcriptomics. Among many advantages, the single-nucleotide resolution of this powerful approach allows mapping on a genome-wide scale of splice junctions and polyadenylation sites, and thus, the precise definition of mature transcript boundaries. This greatly facilitated the transcriptome annotation of the human pathogen Trypanosoma brucei, a protozoan organism in which all mRNA molecules are matured by spliced leader (SL) trans-splicing from longer polycistronic precursors. The protocols described here for the generation of three types of libraries for Illumina RNA-Seq, 5'-SL enriched, 5'-triphosphate-end enriched, and 3'-poly(A) enriched, enabled the discovery of an unprecedented heterogeneity of pre-mRNA-processing sites, a large number of novel coding and noncoding transcripts from previously unannotated genes, and quantify the cellular abundance of RNA molecules. The method for producing 5'-triphosphate-end-enriched libraries was instrumental for obtaining evidence that transcription initiation by RNA polymerase II in trypanosomes is bidirectional and biosynthesis of mRNA precursors is primed not only at the beginning of unidirectional gene clusters, but also at specific internal sites.

ABSTRACT Parasitic protozoa of the flagellate order Kinetoplastida represent one of the deepest branches of the eukaryotic tree. Among this group of organisms, the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser degree in Leishmania (Viannia) spp. The pathway is triggered by long double-stranded RNA (dsRNA) and in T. brucei requires a set of five core genes, including a single Argonaute (AGO) protein, T. brucei AGO1 (TbAGO1). The five genes are conserved in Leishmania (Viannia) spp. but are absent in other major kinetoplastid species, such as Trypanosoma cruzi and Leishmania major. In T. brucei small interfering RNAs (siRNAs) are methylated at the 3′ end, whereas Leishmania (Viannia) sp. siRNAs are not. Here we report that T. brucei HEN1, an ortholog of the metazoan HEN1 2′-O-methyltransferases, is required for methylation of siRNAs. Loss of TbHEN1 causes a reduction in the length of siRNAs. The shorter siRNAs in hen1−/− parasites are single stranded and associated with TbAGO1, and a subset carry a nontemplated uridine at the 3′ end. These findings support a model wherein TbHEN1 methylates siRNA 3′ ends after they are loaded into TbAGO1 and this methylation protects siRNAs from uridylation and 3′ trimming. Moreover, expression of TbHEN1 in Leishmania (Viannia) panamensis did not result in siRNA 3′ end methylation, further emphasizing mechanistic differences in the trypanosome and Leishmania RNAi mechanisms.

One of the key questions in understanding the biology of an organism is how to correlate cellular fate and function with gene expression patterns. This is particularly relevant for pathogenic organisms, like the parasitic protozoa Trypanosoma brucei, who often cycle between different hosts, thereby encountering vastly different environments. Survival in and adaptation to new surroundings requires activation of specific gene networks, which is most often achieved by regulatory mechanisms embedded in the transcriptional machinery. However, in T. brucei and related trypanosomatids these responses appear to be accomplished mainly by post-transcriptional mechanisms. Although an understanding of how this parasite modulates gene regulatory networks is in the early stages, RNA-binding proteins (RBPs) are beginning to take centre stage. Here, we discuss recent progress in the identification of RBPs with crucial roles in different stages of the T. brucei life cycle, and in elucidating targets of RBPs.

On the role of the Trypanosoma brucei HEN1-family methyltransferase in siRNA 1 modification 2 3 Huafang Shi a , Rebecca L. Barnes a* , Nicholas Carriero b , Vanessa D. Atayde a* , Christian 4 Tschudi c# , Elisabetta Ullu a,d# 5 6 Department of Internal Medicine, School of Medicine, Yale University, New Haven, 7 Connecticut, USA a ; Department of Computer Science, Yale University, New Haven, 8 Connecticut, USA b ; Department of Epidemiology of Microbial Diseases, School of Public 9 Health, Yale University, New Haven, Connecticut, USA c ; Department of Cell Biology, School 10 of Medicine, Yale University, New Haven, Connecticut, USA d . 11 12 Running Head: Trypanosoma brucei HEN1 13 14 #Address correspondence to Christian Tschudi, christian.tschudi@yale.edu, or Elisabetta Ullu, 15 elisabetta.ullu@yale.edu. 16 *Present addresses: Rebecca L. Barnes, Department of Molecular Biology and Biotechnology, 17 University of Sheffield, Sheffield, United Kingdom; Vanessa D. Atayde, Department of 18 Microbiology and Immunology, McGill University, Montreal, Canada 19 20 H.S. and R.L.B. contributed equally to this work. 21 22 EC Accepts, published online ahead of print on 1 November 2013 Eukaryotic Cell doi:10.1128/EC.00233-13 Copyright © 2013, American Society for Microbiology. All Rights Reserved.

RNA interference (RNAi), used as a tool, has revolutionized the studies of gene function. Long stem-loop dsRNA has been proven the most effective trigger for down-regulating target transcripts in RNAi-positive trypanosomatid parasites. Here we describe a protocol for constructing plasmids that produce long stem-loops by using a single cloning step. Inverted repeats are first obtained by self-ligation of PCR products that contain a randomized segment at one of their ends and then inserted in a plasmid vector. The random sequences create the loop (or "stuffer") of the hairpin. This methodology was tested in Leishmania (Viannia) braziliensis to constitutively knock down the mRNAs for the well-studied paraflagellar rod protein 1 and 2 (PFR1 and PFR2) genes and revealed that mRNA cleavage products are unusually stable in these parasites. The protocol is suitable for any plasmid (for constitutive or inducible expression) and for any organism in which long stem-loops can be used to elicit RNAi.

In most eukaryotic cells expression or delivery of long double‐stranded RNA (dsRNA) signals the presence of foreign and/or potentially dangerous nucleic acids, such as viruses or transcripts derived from retroposons and transposons. As a consequence cells go on red alert and activate specific defense mechanisms to eliminate the invaders. Among such mechanisms is the RNA interference (RNAi) pathway, which is triggered by long dsRNAs, destroys target transcripts in a sequence‐specific manner and is widespread throughout eukaryotic evolution. In the late 90s our laboratory reported the discovery of RNAi in Trypanosoma brucei , a protozoan parasite that causes devastating diseases in sub‐Saharan Africa. Since then the way experiments are carried out in T. brucei has undergone a major revolution, and gene silencing by RNAi has found wide applications, including forward genetic screens and global analysis of gene function. I will present a historical recount of the discovery and mechanistic understanding of the trypanosome RNAi pathway with emphasis on its biological significance.

Unraveling the intricate interactions between Trypanosoma brucei, the protozoan parasite causing African trypanosomiasis, and the tsetse (Glossina) vector remains a challenge. Metacyclic trypanosomes, which inhabit the tsetse salivary glands, transmit the disease and are produced through a complex differentiation and unknown program. By overexpressing a single RNA-binding protein, TbRBP6, in cultured noninfectious trypanosomes, we recapitulated the developmental stages that have been observed in tsetse, including the generation of infective metacyclic forms expressing the variant surface glycoprotein. Thus, events leading to acquisition of infectivity in the insect vector are now accessible to laboratory investigation, providing an opening for new intervention strategies.

SummaryAmong trypanosomatid protozoa the mechanism of RNA interference (RNAi) has been investigated in Trypanosoma brucei and to a lesser extent in Leishmania braziliensis. Although these two parasitic organisms belong to the same family, they are evolutionarily distantly related raising questions about the conservation of the RNAi pathway. Here we carried out an in‐depth analysis of small interfering RNAs (siRNAs) associated with L. braziliensis Argonaute1 (LbrAGO1). In contrast to T. brucei, Leishmania siRNAs are sensitive to 3′ end oxidation, indicating the absence of blocking groups, and the Leishmania genome does not code for a HEN1 RNA 2′‐O‐methyltransferase, which modifies small RNA 3′ ends. Consistent with this observation, ∼ 20% of siRNA 3′ ends carry non‐templated uridines. Thus siRNA biogenesis, and most likely their metabolism, is different in these organisms. Similarly to T. brucei, putative mobile elements and repeats constitute the major Leishmania siRNA‐producing loci and AGO1 ablation leads to accumulation of long transcripts derived from putative mobile elements. However, contrary to T. brucei, no siRNAs were detected from other genomic regions with the potential to form double‐stranded RNA, namely sites of convergent transcription and inverted repeats. Thus, our results indicate that organism‐specific diversification has occurred in the RNAi pathway during evolution of the trypanosomatid lineage.

Background At the core of the RNA interference (RNAi) pathway in Trypanosoma brucei is a single Argonaute protein, Tb AGO1, with an established role in controlling retroposon and repeat transcripts. Recent evidence from higher eukaryotes suggests that a variety of genomic sequences with the potential to produce double-stranded RNA are sources for small interfering RNAs (siRNAs). Results To test whether such endogenous siRNAs are present in T. brucei and to probe the individual role of the two Dicer-like enzymes, we affinity purified Tb AGO1 from wild-type procyclic trypanosomes, as well as from cells deficient in the cytoplasmic ( Tb DCL1) or nuclear ( Tb DCL2) Dicer, and subjected the bound RNAs to Illumina high-throughput sequencing. In wild-type cells the majority of reads originated from two classes of retroposons. We also considerably expanded the repertoire of trypanosome siRNAs to encompass a family of 147-bp satellite-like repeats, many of the regions where RNA polymerase II transcription converges, large inverted repeats and two pseudogenes. Production of these newly described siRNAs is strictly dependent on the nuclear DCL2. Notably, our data indicate that putative centromeric regions, excluding the CIR147 repeats, are not a significant source for endogenous siRNAs. Conclusions Our data suggest that endogenous RNAi targets may be as evolutionarily old as the mechanism itself.

The introduction ten years ago of RNA interference (RNAi) as a tool for molecular exploration in Trypanosoma brucei has led to a surge in our understanding of the pathogenesis and biology of this human parasite. In particular, a genome-wide RNAi screen has recently been combined with next-generation Illumina sequencing to expose catalogues of genes associated with loss of fitness in distinct developmental stages. At present, this technology is restricted to RNAi-positive protozoan parasites, which excludes T. cruzi, Leishmania major, and Plasmodium falciparum. Therefore, elucidating the mechanism of RNAi and identifying the essential components of the pathway is fundamental for improving RNAi efficiency in T. brucei and for transferring the RNAi tool to RNAi-deficient pathogens. Here we used comparative genomics of RNAi-positive and -negative trypanosomatid protozoans to identify the repertoire of factors in T. brucei. In addition to the previously characterized Argonaute 1 (AGO1) protein and the cytoplasmic and nuclear Dicers, TbDCL1 and TbDCL2, respectively, we identified the RNA Interference Factors 4 and 5 (TbRIF4 and TbRIF5). TbRIF4 is a 3'-5' exonuclease of the DnaQ superfamily and plays a critical role in the conversion of duplex siRNAs to the single-stranded form, thus generating a TbAGO1-siRNA complex required for target-specific cleavage. TbRIF5 is essential for cytoplasmic RNAi and appears to act as a TbDCL1 cofactor. The availability of the core RNAi machinery in T. brucei provides a platform to gain mechanistic insights in this ancient eukaryote and to identify the minimal set of components required to reconstitute RNAi in RNAi-deficient parasites.

The discovery of a plethora of small non-coding RNAs (ncRNAs) has fundamentally changed our understanding of how genes are regulated. In this study, we employed the power of deep sequencing of RNA (RNA-seq) to examine the repertoire of ncRNAs present in small ribonucleoprotein particles (RNPs) of Trypanosoma brucei, an important protozoan parasite. We identified new C/D and H/ACA small nucleolar RNAs (snoRNAs), as well as tens of putative novel non-coding RNAs; several of these are processed from trans-spliced and polyadenylated transcripts. The RNA-seq analysis provided information on the relative abundance of the RNAs, and their 5'- and 3'-termini. The study demonstrated that three highly abundant snoRNAs are involved in rRNA processing and highlight the unique trypanosome-specific repertoire of these RNAs. Novel RNAs were studied using in situ hybridization, association in RNP complexes, and 'RNA walk' to detect interaction with their target RNAs. Finally, we showed that the abundance of certain ncRNAs varies between the two stages of the parasite, suggesting that ncRNAs may contribute to gene regulation during the complex parasite's life cycle. This is the first study to provide a whole-genome analysis of the large repertoire of small RNPs in trypanosomes.

ABSTRACT Protozoan parasites that profoundly affect mankind represent an exceptionally diverse group of organisms, including Plasmodium , Toxoplasma , Entamoeba , Giardia , trypanosomes, and Leishmania . Despite the overwhelming impact of these parasites, there remain many aspects to be discovered about mechanisms of pathogenesis and how these organisms survive in the host. Combined with the ever-increasing availability of sequenced genomes, RNA interference (RNAi), discovered a mere 13 years ago, has enormously facilitated the analysis of gene function, especially in organisms that are not amenable to classical genetic approaches. Here we review the current status of RNAi in studies of parasitic protozoa, with special emphasis on its use as a postgenomic tool.

In most eukaryotic cells, expression or delivery of long double-stranded RNA (dsRNA) signals the presence of foreign and/or potentially dangerous nucleic acids, such as viruses or transcripts derived from retroposons and transposons. As a consequence cells go on red alert and activate specific defense mechanisms to eliminate the invaders. Among such mechanisms is the RNA interference (RNAi) pathway, which is triggered by long dsRNAs, destroys target transcripts in a sequence-specific manner, and is widespread throughout eukaryotic evolution. Here we summarize our current understanding of the RNAi mechanism in Trypanosoma brucei, a protozoan parasite and early divergent eukaryote, and highlight similarities and differences with the RNAi machinery and its function in higher eukaryotes.

A new RNA world has emerged in the past 10 years with the discovery of a plethora of 20- to 30-nucleotide long small RNAs that are involved in various gene silencing mechanisms. These small RNAs have considerably changed our view of the regulation of gene expression in eukaryotic organisms, with a major shift towards epigenetic and post-transcriptional mechanisms. In this article, we focus on the striking diversity of small silencing RNAs that have been identified in several protozoan parasites and their potential biological role.