Search

Background: At the core of the RNA interference (RNAi) pathway in Trypanosoma brucei is a single Argonaute protein, TbAGO1, 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 TbAGO1 from wild-type procyclic trypanosomes, as well as from cells deficient in the cytoplasmic (TbDCL1) or nuclear (TbDCL2) 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

One of the unique aspects of RNA processing in trypanosomatid protozoa is the presence of a cap 4 structure (m7Gpppm26AmpAmpCmpm³Um) at the 5' end of all mRNAs. The cap 4 becomes part of the mRNA through trans-splicing of a 39-nucleotide-long sequence donated by the spliced leader RNA. Although the cap 4 modifications are required for trans-splicing to occur, the underlying mechanism remains to be determined. We now describe an unconventional nuclear cap binding complex (CBC) in Trypanosoma brucei with an apparent molecular mass of 300 kDa and consisting of five protein components: the known CBC subunits CBP20 and importin-α and three novel proteins that are only present in organisms featuring a cap 4 structure and trans-splicing. Competitive binding studies are consistent with a specific interaction between the CBC and the cap 4 structure. Downregulation of several individual components of the T. brucei CBC by RNA interference demonstrated an essential function at an early step in trans-splicing. Thus, our studies are consistent with the CBC providing a mechanistic link between cap 4 modifications and trans-splicing. [ABSTRACT FROM AUTHOR]

RNA interference or RNAi is defined as the mechanism through which gene-specific, double-stranded RNA (dsRNA) triggers degradation of homologous transcripts. Besides providing an invaluable tool to downregulate gene expression in a variety of organisms, it is now evident that RNAi extends its tentacles into both the nucleus and the cytoplasm and is involved in a variety of gene silencing phenomena. Here we review the current status of RNAi in protozoan parasites that cause diseases of considerable medical and veterinary importance throughout Africa, Asia and the Americas. RNAi was first discovered in Trypanosoma brucei, a species of the family Trypanosomatidae, and it rapidly became the method of choice to downregulate gene expression in these organisms. At the same time, mechanistic studies exposed a role for RNAi in the control of retroposon transcript abundance. Whereas RNAi is also present in T. congolense, other members of the same family of organisms, namely T. cruzi and Leishmania major, are RNAi-negative. In apicomplexan parasites, there is experimental evidence for RNAi in Plasmodium, but this is not supported by their genetic make up. In contrast, the genome of Toxoplasma gondii harbours gene candidates with convincing similarity to 'classical' RNAi genes. Thus, as previously shown in fungi, protozoan parasites are genetically heterogeneous as far as the RNAi pathway is concerned. Finally, data- base mining predicts that Entamoeba histolytica and Giardia intestinalis have an RNAi pathway and the presence of RNAi genes in Giardia supports the view that gene silencing by dsRNA appeared very early during evolution of the eukaryotic lineage. [ABSTRACT FROM AUTHOR]

Flagellate protozoa of the family Trypanosomatidae, which includes various members of the genera Leishmania and Trypanosoma, are model systems for unicellular pathogens to study fundamentally important biological phenomena. Recently, ablation of gene expression by RNA interference (RNAi) has become the method of choice to study gene function in Trypanosoma brucei, an early divergent eukaryote that infects humans and animals. As has been shown in multicellular organisms, the RNAi mechanism in T. brucei involves processing of double-stranded RNA 24- to 26-nt RNAs, termed small interfering RNAs (siRNAs), which guide degradation of the target mRNA. In this article, we describe some of the methods we employ for the analysis of the RNAi mechanism in T. brucei with particular emphasis on detection, cloning, and fractionation of siRNAs and siRNA complexes. [Copyright &y& Elsevier]

Deals with a study which presented the various experimental approaches to elicit RNAi in the parasitic protozoan Trypanosoma brucei. Description of the RNAi in Trypanosoma brucei; Applications of RNAi.

Presents information on a study that was conducted to determine the effects of the introduction of a gene-specific double-stranded RNA to a specific homologous mRNA. Materials and methods; Results; Discussion.

Graphical abstract Highlights • Metacyclic VSG expression site (ES) promoters are insensitive to changes in temperature. • Bloodstream VSG ES (BES) promoters are repressed at ambient temperature. • BES promoters are activated at mammalian body temperature. Abstract Trypanosoma brucei relies on two types of variant surface glycoprotein (VSG) expression sites (ESs) for RNA polymerase I (Pol I) transcription of VSG pre-mRNA. Trypanosomes developing into infectious metacyclic cells in the tsetse vector use metacyclic VSG ESs (MESs) and proliferating parasites in the mammalian host deploy bloodstream VSG ESs (BESs). Unlike the monocistronic MESs, BESs are polycistronic and their highly conserved promoters differ considerably from the MES promoters. The significance of the divergent sequences of MES and BES promoters remains to be determined. We used a reporter system to specifically test the effect of temperature on the activity of MES and BES promoters in procyclic trypanosomes and our results demonstrate that transcription from the MES promoter is largely insensitive to changes in temperature. In contrast, the BES promoter drives rapid activation of transcription upon a change of temperature from 28 °C to 37 °C. Additionally, endogenous BESs respond similarly to the elevation of temperature and initiate increased production of BES pre-mRNA and mRNA. Our results indicate that the sequence of the BES promoter is a specificity signal which triggers BES activation in vivo upon entry into the mammalian host. [ABSTRACT FROM AUTHOR]

Graphical abstract Overexpression of a single point mutation in the Trypanosoma brucei RBP6 (Q109 K) generates metacyclics that further differentiate in vitro to bloodstream forms, expressing VSG-2. Abstract We previously established an in vitro differentiation system based on the inducible expression of the RNA binding protein 6 (RBP6), which initiated differentiation of Trypanosoma brucei non-infectious procyclics to infectious metacyclics (MFs). However, further differentiation to bloodstream forms (BFs) required infection of mice. Here we report the serendipitous isolation of a single point mutation in RBP6 (Q109K), whose expression not only generated MFs, but purified MFs continued the developmental cycle in vitro to BFs expressing variant surface glycoprotein-2 (VSG-2), formerly known as VSG 221. This transition occurred over a period of 11 days and by RNA-Seq, VSG-2 was first measureable on day 1, whereas metacyclic VSGs were detected up to 8 days. We further showed that inducible expression of mutant RBP6 appeared to skip the intermediate epimastigote stage and we highlight the potential involvement of RBP33 in the establishment of metacyclics and in particular in the generation of metacyclics uncharacteristically arrested at the G2/M checkpoint. [ABSTRACT FROM AUTHOR]

Cold shock proteins are members of a family of DNA- and RNA-binding proteins with one or more evolutionarily conserved cold shock domain (CSD). These proteins have a wide variety of biological functions, including DNA-damage repair, mRNA stability, and regulation of transcription, splicing and translation. We previously identified two CSD containing proteins, CSD1 and CSD2, in the protozoan parasite Trypanosoma brucei to be required for RBP6-driven metacyclic production, albeit at different steps of the developmental program. During metacyclogenesis T. brucei undergoes major morphological and metabolic changes that culminate in the establishment of quiescent metacyclic parasites and the acquisition of mammalian infectivity. To investigate the specific role of CSD1 and CSD2 in this process, we ectopically expressed CSD1 or CSD2 in non-infectious procyclic parasites and discovered that each protein is sufficient to produce infectious metacyclic parasites in 24 hours. Domain truncation assays determined that the N-terminal domain, but not the C-terminal domain, of CSD1 and CSD2 was required for metacyclic development. Furthermore, conserved amino acid residues in the CSD of CSD1 and CSD2, known to be important for binding nucleic acids, were found to be necessary for metacyclic production. Using single-end enhanced crosslinking and immunoprecipitation (seCLIP) we identified the specific binding motif of CSD1 and CSD2 as "ANACAU" and the bound mRNAs were enriched for biological processes, including lipid metabolism, microtubule-based movement and nucleocytoplasmic transport that are likely involved in the transition to bloodstream form-like cells. Author summary: Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness in humans and together with T. congolense and T. vivax remains a public health concern in sub-Saharan Africa by afflicting livestock with nagana. During its complex life cycle between the mammalian host and the blood-feeding tsetse fly vector (Glossina sp.), T. brucei relies on several differentiation steps, where parasites transition between replicative and non-replicative forms specialized for infectivity and survival in mammalian and tsetse fly hosts. The transmission cycle begins with the tsetse fly taking a bloodmeal from an infected mammal. Following establishment of non-infective midgut procyclics, trypanosomes must find their way to the salivary glands where they develop into quiescent and infective metacyclics. We previously identified two cold shock domain containing proteins, CSD1 and CSD2, to be required for this developmental process. In this study, we determined that each protein is sufficient to trigger the development of infectious metacyclic parasites in 24 hours. The N-terminal domain of both proteins and conserved amino acids in the cold shock domain known to bind amino acids were required for this process. In addition, CSD1 and CSD2 bind mRNAs through the sequence ANACAU and preferentially target mRNAs that help to establish parasites capable of surviving in the mammalian host. [ABSTRACT FROM AUTHOR]

Infectious metacyclic Trypanosoma brucei cells develop in the salivary glands of tsetse flies. A critical aspect of the developmental program leading to acquisition of infectivity is the synthesis of a variant surface glycoprotein (VSG) coat. Metacyclic VSG genes are transcribed from a set of specialized VSG expression sites (ESs) that differ from bloodstream VSG ESs by being monocistronic, being significantly shorter, lacking long stretches of 70-bp repeats, and having distinct promoter sequences. Both metacyclic and bloodstream VSG ESs are transcribed by the multifunctional T. brucei RNA polymerase I (Pol I), however the factor that recognizes the divergent metacyclic VSG ES promoters and recruits Pol I during the development to infectious cells remains unknown. We used an in vitro assay to show that the promoters for both metacyclic and bloodstream VSG ESs are recognized by the same class I transcription factor A (CITFA). This general Pol I transcription initiation factor was previously shown to be essential for the transcription of bloodstream VSG genes, procyclin genes and rRNA genes, and was demonstrated to have distinct binding affinities for these three types of promoters. We now show that differences in the sequence of individual metacyclic VSG ESs promoters determine different affinities for CITFA. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (s) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T. brucei. We show using HydraPsiSeq and RiboMeth-seq that the on C/D snoRNA guiding 20-O-methylation increased the efficiency of the guided modification on its target, rRNA. We found differential levels of s on these noncoding RNAs in the two life stages (insect host and mammalian host) of the parasite. Furthermore, tRNA isoform abundance and modifications were characterized in these two life stages demonstrating stage-specific regulation. We conclude that the differential modifications identified here may contribute to modulating the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of the parasite between its two hosts. [ABSTRACT FROM AUTHOR]

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 T rypanosoma 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. [ABSTRACT FROM AUTHOR]

Abstract: Many U-snRNAs contain a hypermodified 2,2,7-trimethylguanosine (TMG) cap structure, which is formed by post-transcriptional methylation of an m7G cap. At present, little is known about the maturation of U-snRNAs in trypanosomes. The current evidence is consistent with the primary transcript containing an m7G moiety, but it is not clear whether the conversion of m7G to TMG takes place in the cytoplasm or in the nucleus. To address this issue, we characterized the Trypanosoma brucei homologue of the trimethylguanosine synthase (TbTgs1), a 28kDa protein, which is mainly composed of the conserved catalytic domain and lacks a large N-terminal domain present in higher eukaryotes. A GFP fusion with TbTgs1 revealed that this protein localizes throughout the nucleoplasm, as well as in one or two dots outside the nucleolus and RNAi-mediated downregulation of TbTgs1 suggests that this protein is responsible for hypermethylation of the m7G cap of both snRNAs and snoRNAs. [Copyright &y& Elsevier]

Argonaute proteins are central components of RNA interference (RNAi) and related phenomena in a wide variety of eukaryotes, including the early diverging protozoan Trypanosoma brucei. The single T. brucei Argonaute protein (TbAG01) is in a complex with small interfering RNAs (siRNAs), and a fraction of this ribonucleoprotein particle is associated with polyribosomes. In this study, we generated a panel of insertion, deletion, and single point mutants of TbAGO1 and assayed them in vivo for their function in RNAi. In addition to the signature domains of Argonaute proteins, PAZ and Piwi, TbAGO1 has an N-terminal domain with a high abundance of RGG repeats. Deletion of the N-terminal domain blocked association of AGO1 with polyribosomes and severely affected mRNA cleavage. Nevertheless, the mutant protein was in a complex with siRNAs. In contrast, deletion of the Piwi domain led to a loss of siRNAs but did not abolish polyribosome association. Site-directed mutagenesis of conserved amino acids in the Piwi domain identified arginine 735 as essential for RNAi. Although the R735A mutant bound siRNAs and associated with polyribosomes, it displayed a severe defect in the cleavage of target mRNA. [ABSTRACT FROM AUTHOR]

Members of the Argonaute protein family have been linked through a combination of genetic and biochemical studies to RNA interference (RNAi) and related phenomena. Here, we describe the characterization of the first Argonaute protein (AGO1) in Trypanosoma brucei, the earliest divergent eukaryote where RNAi has been described so far. AGO1 is predominantly cytoplasmic and is found in a ribonucleoprotein particle with small interfering RNAs (siRNAs), and this particle is present in a soluble form, as well as associated with polyribosomes. A genetic knockout of AGO1 leads to a loss of RNAi, and concomitantly, endogenous retroposonderived siRNAs as well as siRNAs derived from transgenic double-stranded RNA are reduced to almost undetectable levels. Furthermore, AGO1 deficiency leads to an increase in retroposon transcript abundance via mechanisms operating at the transcriptional level and at the RNA stability level. Our results suggest that AGO1 function is required for production and/or stabilization of siRNAs and provide the first evidence for an Argonaute protein being involved in the regulation of retroposon transcript levels. [ABSTRACT FROM AUTHOR]

The parasite Trypanosoma brucei cycles between an insect and a mammalian host and is the causative agent of sleeping sickness. Here, we performed high‐throughput mapping of pseudouridines (Ψs) on mRNA from two life stages of the parasite. The analysis revealed ~273 Ψs, including developmentally regulated Ψs that are guided by homologs of pseudouridine synthases (PUS1, 3, 5, and 7). Mutating the U that undergoes pseudouridylation in the 3′ UTR of valyl‐tRNA synthetase destabilized the mRNA level. To investigate the mechanism by which Ψ affects the stability of this mRNA, proteins that bind to the 3′ UTR were identified, including the RNA binding protein RBSR1. The binding of RBSR1 protein to the 3′ UTR was stronger when lacking Ψ compared to transcripts carrying the modification, suggesting that Ψ can inhibit the binding of proteins to their target and thus affect the stability of mRNAs. Consequently, Ψ modification on mRNA adds an additional level of regulation to the dominant post‐transcriptional control in these parasites. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei is a protozoan parasite that causes important human and livestock diseases in sub-Saharan Africa. By overexpressing a single RNA-binding protein, RBP6, in non-infectious procyclics trypanosomes, we previously recapitulated in vitro the events occurring in the tsetse fly vector, namely the development of epimastigotes and infectious, quiescent metacyclic parasites. To identify genes involved in this developmental progression, we individually targeted 86 transcripts by RNAi in the RBP6 overexpression cell line and assessed the loss-of-function phenotypes on repositioning the kinetoplast, an organelle that contains the mitochondrial genome, the expression of BARP or brucei alanine rich protein, a marker for epimastigotes, and metacyclic variant surface glycoprotein. This screen identified 22 genes that positively or negatively regulate the stepwise progression towards infectivity at different stages. Two previously uncharacterized putative nucleic acid binding proteins emerged as potent regulators, namely the cold shock domain-containing proteins CSD1 and CSD2. RNA-Seq data from a selected group of cell lines further revealed that the components of gene expression regulatory networks identified in this study affected the abundance of a subset of transcripts in very similar fashion. Finally, our data suggest a considerable overlap between the genes that regulate the formation of stumpy bloodstream form trypanosomes and the genes that govern the development of metacyclic form parasites. [ABSTRACT FROM AUTHOR]

The vault ribonucleoprotein (RNP), comprising vault RNA (vtRNA) and telomerase-associated protein 1 (TEP1), is found in many eukaryotes. However, previous studies of vtRNAs, for example in mammalian cells, have failed to reach a definitive conclusion about their function. vtRNAs are related to Y RNAs, which are complexed with Ro protein and influence Ro's function in noncoding RNA (ncRNA) quality control and processing. In Trypanosoma brucei, the small noncoding TBsRNA-10 was first described in a survey of the ncRNA repertoire in this organism. Here, we report that TBsRNA-10 in T. brucei is a vtRNA, based on its association with TEP1 and sequence similarity to those of other known and predicted vtRNAs. We observed that like vtRNAs in other species, TBsRNA-10 is transcribed by RNA polymerase III, which in trypanosomes also generates the spliceosomal U-rich small nuclear RNAs. In T. brucei, spliced leader (SL)-mediated trans-splicing of pre-mRNAs is an obligatory step in gene expression, and we found here that T. brucei's vtRNA is highly enriched in a non-nucleolar locus in the cell nucleus implicated in SL RNP biogenesis. Using a newly developed permeabilized cell system for the bloodstream form of T. brucei, we show that down-regulated vtRNA levels impair trans-spliced mRNA production, consistent with a role of vtRNA in trypanosome mRNA metabolism. Our results suggest a common theme for the functions of vtRNAs and Y RNAs. We conclude that by complexing with their protein-binding partners TEP1 and Ro, respectively, these two RNA species modulate the metabolism of various RNA classes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals, have a complex digenetic life cycle between a mammalian host and an insect vector, the blood-feeding tsetse fly. Although the importance of the insect vector to transmit the disease was first realized over a century ago, many aspects of trypanosome development in tsetse have not progressed beyond a morphological analysis, mainly due to considerable challenges to obtain sufficient material for molecular studies. Here, we used high-throughput RNA-Sequencing (RNA-Seq) to profile Trypanosoma brucei transcript levels in three distinct tissues of the tsetse fly, namely the midgut, proventriculus and salivary glands. Consistent with current knowledge and providing a proof of principle, transcripts coding for procyclin isoforms and several components of the cytochrome oxidase complex were highly up-regulated in the midgut transcriptome, whereas transcripts encoding metacyclic VSGs (mVSGs) and the surface coat protein brucei alanine rich protein or BARP were extremely up-regulated in the salivary gland transcriptome. Gene ontology analysis also supported the up-regulation of biological processes such as DNA metabolism and DNA replication in the proventriculus transcriptome and major changes in signal transduction and cyclic nucleotide metabolism in the salivary gland transcriptome. Our data highlight a small repertoire of expressed mVSGs and potential signaling pathways involving receptor-type adenylate cyclases and members of a surface carboxylate transporter family, called PADs (Proteins Associated with Differentiation), to cope with the changing environment, as well as RNA-binding proteins as a possible global regulators of gene expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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, like 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-Seq 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 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. [ABSTRACT FROM AUTHOR]

Among trypanosomatid protozoa the mechanism of RNA interference ( RNAi) has been investigated in T rypanosoma brucei and to a lesser extent in L eishmania 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 ( Lbr AGO1). In contrast to T . brucei, L eishmania siRNAs are sensitive to 3′ end oxidation, indicating the absence of blocking groups, and the L eishmania 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 L eishmania 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. [ABSTRACT FROM AUTHOR]

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 39-59 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. [ABSTRACT FROM AUTHOR]

Argonaute proteins (AGOs) are central to RNA interference (RNAi) and related silencing pathways. At the core of the RNAi pathway in the ancient parasitic eukaryote Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in the destruction of potentially harmful retroposon transcripts. One notable feature of TbAGO1 is that a fraction sediments with polyribosomes, and this association is facilitated by an arginine/glycine-rich domain (RGG domain) at the N terminus of the protein. Here we report that reducing the size of the RGG domain and, in particular, mutating all arginine residues severely reduced the association of TbAGO1 with polyribosomes and RNAi-induced cleavage of mRNA. However, these mutations did not change the cellular localization of Argonaute and did not affect the accumulation of single-stranded siRNAs, an essential step in the activation of the RNA-induced silencing complex. We further show that mRNA on polyribosomes can be targeted for degradation, although this alliance is not a pre-requisite. Finally, sequestering tubulin mRNAs from translation with antisense morpholino oligonucleotides reduced the RNAi response indicating that mRNAs not engaged in translation may be less accessible to the RNAi machinery. We conclude that the association of the RNAI machinery and target mRNA on polyribosomes promotes an efficient RNAi response. This mechanism may represent an ancient adaptation to ensure that retroposon transcripts are efficiently destroyed, if they become associated with the translational apparatus. [ABSTRACT FROM AUTHOR]

Trypanosoma brucei is one of the most ancient eukaryotes where RNA interference (RNAi) is operational and is the only single-cell pathogen where RNAi has been extensively studied and used as a tool for functional analyses. Here, we report that the T. brucei RNAi pathway, although relying on a single Argonaute protein (AGO1), is initiated by the activities of two distinct Dicer-like enzymes. Both TbDCL1, a mostly cytoplasmic protein, and the previously undescribed nuclear enzyme TbDCL2 contribute to the biogenesis of 5IRNAs from retroposons. However, TbDCL2 has a predominant role in generating siRNAs from chromosomal internal repeat transcripts that accumulate at the nucleolus in RNAi-deficient cells and in initiating the endogenous RNAi response against retroposons and repeats alike. Moreover, siRNAs generated by both TbDCL1 and TbDCL2 carry a 5'-monophosphate and a blocked 3' terminus, suggesting that 3' end modification is an ancient trait of siRNAs. We thus propose a model whereby TbDCL2 fuels the T. bruce! nuclear RNAi pathway and TbDCL1 patrols the cytoplasm, posttranscriptionally silencing potentially harmful nucleic acid parasites that may access the cytoplasm. Nevertheless, we also provide evidence for cross-talk between the two Dicer-like enzymes, because TbDCL2 is implicated in the generation of 35 to 65-nucleotide intermediate transcripts that appear to be substrates for TbDCL1. Our finding that dcl2KO cells are more sensitive to RNAi triggers than wild-type cells has significant implications for reverse genetic analyses in this important human pathogen. [ABSTRACT FROM AUTHOR]

The Trypanosoma brucei genome is colonized by the site-specific non-LTR retrotransposon SLACS, or spliced leader-associated conserved sequence, which integrates exclusively into the spliced leader (SL) RNA genes. Although there is evidence that the RNA interference (RNAi) machinery regulates SLACS transcript levels, we do not know whether RNAi deficiency affects the genomic stability of SLACS, nor do we understand the mechanism of SLACS transcription. Here, we report that prolonged culturing of RNAi-deficient T. brucei cells, but not wild-type cells, results in genomic rearrangements of SLACS. Furthermore, two populations of SLACS transcripts persist in RNAi-deficient cells: a full-length transcript of approximately 7 kb and a heterogeneous population of small SLACS transcripts ranging in size from 450 to 550 nt. We provide evidence that SLACS transcription initiates at the +1 of the interrupted SL RNA gene and proceeds into the 5′ UTR and open reading frame 1 (ORF1). This transcription is carried out by an RNA polymerase with α-amanitin sensitivity reminiscent of SL RNA synthesis and is dependent on the SL RNA promoter. Additionally, we show that both sense and antisense small SLACS transcripts originate from ORF1 and that they are associated with proteins in vivo. We speculate that the small SLACS transcripts serve as substrates for the production of siRNAs to regulate SLACS expression. [ABSTRACT FROM AUTHOR]

Abstract: Capping of the pre-mRNA 5′ end by addition a monomethylated guanosine cap (m7G) is an essential and the earliest modification in the biogenesis of mRNA. The reaction is catalyzed by three enzymes: triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. Whereas this modification occurs co-transcriptionally in most eukaryotic organisms, trypanosomatid protozoa mRNAs acquire the m7G cap by trans-splicing, which entails the transfer of the capped spliced leader (SL) from the SL RNA to the mRNA. Intriguingly, the genomes of all trypanosomatid protozoa sequenced to date possess two distinct proteins with the signature motifs of guanylyltransferases: TbCGM1 and the previously characterized TbCE1. Here we provide biochemical evidence that TbCgm1 is a capping enzyme. Whereas RNAi-induced downregulation of TbCe1 had no phenotypic consequences, we found that TbCGM1 is essential for trypanosome viability and is required for SL RNA capping. Furthermore, consistent with co-transcriptional addition of the m7G cap, chromatin immunoprecipitation revealed recruitment of TbCgm1 to the SL RNA genes. [Copyright &y& Elsevier]

Abstract: In trypanosomatids all mRNAs undergo trans-splicing, whereas cis-splicing is restricted to a few transcripts. Trans-splicing is mechanistically similar to cis-splicing, however, little is known about the trans-splicing machinery and its underlying mechanism. In this study, we examined the involvement of splicing factors in cis- and trans-splicing by RNA interference (RNAi). Two factors (Prp31 and Prp43) were found to be essential for both pathways, suggesting that splicing factors are shared by these two reactions. We identified a 45S complex carrying pre-mRNA and all the U-snRNAs, including U1 and the SL RNA, suggesting that a single spliceosomal complex may potentially conduct both trans- and cis-splicing. [Copyright &y& Elsevier]

Transcriptional mechanisms remain poorly understood in trypanosomatid protozoa. In particular, there is no knowledge about the function of basal transcription factors, and there is an apparent rarity of promoters for protein-coding genes transcribed by RNA polymerase (Pol) II. Here we describe a Trypanosoma brucei factor related to the TATA-binding protein (TBP). Although this TBP-related factor (TBP-related factor 4 [TRF4]) has about 31% identity to the TBP core domain, several key residues involved in TATA box binding are not conserved. Depletion of the T. brucei TRF4 (TbTRF4) by RNA interference revealed an essential role in RNA Pol I, II, and HI transcription. Using chromatin immunoprecipitation, we further showed that TRF4 is recruited to the Pol I-transcribed procyclic acidic repetitive genes, Pol II-transcribed spliced leader RNA genes, and Pol III-transcribed U-snRNA and 7SL RNA genes, thus supporting a role for TbTRF4 in transcription performed by all three nuclear RNA polymerases. Finally, a search for TRF4 binding sites in the T. brucei genome led to the identification of such sites in the 3′ portion of certain protein-coding genes, indicating a unique aspect of Pol II transcription in these organisms. [ABSTRACT FROM AUTHOR]

Ixodes scapularis ticks transmit many pathogens, including Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. Vaccines directed against arthropod proteins injected into the host during tick engorgement could prevent numerous infectious diseases. Salp14, a salivary anticoagulant poses a key target for such intervention. Salp14 is the prototypic member of a family of potential l. scapularis anticoagulants, expressed and secreted in tick saliva during tick feeding. RNA interference was used to assess the role of Salp14 in tick feeding. Salp14 and its paralogs were silenced, as demonstrated by the reduction of mRNA and protein specific for these antigens. Tick salivary glands lacking Salp14 had reduced anticoagulant activity, as revealed by a 60-80% reduction of anti-factor Xa activity. Silencing the expression of salp14 and its paralogs also reduced the ability of l. scapularis to feed, as demonstrated by a 50-70% decline in the engorgement weights. Because ticks have several anticoagulants, it is likely that the expression of multiple anticoagulants in I. scapularis saliva would have to be ablated simultaneously to abolish tick feeding. These studies demonstrate that RNA interference can silence I. scapularis genes and disrupt their physiologic function in vivo, and they identify vaccine candidates that can alter vector engorgement. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]