• Alba domain
• RNA stability
• heat stress
• male fertility
• pollen

• Arabidopsis/genetics
• Arabidopsis/physiology
• Arabidopsis/metabolism
• Heat-Shock Response/genetics
• Heat-Shock Response/physiology
• Arabidopsis Proteins/metabolism
• Arabidopsis Proteins/genetics
• Pollen/metabolism
• Pollen/genetics
• Pollen/physiology
• Gene Expression Regulation, Plant
• Fertility
• RNA, Messenger/metabolism
• RNA, Messenger/genetics

• 32170285 National Natural Science Foundation of China
• 32270288 National Natural Science Foundation of China

• Apicomplexans
• Gene regulation
• Kinetoplastids
• Leishmania
• Plasmodium
• Protein regulation
• Toxoplasma
• Trypanosomes

• Alba-domain proteins
• RNA motifs
• RNA-binding proteins
• Trypanosomatidae
• cis-acting elements

• Trypanosoma cruzi/metabolism
• Trypanosoma cruzi/genetics
• RNA-Binding Proteins/metabolism
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/chemistry
• Protozoan Proteins/metabolism
• Protozoan Proteins/genetics
• Protozoan Proteins/chemistry
• Phylogeny
• Protein Binding
• Amino Acid Sequence
• Conserved Sequence

• parasite biology
• rna decay
• parasite development

• DEAD-box RNA Helicases/metabolism
• DEAD-box RNA Helicases/genetics
• Plasmodium falciparum/genetics
• Plasmodium falciparum/metabolism
• Plasmodium falciparum/growth & development
• RNA, Messenger/metabolism
• RNA, Messenger/genetics
• Protozoan Proteins/metabolism
• Protozoan Proteins/genetics
• Ribonucleoproteins/metabolism
• Ribonucleoproteins/genetics
• Life Cycle Stages/genetics
• RNA, Protozoan/metabolism
• RNA, Protozoan/genetics
• RNA Stability
• Humans
• Malaria, Falciparum/parasitology

• R01AI104946 U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)
• 81902089 National Natural Science Foundation of China (National Science Foundation of China)
• 81902089 National Natural Science Foundation of China (National Science Foundation of China)
• U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)

• Biochemistry
• Drugs
• Microbiology parasite
• Structural biology

• parasite biology
• parasite development
• stress signalling
• organelles
• differentiation

• Animals
• Trypanosoma
• Trypanosoma brucei brucei/genetics
• Trypanosoma brucei brucei/metabolism
• Mammals

• Wellcome Trust
• 108504 Wellcome Trust
• RCUK | Medical Research Council (MRC)
• Wellcome Trust (Wellcome)
• Wolfson Foundation

• Animals
• Trypanosoma brucei brucei/metabolism
• Immune Evasion/genetics
• RNA/genetics
• Antigens, Surface
• Antigenic Variation/genetics
• DNA/genetics
• Mammals/genetics
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism

• Wellcome Trust
• Philippine Council for Health Research and Development
• Welcome Trust Sir Henry Wellcome fellowship
• University of Glasgow
• BBSRC
• Wellcome Trust Strategic Award
• UKRI

• Trypanosoma brucei
• bromodomain proteins
• epigenetics
• gene regulation
• molecular parasitology
• parasite

• Humans
• Animals
• Trypanosomiasis, African/parasitology
• Transcriptome
• Membrane Glycoproteins
• Nuclear Proteins/genetics
• Transcription Factors/genetics
• Trypanosoma/genetics
• Trypanosoma brucei brucei/genetics
• Tsetse Flies/genetics
• Tsetse Flies/parasitology
• Membrane Proteins/genetics
• Mammals
• Chromatin
• Variant Surface Glycoproteins, Trypanosoma/genetics
• Variant Surface Glycoproteins, Trypanosoma/pharmacology
• Protozoan Proteins/genetics

• National Science Foundation (NSF)

• RNA regulon
• RNA-binding protein
• RNA-protein interaction
• gene regulation
• trypanosome

• gene expression regulation
• infective forms
• parasite differentiation
• surface proteins
• trypanosome

• evolution
• gene expression
• mRNA export
• proteomics
• trypanosomes

• Active Transport, Cell Nucleus
• Proteomics
• RNA
• RNA Splicing
• RNA Transport
• Trypanosoma cruzi

• Wellcome Trust
• MR/N010558/1 Medical Research Council
• 204697/Z/16/Z Wellcome Trust

The long and complex Trypanosoma brucei development in the tsetse fly vector culminates when parasites gain mammalian infectivity in the salivary glands. A key step in this process is the establishment of monoallelic variant surface glycoprotein (VSG) expression and the formation of the VSG coat. The establishment of VSG monoallelic expression is complex and poorly understood, due to the multiple parasite stages present in the salivary glands. Therefore, we sought to further our understanding of this phenomenon by performing single-cell RNA-sequencing (scRNA-seq) on these trypanosome populations. We were able to capture the developmental program of trypanosomes in the salivary glands, identifying populations of epimastigote, gamete, pre-metacyclic and metacyclic cells. Our results show that parasite metabolism is dramatically remodeled during development in the salivary glands, with a shift in transcript abundance from tricarboxylic acid metabolism to glycolytic metabolism. Analysis of VSG gene expression in pre-metacyclic and metacyclic cells revealed a dynamic VSG gene activation program. Strikingly, we found that pre-metacyclic cells contain transcripts from multiple VSG genes, which resolves to singular VSG gene expression in mature metacyclic cells. Single molecule RNA fluorescence in situ hybridisation (smRNA-FISH) of VSG gene expression following in vitro metacyclogenesis confirmed this finding. Our data demonstrate that multiple VSG genes are transcribed before a single gene is chosen. We propose a transcriptional race model governs the initiation of monoallelic expression.

African trypanosomes are parasitic protists which cause endemic disease in sub-Saharan Africa. To evade mammalian immune responses the parasite has developed a system of antigenic variation, where the surface of the cell is covered in a tightly packed coat of variant surface glycoproteins (VSGs). Each cell expresses only one variant surface glycoprotein at a time, and this is periodically switched to evade new antibodies. The process of singular gene expression is termed monoallelic expression and this has two components, establishment and maintenance, i.e. how a single gene is selected for expression and how its singular expression is maintained throughout successive generations. The establishment of monoallelic VSG gene expression occurs in the salivary gland of the tsetse fly vector, although this process is not well understood. We used single cell gene expression profiling applied to thousands of single cells in the salivary gland of the fly. We show that in order to select a single gene, trypanosomes initially transcribe multiple VSGs before a single gene is selected for high-level expression. We propose a model where this process is driven by a race to accumulate transcription factors at a single VSG gene.

• Animals
• Gene Expression Regulation/genetics
• Insect Vectors/parasitology
• RNA-Seq
• Salivary Glands/parasitology
• Trypanosoma brucei brucei/genetics
• Tsetse Flies/parasitology
• Variant Surface Glycoproteins, Trypanosoma/genetics

• Horizon 2020
• Institut Pasteur
• Agence Nationale de la Recherche
• Site de Recherche Intégrée sur le Cancer

• Alba domain
• Crystal structure
• T. brucei
• TbAlba1

• CFB2
• F-box
• RNA-binding proteins
• Trypanosoma brucei
• VSG
• antigenic variation
• infectious disease
• microbiology

• Proteome/chemistry
• Protozoan Proteins/chemistry
• RNA Stability
• RNA, Messenger/chemistry
• Trypanosoma brucei brucei/chemistry
• Variant Surface Glycoproteins, Trypanosoma/chemistry

• 649019 H2020 European Research Council
• ND Core Funding University of Heidelberg
• ND University of Heidelberg

• ALBA
• Arabidopsis thaliana
• PABP3
• confocal microscopy
• expression analysis
• flowering
• heat stress
• pollen development
• protein localization

• Arabidopsis/embryology
• Arabidopsis/metabolism
• Arabidopsis Proteins/genetics
• Arabidopsis Proteins/metabolism
• Flowers/growth & development
• Gene Expression Regulation, Plant/genetics
• Heat-Shock Response/physiology
• Microscopy, Confocal
• Pollen/embryology
• Poly(A)-Binding Proteins/metabolism
• Promoter Regions, Genetic/genetics
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism
• Stress, Physiological/genetics

• 18-07027S Grantová Agentura České Republiky
• LM2018129 Czech-BioImaging Ministry of Education, Youth and Science

The transition between hosts is a challenge for digenetic parasites as it is unpredictable. For Trypanosoma brucei subspecies, which are disseminated by tsetse flies, adaptation to the new host requires differentiation of stumpy forms picked up from mammals to procyclic forms in the fly midgut. Here we show that the Alba-domain protein Alba3 is not essential for mammalian slender forms, nor is it required for differentiation of slender to stumpy forms in culture or in mice. It is crucial, however, for the development of T. brucei procyclic forms during the host transition. While steady state levels of mRNAs in differentiating cells are barely affected by the loss of Alba3, there are major repercussions for the proteome. Mechanistically, Alba3 aids differentiation by rapidly releasing stumpy forms from translational repression and stimulating polysome formation. In its absence, parasites fail to remodel their proteome appropriately, lack components of the mitochondrial respiratory chain and show reduced infection of tsetse. Interestingly, Alba3 and the closely related Alba4 are functionally redundant in slender forms, but Alba4 cannot compensate for the lack of Alba3 during differentiation from the stumpy to the procyclic form. We postulate that Alba-domain proteins play similar roles in regulating translation in other protozoan parasites, in particular during life-cycle and host transitions.

Trypanosoma brucei is a unicellular eukaryotic parasite that is responsible for African trypanosomiasis. The parasite needs two hosts, mammals and tsetse flies, in order to complete its life cycle. Throughout its developmental cycle, T. brucei encounters diverse environments to which it has to adapt in order to maintain its transmission and infectivity. Successful adaptation to the new environment and transition to different life-cycle stages are the general challenges faced by many digenetic parasites. In this study we show that the Alba-domain protein Alba3 is essential for differentiation of the mammalian stumpy form (transition form) to the procyclic form in the tsetse host. An Alba3 deletion mutant infects mice and shows characteristic waves of parasitaemia, but is severely compromised in its ability to infect tsetse flies. Stumpy forms are translationally repressed, but are poised to resume protein synthesis during differentiation. We show that Alba3 is key to efficient escape from translation repression; in its absence, there is a delay in the formation of polysomes and resumption of protein synthesis. This impacts the formation of procyclic-specific mitochondrial respiratory complex proteins as well as the repression of some bloodstream-specific proteins. This is the first time that a single protein has been shown to have a major influence on translation as an adaptive response to changing hosts. It is also the first time that a mechanism has been established for Alba-domain proteins in parasites.

• Adaptation, Physiological
• Animals
• Cell Cycle
• Cell Differentiation
• Female
• Gene Knockout Techniques
• Life Cycle Stages
• Mammals
• Mice
• Polyribosomes/metabolism
• Protein Domains
• Proteome/metabolism
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism
• Trypanosoma brucei brucei/genetics
• Trypanosoma brucei brucei/physiology
• Tsetse Flies/parasitology

• Swiss National Science Foundation

• Low-complexity regions (LCRs)
• granules
• liquid-liquid phase separation
• nucleic acid binding proteins
• phosphorylation
• proteome

• Wellcome Trust

• Low-complexity regions (LCRs)
• granules
• liquid-liquid phase separation
• nucleic acid binding proteins
• phosphorylation
• proteome

• P-bodies
• RIP-seq
• cajal bodies
• cytoplasmic bodies
• mRNP
• stress granules

• Cytoplasm/metabolism
• Gene Expression Regulation, Plant
• Larix/metabolism
• Plant Proteins/metabolism
• RNA, Messenger/metabolism
• RNA, Plant/metabolism
• RNA, Small Cytoplasmic/metabolism
• RNA-Binding Proteins/metabolism
• Ribonucleoproteins/metabolism
• Spliceosomes/metabolism

• Leishmania
• adaptation
• mRNA
• parasitology
• posttranscriptional regulation
• purine
• starvation
• stress response
• transporter

• 3' Untranslated Regions
• Base Sequence
• Culture Media/chemistry
• Genomic Instability
• Leishmania donovani/genetics
• Leishmania donovani/metabolism
• Mutagenesis
• Nucleic Acid Conformation
• Nucleobase Transport Proteins/genetics
• Nucleobase Transport Proteins/metabolism
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism
• Purines/metabolism
• RNA, Messenger/chemistry
• RNA, Messenger/metabolism
• Trypanosoma brucei brucei/metabolism

• R03 AI137636 NIAID NIH HHS
• T32 AI007472 NIAID NIH HHS
• HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)

Leishmaniasis represents a serious health problem worldwide and drug resistance is a growing concern. Leishmania parasites use unusual mechanisms to control their gene expression. In contrast to many other species, they do not have transcriptional regulation. The lack of transcriptional control is mainly compensated by post-transcriptional mechanisms, including tight translational control and regulation of mRNA stability/translatability by RNA-binding proteins. Modulation of translation plays a major role in parasite survival and adaptation to dramatically different environments during change of host; however, our knowledge of fine molecular mechanisms of translation in Leishmania remains limited. Here, we review the current progress in our understanding of how changes in the translational machinery promote parasite differentiation during transmission from a sand fly to a mammalian host, and discuss how translational reprogramming can contribute to the development of drug resistance.

• Leishmania parasites
• RNA-binding proteins
• drug resistance
• parasite differentiation
• protein synthesis
• ribosome
• selective translation
• translational control
• translational reprogramming
• unicellular protozoa

The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. In the case of Trypanosoma cruzi, the characterization of messenger ribonucleoprotein (mRNP) particles has allowed the identification of several classes of RNA binding proteins (RBPs), as well as non-canonical RBPs, associated with mRNA molecules. The protein composition of the mRNPs as well as the localization and functionality of the mRNAs depend on their associated proteins. mRNPs can also be organized into larger complexes forming RNA granules, which function as stress granules or P-bodies depending on the associated proteins. The fate of mRNAs in the cell, and consequently the genes expressed, depends on the set of proteins associated with the messenger molecule. These proteins allow the coordinated expression of mRNAs encoding proteins that are related in function, resulting in the formation of post-transcriptional operons. However, the puzzle posed by the combinatorial association of sets of RBPs with mRNAs and how this relates to the expressed genes remain to be elucidated. One important tool in this endeavor is the use of the CRISPR/CAS system to delete genes encoding RBPs, allowing the evaluation of their effect on the formation of mRNP complexes and associated mRNAs in the different compartments of the translation machinery. Accordingly, we recently established this methodology for T. cruzi and deleted the genes encoding RBPs containing zinc finger domains. In this manuscript, we will discuss the data obtained and the potential of the CRISPR/CAS methodology to unveil the role of RBPs in T. cruzi gene expression regulation.

• CRISPR/CAS
• RNA binding proteins
• RNA granules
• Trypanosoma cruzi
• gene expression regulation
• zinc finger protein

• Trypanosoma brucei
• Trypanosoma congolense
• development and differentiation
• gene expression
• signaling
• tsetse fly

• ALBA30
• Nutritional stress
• RNA-binding proteins
• Trypanosoma cruzi

• Animals
• Cell Cycle
• Chagas Disease/parasitology
• Cytoplasm/metabolism
• Gene Expression Regulation/genetics
• Life Cycle Stages/physiology
• Protozoan Proteins/genetics
• RNA, Messenger/metabolism
• RNA-Binding Proteins/genetics
• Starvation
• Stress, Physiological/physiology
• Trypanosoma cruzi/genetics

• Animals
• Drosophila melanogaster/genetics
• Drosophila melanogaster/parasitology
• Euglenozoa Infections/genetics
• Euglenozoa Infections/parasitology
• Euglenozoa Infections/transmission
• Host-Parasite Interactions/genetics
• Humans
• Insect Vectors/genetics
• Leishmania/genetics
• Leishmania/pathogenicity
• Leishmaniasis/genetics
• Leishmaniasis/parasitology
• Leishmaniasis/transmission
• Psychodidae/genetics
• Psychodidae/parasitology
• Trypanosomatina/genetics
• Trypanosomatina/pathogenicity

• Wellcome Trust
• BB/K003569 Biotechnology and Biological Sciences Research Council
• 206194 Wellcome Trust

• VIGS and wild cotton
• cis-regulatory elements
• cotton Alba genes
• miRNAs
• oxidant and antioxidant enzymes
• virus-induced gene silencing (VIGS)
• water deficit and salt stresses

• Ciliates
• Epigenetics
• Genome Rearrangement

• Chromosomes, Artificial
• Fluorescent Antibody Technique
• Gene Expression Regulation
• Gene Rearrangement
• Genes, Protozoan
• Genome, Protozoan
• Oxytricha/genetics
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism

• R01 GM059708 NIGMS NIH HHS
• R01 GM111933 NIGMS NIH HHS
• R35 GM122555 NIGMS NIH HHS

RNA-binding proteins (RBPs) are well known as key factors in gene expression regulation in eukaryotes. These proteins associate with mRNAs and other proteins to form mRNP complexes that ultimately determine the fate of target transcripts in the cell. This association is usually mediated by an RNA-recognition motif (RRM). In the case of trypanosomatids, these proteins play a paramount role, as gene expression regulation is mostly posttranscriptional. Despite their relevance in the life cycle of Trypanosoma cruzi, the causative agent of Chagas’ disease, to date, few RBPs have been characterized in this parasite.

We investigated the role of DRBD2 in T. cruzi, an RBP with two RRM domains that is associated with cytoplasmic translational complexes. We show that DRBD2 is an ortholog of the Gbp2 in yeast, an SR-rich protein involved in mRNA quality control and export. We used an immunoprecipitation assay followed by shotgun proteomics and RNA-seq to assess the interaction partners of the DRBD2-mRNP complex in epimastigotes. The analysis identified mostly proteins involved in RNA metabolism and regulation, such as ALBA1, ALBA3, ALBA4, UBP1, UBP2, DRBD3, and PABP2. The RNA-seq results showed that most of the transcripts regulated by the DRBD2 complex mapped to hypothetical proteins related to multiple processes, such as to biosynthetic process, DNA metabolic process, protein modification, and response to stress.

The identification of regulatory proteins in the DRBD2-mRNP complex corroborates the important role of DRBD2 in gene expression regulation in T. cruzi. We consider these results an important contribution to future studies regarding gene expression regulation in T. cruzi, especially in the field of RNA-binding proteins.

The online version of this article (10.1186/s12866-019-1505-8) contains supplementary material, which is available to authorized users.

• Gene expression regulation
• RNA metabolism
• RNA-binding protein
• Trypanosoma cruzi

• RNA-binding protein
• Trypanosoma cruzi
• gene expression regulation
• translation regulation

• Amino Acid Sequence
• Gene Expression Regulation
• High-Throughput Nucleotide Sequencing/methods
• Proteomics/methods
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism
• Ribonucleoproteins/genetics
• Ribonucleoproteins/metabolism
• Sequence Homology
• Trypanosoma cruzi/genetics
• Trypanosoma cruzi/growth & development
• Trypanosoma cruzi/metabolism

The tsetse transmitted parasitic flagellate Trypanosoma congolense causes animal African trypanosomosis (AAT) across sub-Saharan Africa. AAT negatively impacts agricultural, economic, nutritional and subsequently, health status of the affected populace. The molecular mechanisms that underlie T. congolense’s developmental program within tsetse are largely unknown due to considerable challenges with obtaining sufficient parasite cells to perform molecular studies.

In this study, we used RNA-seq to profile T. congolense gene expression during development in two distinct tsetse tissues, the cardia and proboscis. Indirect immunofluorescent antibody test (IFA) and confocal laser scanning microscope was used to localize the expression of a putative protein encoded by the hypothetical protein (TcIL3000_0_02370).

Consistent with current knowledge, genes coding several variant surface glycoproteins (including metacyclic specific VSGs), and the surface coat protein, congolense epimastigote specific protein, were upregulated in parasites in the proboscis (PB-parasites). Additionally, our results indicate that parasites in tsetse’s cardia (C-parasites) and PB employ oxidative phosphorylation and amino acid metabolism for energy. Several genes upregulated in C-parasites encoded receptor-type adenylate cyclases, surface carboxylate transporter family proteins (or PADs), transport proteins, RNA-binding proteins and procyclin isoforms. Gene ontology analysis of products of genes upregulated in C-parasites showed enrichment of terms broadly associated with nucleotides, microtubules, cell membrane and its components, cell signaling, quorum sensing and several transport activities, suggesting that the parasites colonizing the cardia may monitor their environment and regulate their density and movement in this tissue. Additionally, cell surface protein (CSP) encoding genes associated with the Fam50 ‘GARP’, ‘iii’ and ‘i’ subfamilies were also significantly upregulated in C-parasites, suggesting that they are important for the long non-dividing trypomastigotes to colonize tsetse’s cardia. The putative products of genes that were upregulated in PB-parasites were linked to nucleosomes, cytoplasm and membrane-bound organelles, which suggest that parasites in this niche undergo cell division in line with prior findings. Most of the CSPs upregulated in PB-parasites were hypothetical, thus requiring further functional characterization. Expression of one such hypothetical protein (TcIL3000_0_02370) was analyzed using immunofluorescence and confocal laser scanning microscopy, which together revealed preferential expression of this protein on the entire surface coat of T. congolense parasite stages that colonize G. m. morsitans’ proboscis.

Collectively, our results provide insight into T. congolense gene expression profiles in distinct niches within the tsetse vector. Our results show that the hypothetical protein TcIL3000_0_02370, is expressed on the entire surface of the trypanosomes inhabiting tsetse’s proboscis. We discuss our results in terms of their relevance to disease transmission processes.

The online version of this article (10.1186/s13071-018-2964-8) contains supplementary material, which is available to authorized users.

• Gene expression analysis
• Glossina morsitans morsitans
• Trypanosoma congolense
• Tsetse cardia
• Tsetse proboscis and confocal microscopy

• Eukaryotic Initiation Factor-4E/genetics
• Eukaryotic Initiation Factor-4E/metabolism
• Poly(A)-Binding Protein I/chemistry
• Poly(A)-Binding Protein I/genetics
• Poly(A)-Binding Protein I/metabolism
• Poly(A)-Binding Protein II/chemistry
• Poly(A)-Binding Protein II/genetics
• Poly(A)-Binding Protein II/metabolism
• Protein Binding
• Proteomics
• Protozoan Proteins/chemistry
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• Trypanosoma brucei brucei/chemistry
• Trypanosoma brucei brucei/genetics
• Trypanosoma brucei brucei/metabolism

• MR/P009018/1 Medical Research Council
• MR/N010558/1 Medical Research Council
• 203134/Z/16/Z Wellcome Trust
• WTI 204697/Z/16/Z Wellcome Trust
• MR/K008749/1 Medical Research Council
• Deutsche Forschungsgemeinschaft
• Bayerische Forschungsallianz

Trypanosoma brucei undergoes life cycle form transitions from trypomastigotes to epimastigotes in the insect vector by re-positioning the mitochondrial genome and re-locating the flagellum and flagellum-associated cytoskeletal structures. The mechanism underlying these dramatic morphology transitions remains poorly understood. Here we report the regulatory role of the orphan kinesin KIN-E in controlling trypanosome morphology transitions. KIN-E localizes to the flagellum and is enriched at the flagellar tip, and this localization depends on the C-terminal m-calpain domain III-like domains. Depletion of KIN-E in the trypomastigote form of T. brucei causes major morphology changes and a gradual increase in the level of EP procyclin, generating epimastigote-like cells. Mechanistically, through its C-terminal importin α-like domain, KIN-E targets FLAM3, a flagellar protein involved in morphology transitions, to the flagellum to promote elongation of the flagellum attachment zone and positioning of the flagellum and flagellum-associated cytoskeletal structure, thereby maintaining trypomastigote cell morphology. Our findings suggest that morphology transitions in trypanosomes require KIN-E-mediated transport of FLAM3 to the flagellum.

Trypanosoma brucei, the causative agent of sleeping sickness in humans and nagana in cattle in sub-Saharan Africa, has a complex life cycle by alternating between the tsetse fly vector and the mammalian hosts. In the gut of tsetse flies, trypanosomes undergo life cycle transitions from the trypomastigote form to the epimastigote form by re-positioning the mitochondrial genome and re-locating the flagellum and flagellum-associated cytoskeletal structures. Previous work demonstrated that elongation of the flagellum attachment zone plays an important role in controlling morphology transitions, but how it is regulated remains poorly understood. This work discovered that an orphan kinesin plays an essential role in regulating trypanosome morphology transitions. This novel kinesin localizes to the flagellum and targets FLAM3, one of the two flagellar proteins involved in morphology transitions, to the flagellum. This work suggests that trypanosome morphology transitions require kinesin-mediated transport of FLAM3 to the flagellum to promote the elongation of the flagellum attachment zone, thereby maintaining flagellum-cell body attachment and positioning the flagellum and flagellum-associated cytoskeletal structures to assume trypomastigote cell morphology.

• Amino Acid Sequence
• Carrier Proteins/physiology
• Cytoskeletal Proteins
• Cytoskeleton/ultrastructure
• Flagella/metabolism
• Flagella/ultrastructure
• Kinesins/chemistry
• Kinesins/physiology
• Membrane Glycoproteins/genetics
• Membrane Glycoproteins/metabolism
• Protein Structure, Secondary
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism
• Protozoan Proteins/physiology
• RNA Interference/physiology
• Sequence Alignment
• Transcription Elongation, Genetic/physiology
• Trypanosoma brucei brucei/metabolism
• Trypanosoma brucei brucei/ultrastructure

• R01 AI101437 NIAID NIH HHS
• R01 AI118736 NIAID NIH HHS
• National Institute of Allergy and Infectious Diseases

• 3D structure
• Alba domain
• architectural proteins
• evolutionary relevant
• phylogenetic relationship
• regulatory elements
• subcellular localization

From synthesis to decay, mRNA associates with RNA-binding proteins (RBPs) establishing dynamic ribonucleoprotein particles (RNPs). Understanding the composition and function of RNPs is fundamental to understanding how eukaryotic mRNAs are controlled. This is especially relevant for trypanosomes and related kinetoplastid parasites, which mostly rely on post-transcriptional mechanisms to control gene expression. Crucial for trypanosome differentiation, development, or even response to heat shock, RBPs are known to be essential modulators of diverse molecular processes. The recent application of large-scale quantitative methods, such as Next-Generation Sequencing (NGS) and quantitative mass spectrometry, has revealed new exciting features about the parasite RNA-related metabolism. Novel proteins carrying RNA-binding activity, including many proteins without RNA-related ontology were discovered setting a necessary groundwork to get in insights into RNA biology.

Conclusion: This review aims to give the reader an understanding of current trypanosome RNP research, highlighting the progress made using high-throughput approaches.

• Gene expression regulation
• High-throughput
• Post-transcriptional control
• RNA biology
• RNA-binding proteins
• mRNA-fate

• Kinetoplastids
• eIF4E
• translation initiation

• Alba protein
• Leishmania
• Leishmaniasis
• Structural biology

• Trypanosoma
• biophysics
• microswimmer
• none
• sleeping sickness
• structural biology
• tsetse fly

The fate of an mRNA is determined by its interaction with proteins and small RNAs within dynamic complexes called ribonucleoprotein complexes (mRNPs). In Trypanosoma brucei and related kinetoplastids, responses to internal and external signals are mainly mediated by post-transcriptional processes. Here, we used proximity-dependent biotin identification (BioID) combined with RNA-seq to investigate the changes resulting from ectopic expression of RBP10 and RBP9, two developmentally regulated RNA-binding proteins (RBPs). Both RBPs have reduced expression in insect procyclic forms (PCFs) compared with bloodstream forms (BSFs). Upon overexpression in PCFs, both proteins were recruited to cytoplasmic foci, co-localizing with the processing body marker SCD6. Further, both RBPs altered the transcriptome from a PCF- to a BSF-like pattern. Notably, upon expression of BirA*-RBP9 and BirA*-RBP10, BioID yielded more than 200 high confidence protein interactors (more than 10-fold enriched); 45 (RBP9) and 31 (RBP10) were directly related to mRNA metabolism. This study validates the use of BioID for investigating mRNP components but also illustrates the complexity of mRNP function.

• RNA
• biotin identification
• differentiation
• gene expression
• kinetoplastid

• Alba-domain protein
• Amastin
• Post transcriptional regulation
• Trypanosoma cruzi
• mRNA

Experimental immunization with radiation attenuated sporozoites (RAS) and genetically attenuated sporozoites has proved to be a promising approach for malaria vaccine development. However, parasite biomarkers of growth attenuation and enhanced immune protection in response to radiation remain poorly understood. Here, we report on the effect of an attenuating dose of γ-irradiation (15 krad) on the Plasmodium falciparum sporozoite (PfSPZ) ultrastructure by electron microscopy, growth rate of liver stage P. falciparum in liver cell cultures, and genome-wide transcriptional profile of liver stage parasites by microarray. We find that γ-irradiation treated PfSPZ retained a normal cellular structure except that they were vacuous with a partially disrupted plasma membrane and inner membrane complex. A similar infection rate was observed by γ-irradiation-treated and untreated PfSPZ in human HCO-4 liver cells (0.47% versus 0.49%, respectively) on day 3 post-infection. In the microarray studies, cumulatively, 180 liver stage parasite genes were significantly transcriptionally altered on day 3 and/or 6 post-infection. Among the transcriptionally altered biomarkers, we identified a signature of seven candidate parasite genes that associated with functionally diverse pathways that may regulate radiation induced cell cycle arrest of the parasite within the hepatocyte. A repertoire of 14 genes associated with protein translation is transcriptionally overexpressed within the parasite by radiation. Additionally, 37 genes encode proteins expressed on the cell surface or exported into the host cell, 4 encode membrane associated transporters, and 10 encode proteins related to misfolding and stress-related protein processing. These results have significantly increased the repertoire of novel targets for 1) biomarkers of safety to define proper attenuation, 2) generating genetically attenuated parasite vaccine candidates, and 3) subunit candidate vaccines against liver stage malaria.

• Animals
• Anopheles
• Biomarkers/metabolism
• Cell Line
• Female
• Gamma Rays
• Gene Expression Regulation/radiation effects
• Humans
• Liver/metabolism
• Liver/parasitology
• Malaria Vaccines/metabolism
• Malaria, Falciparum/metabolism
• Malaria, Falciparum/prevention & control
• Plasmodium falciparum/metabolism
• Protozoan Proteins/metabolism
• Sporozoites/metabolism
• Vaccines, Attenuated/metabolism

• Arabidopsis/genetics
• Arabidopsis/metabolism
• Arabidopsis Proteins/genetics
• Arabidopsis Proteins/metabolism
• Proteome/metabolism
• RNA, Messenger/metabolism
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism
• Seedlings/genetics
• Seedlings/metabolism

• (co-)evolution
• drug resistance
• escape
• insect vector
• parasites