• Leishmania infantum
• Chromosome clustering
• Developmental gene regulation
• Evolutionary divergence
• Functional bias
• Low expression
• SIDER2 retroposons
• SIDER2 transcriptome
• Virulence

• Retroelements/genetics
• Leishmania/genetics
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• Evolution, Molecular
• Genome, Protozoan
• Gene Expression Regulation, Developmental
• RNA, Protozoan/genetics
• RNA, Protozoan/metabolism

• MOP-12182 CIHR
• MOP-12182 CIHR
• MOP-12182 CIHR
• MOP-12182 CIHR
• MOP-12182 CIHR
• Canadian Institutes of Health Research

• drug resistance
• genome organization
• leishmania
• parasite survival
• plasticity

• Acetylation
• CCR4-NOT complex
• CPEB4
• Cytoplasmic polyadenylation element
• Deadenylation
• Immediate early genes
• mRNA turnover

• 3' Untranslated Regions
• Genes, Immediate-Early
• HeLa Cells
• Humans
• RNA Stability
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism

• Deutsche Forschungsgemeinschaft
• Ruprecht-Karls-Universität Heidelberg (1026)

• Leishmania/genetics
• Leishmania/metabolism
• Poly(A)-Binding Proteins/genetics
• Poly(A)-Binding Proteins/metabolism
• Protein Binding
• Protein Biosynthesis
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism
• Ribosomal Proteins/genetics
• Ribosomal Proteins/metabolism

• FACEPE
• CNPq
• CAPES
• FIOTEC

The nuclear pore complex is an ancient component of the eukaryotic cell. We show here that an FG nucleoporin, TbNup53b, in trypanosomes has an association with the splicing machinery and roles in gene expression, indicating that moonlighting roles for nucleoporins are highly ancient and present in the earliest eukaryotes.

Components of the nuclear periphery coordinate a multitude of activities, including macromolecular transport, cell-cycle progression, and chromatin organization. Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport, mRNA processing, and transcriptional regulation, and NPC components can define regions of high transcriptional activity in some organisms at the nuclear periphery and nucleoplasm. Lineage-specific features underpin several core nuclear functions and in trypanosomatids, which branched very early from other eukaryotes, unique protein components constitute the lamina, kinetochores, and parts of the NPCs. Here we describe a phenylalanine-glycine (FG)-repeat nucleoporin, TbNup53b, that has dual localizations within the nucleoplasm and NPC. In addition to association with nucleoporins, TbNup53b interacts with a known trans-splicing component, TSR1, and has a role in controlling expression of surface proteins including the nucleolar periphery-located, procyclin genes. Significantly, while several nucleoporins are implicated in intranuclear transcriptional regulation in metazoa, TbNup53b appears orthologous to components of the yeast/human Nup49/Nup58 complex, for which no transcriptional functions are known. These data suggest that FG-Nups are frequently co-opted to transcriptional functions during evolution and extend the presence of FG-repeat nucleoporin control of gene expression to trypanosomes, suggesting that this is a widespread and ancient eukaryotic feature, as well as underscoring once more flexibility within nucleoporin function.

• Leishmania
• phosphorylation
• polyadenylate-binding protein
• protein-protein interaction
• translation
• translation initiation factor

• Leishmania
• MS2 coat protein tethering system
• PUF6
• Pumilio proteins
• SIDER2 retroposons
• mRNA decay

• 3' Untranslated Regions/genetics
• Gene Expression Regulation
• Genome, Protozoan
• Leishmania infantum/genetics
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism
• RNA Stability
• RNA, Protozoan/genetics
• Retroelements/genetics

• STP-53924 CIHR
• MOP-12182 CIHR
• Canadian Institutes of Health Research (CIHR)
• Fonds de Recherche du Québec-Nature et Technologies
• Canadian Institutes of Health Research

• ABC transporter
• Aquaporin
• Leishmania Viannia panamensis
• Macrophage
• Meglumine antimoniate

• Kinetoplastids
• eIF4E
• translation initiation

Mammalian mitochondrial RNAs are unique as they are derived from primary transcripts that encompass almost the entire mitochondrial genome. This necessitates extensive processing to release the individual mRNAs, rRNAs and tRNAs required for gene expression. Recent studies have revealed many of the proteins required for mitochondrial RNA processing, however the rapid turnover of precursor RNAs has made it impossible to analyze their composition and the hierarchy of processing. Here, we find that circularization of RNA prior to deep sequencing enables the discovery and characterization of unprocessed RNAs. Using this approach, we identify the most stable processing intermediates and the presence of intermediate processing products that are partially degraded and polyadenylated. Analysis of libraries constructed using RNA from mice lacking the nuclease subunit of the mitochondrial RNase P reveals the identities of stalled processing intermediates, their order of cleavage, and confirms the importance of RNase P in generating mature mitochondrial RNAs. Using RNA circularization prior to library preparation should provide a generally useful approach to studying RNA processing in many different biological systems.

• Animals
• Computational Biology
• Mice, Inbred C57BL
• Mice, Transgenic
• Polyadenylation
• RNA/genetics
• RNA/metabolism
• RNA Processing, Post-Transcriptional
• RNA Stability
• RNA, Circular
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• RNA, Mitochondrial
• Ribonuclease P/metabolism
• Sequence Analysis, RNA/methods

We have reported previously that Short Interspersed Degenerate Retroposons of the SIDER2 subfamily, largely located within 3'UTRs of Leishmania transcripts, promote rapid turnover of mRNAs through endonucleolytic cleavage within the highly conserved second tandem 79-nt hallmark sequence (79-nt SII). Here, we used site-directed mutagenesis and in silico RNA structural studies to delineate the cis-acting requirements within 79-nt SII for cleavage and mRNA degradation. The putative cleavage site(s) and other nucleotides predicted to alter the RNA secondary structure of 79-nt SII were either deleted or mutated and their effect on mRNA turnover was monitored using a gene reporter system. We found that short deletions of 8-nt spanning the two predicted cleavage sites block degradation of SIDER2-containing transcripts, leading to mRNA accumulation. Furthermore, single or double substitutions of the dinucleotides targeted for cleavage as well as mutations altering the predicted RNA secondary structure encompassing both cleavage sites also prevent mRNA degradation, confirming that these dinucleotides are the bona fide cleavage sites. In line with these results, we show that stage-regulated SIDER2 inactivation correlates with the absence of endonucleolytic cleavage. Overall, these data demonstrate that both cleavage sites within the conserved 79-nt SII as well as RNA folding in this region are essential for SIDER2-mediated mRNA decay, and further support that SIDER2-harboring transcripts are targeted for degradation by endonucleolytic cleavage.

• Base Sequence
• Computer Simulation
• Conserved Sequence
• Leishmania/genetics
• Models, Molecular
• Mutagenesis, Site-Directed
• Nucleic Acid Conformation
• RNA Stability
• RNA, Messenger/chemistry
• RNA, Protozoan/chemistry
• Sequence Deletion
• Short Interspersed Nucleotide Elements

• Canadian Institutes of Health Research (CIHR)

Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, with regulation of mRNA processing, translation and degradation by RNA-binding proteins. ZC3H32 is a cytosolic mRNA-binding protein with three non-canonical CCCH zinc finger domains. It is much more abundant in bloodstream-form Trypanosoma brucei than in procyclic forms. Tethering of ZC3H32 to a reporter mRNA suppressed translation and resulted in mRNA degradation, and deletion analysis suggested that this activity was present in both the N- and C-terminal domains, but not the central zinc finger-containing domain. Tandem affinity purification, however, revealed no interaction partners that might account for this activity. RNASeq analyses did not yield any evidence for sequence-specific binding or regulation of specific mRNAs. The presence of ZC3H32 homologues in monogenetic and free-living Euglenids also argues against a role in developmental regulation, although its function may have diverged in evolution. T. brucei ZC3H32 might be implicated in basal mRNA metabolism, with this role perhaps being taken over by another protein in procyclic forms.

• Binding Sites
• Euglenida/metabolism
• Gene Expression Regulation
• Protein Domains
• Protozoan Proteins/chemistry
• Protozoan Proteins/metabolism
• RNA, Messenger/metabolism
• RNA, Protozoan/metabolism
• Sequence Analysis, RNA
• Trypanosoma brucei brucei/growth & development
• Trypanosoma brucei brucei/metabolism
• Zinc Fingers

• Deutsche Forschungsgemeinschaft

Leishmania are unicellular eukaryotes responsible for leishmaniasis in humans. Like other trypanosomatids, leishmania regulate protein coding gene expression almost exclusively at the post-transcriptional level with the help of RNA binding proteins (RBPs). Due to the presence of polycystronic transcription units, leishmania do not regulate RNA polymerase II-dependent transcription initiation. Recent evidence suggests that the main control points in gene expression are mRNA degradation and translation. Protein-RNA interactions are involved in every aspect of RNA biology, such as mRNA splicing, polyadenylation, localization, degradation, and translation. A detailed picture of these interactions would likely prove to be highly informative in understanding leishmania biology and virulence. We developed a strategy involving covalent UV cross-linking of RBPs to mRNA in vivo, followed by interactome capture using oligo(dT) magnetic beads to define comprehensively the mRNA interactome of growing L. donovani amastigotes. The protein mass spectrometry analysis of captured proteins identified 79 mRNA interacting proteins which withstood very stringent washing conditions. Strikingly, we found that 49 of these mRNA interacting proteins had no orthologs or homologs in the human genome. Consequently, these may represent high quality candidates for selective drug targeting leading to novel therapeutics. These results show that this unbiased, systematic strategy has the promise to be applicable to study the mRNA interactome during various biological settings such as metabolic changes, stress (low pH environment, oxidative stress and nutrient deprivation) or drug treatment.

• Gene Ontology
• Humans
• Leishmania donovani/metabolism
• Mass Spectrometry
• Protein Domains
• Protein Interaction Mapping/methods
• Proteome/metabolism
• Protozoan Proteins/chemistry
• Protozoan Proteins/metabolism
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• RNA-Binding Proteins/chemistry
• RNA-Binding Proteins/metabolism

• Canadian Institutes of Health Research

Leishmania has a plastic genome, and drug pressure can select for gene copy number variation (CNV). CNVs can apply either to whole chromosomes, leading to aneuploidy, or to specific genomic regions. For the latter, the amplification of chromosomal regions occurs at the level of homologous direct or inverted repeated sequences leading to extrachromosomal circular or linear amplified DNAs. This ability of Leishmania to respond to drug pressure by CNVs has led to the development of genomic screens such as Cos-Seq, which has the potential of expediting the discovery of drug targets for novel promising drug candidates.

• Cos-Seq
• Drug Resistance
• Leishmania
• Mode of action
• Ploidy

Gene expression is controlled at multiple levels, including transcription, stability, translation, and degradation. Over the years, it has become apparent that Plasmodium falciparum exerts limited transcriptional control of gene expression, while at least part of Plasmodium’s genome is controlled by post-transcriptional mechanisms. To generate insights into the mechanisms that regulate gene expression at the post-transcriptional level, we undertook complementary computational, comparative genomics, and experimental approaches to identify and characterize mRNA-binding proteins (mRBPs) in P. falciparum.

Close to 1000 RNA-binding proteins are identified by hidden Markov model searches, of which mRBPs encompass a relatively large proportion of the parasite proteome as compared to other eukaryotes. Several abundant mRNA-binding domains are enriched in apicomplexan parasites, while strong depletion of mRNA-binding domains involved in RNA degradation is observed. Next, we experimentally capture 199 proteins that interact with mRNA during the blood stages, 64 of which with high confidence. These captured mRBPs show a significant overlap with the in silico identified candidate RBPs (p < 0.0001). Among the experimentally validated mRBPs are many known translational regulators active in other stages of the parasite’s life cycle, such as DOZI, CITH, PfCELF2, Musashi, and PfAlba1–4. Finally, we also detect several proteins with an RNA-binding domain abundant in Apicomplexans (RAP domain) that is almost exclusively found in apicomplexan parasites.

Collectively, our results provide the most complete comparative genomics and experimental analysis of mRBPs in P. falciparum. A better understanding of these regulatory proteins will not only give insight into the intricate parasite life cycle but may also provide targets for novel therapeutic strategies.

The online version of this article (doi:10.1186/s13059-016-1014-0) contains supplementary material, which is available to authorized users.

• Gene expression
• Post-transcriptional regulation
• Protein domains
• RNA-binding proteins
• Translation

Intracellular colonization and persistent infection by the kinetoplastid protozoan parasite, Trypanosoma cruzi, underlie the pathogenesis of human Chagas disease. To obtain global insights into the T. cruzi infective process, transcriptome dynamics were simultaneously captured in the parasite and host cells in an infection time course of human fibroblasts. Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. Contrasting this early response, few additional changes in steady state mRNA levels were detected once mature T. cruzi amastigotes were formed. Our findings suggest that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Despite complex mechanisms for regulation of phenotypic expression in T. cruzi, transcriptomic signatures derived from distinct developmental stages mirror known or projected characteristics of T. cruzi biology. Focusing on energy metabolism, we were able to validate predictions forecast in the mRNA expression profiles. We demonstrate measurable differences in the bioenergetic properties of the different mammalian-infective stages of T. cruzi and present additional findings that underscore the importance of mitochondrial electron transport in T. cruzi amastigote growth and survival. Consequences of T. cruzi colonization for the host include dynamic expression of immune response genes and cell cycle regulators with upregulation of host cholesterol and lipid synthesis pathways, which may serve to fuel intracellular T. cruzi growth. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and host, our comprehensive, high resolution transcriptomic dataset provides a substantially more detailed interpretation of T. cruzi infection biology and offers a basis for future drug and vaccine discovery efforts.

In-depth knowledge of the functional processes governing host colonization and transmission of pathogenic microorganisms is essential for the advancement of effective intervention strategies. This study focuses on Trypanosoma cruzi, the vector-borne protozoan parasite responsible for human Chagas disease and the leading cause of infectious myocarditis worldwide. To gain global insights into the biology of this parasite and its interaction with mammalian host cells, we have exploited a deep-sequencing approach to generate comprehensive, high-resolution transcriptomic maps for mammalian-infective stages of T. cruzi with the simultaneous interrogation of the human host cell transcriptome across an infection time course. We demonstrate that the establishment of intracellular T. cruzi infection in mammalian host cells is accompanied by extensive remodeling of the parasite and host cell transcriptomes. Despite the lack of transcriptional control mechanisms in trypanosomatids, our analyses identified functionally-enriched processes within sets of developmentally-regulated transcripts in T. cruzi that align with known or predicted biological features of the parasite. The novel insights into the biology of intracellular T. cruzi infection and the regulation of amastigote development gained in this study establish a unique foundation for functional network analyses that will be instrumental in providing functional links between parasite dependencies and host functional pathways that have the potential to be exploited for intervention.

Posttranscriptional mechanisms have a critical role in the overall outcome of gene expression. These mechanisms are especially relevant in protozoa from the genus Trypanosoma, which is composed by death threatening parasites affecting people in Sub-saharan Africa or in the Americas. In these parasites the classic view of regulation of transcription initiation to modulate the products of a given gene cannot be applied. This is due to the presence of transcription start sites that give rise to long polycistronic units that need to be processed costranscriptionally by trans-splicing and polyadenylation to give mature monocistronic mRNAs. Posttranscriptional mechanisms such as mRNA degradation and translational repression are responsible for the final synthesis of the required protein products. In this context, RNA-binding proteins (RBPs) in trypanosomes have a relevant role as modulators of mRNA abundance and translational repression by associating to the 3’ untranslated regions in mRNA. Many different RBPs have been proposed to modulate cohorts of mRNAs in trypanosomes. However, the current understanding of their functions lacks a dynamic view on the different steps at which these RBPs are regulated. Here, we discuss different evidences to propose regulatory events for different RBPs in these parasites. These events vary from regulated developmental expression, to biogenesis of cytoplasmic ribonucleoprotein complexes in the nucleus, and condensation of RBPs and mRNA into large cytoplasmic granules. Finally, we discuss how newly identified posttranslational modifications of RBPs and mRNA metabolism-related proteins could have an enormous impact on the modulation of mRNA abundance. To understand these modifications is especially relevant in these parasites due to the fact that the enzymes involved could be interesting targets for drug therapy.

• Trypanosoma
• Posttranscriptional gene expression
• Ribonucleoprotein complexes
• RNA-binding protein
• Developmental regulation
• Sleeping sickness
• Posttranslational modification
• Phosphorylation
• Chagas disease

• GST, Glutathione-S-Transferase
• HEAT domain, Huntingtin, elongation factor 3 (EF3)
• Leishmania major
• MIF4G, Middle domain of eukaryotic Initiation Factor 4G
• PABP, Poly(A) Binding Protein
• Trypanosoma brucei
• cap binding complex
• eIF, eukaryotic Initiation Factor
• protein phosphatase 2A (PP2A) and the yeast kinase TOR1
• protein synthesis
• translation initiation

• C1QBP
• HABP1
• P32
• Protein–protein interactions
• RNA Recognition Motif
• RNA-binding proteins
• Trypanosoma
• Trypanosomes

• Leishmania
• cap binding protein
• eIF4E
• eIF4G
• phosphorylation
• protein synthesis
• translation initiation PABP

RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released.

RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation.

The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function.

Regulation of gene expression in trypanosomatids is mainly posttranscriptional. Tight regulation of mRNA stability and access to polysomes allows Trypanosoma cruzi to adapt to different environmental conditions during its life cycle. Posttranscriptional regulation requires association between mRNAs and specific proteins to form mRNP complexes. Proteins that lack a canonical RNA-binding domain, such as eukaryotic elongation factor-1α (EF-1α), may also associate with mRNPs. EF-1α is conserved in many organisms, and it plays roles in many cellular processes other than translation, including RNA transport, the cell cycle, and apoptosis.

In a previous study, EF-1α was found associated with mRNP-forming mRNAs in polysome-free fractions both in epimastigotes growing under normal conditions and in nutritionally stressed parasites. This finding suggested the possibility that EF-1α has a non-canonical function. Thus, we investigated the dynamics of EF-1α in association with T. cruzi epimastigote mRNAs under normal and stressed nutritional conditions. EF-1α is expressed throughout the parasite life cycle, but it shows a slight decrease in protein levels in the metacyclic trypomastigote form. The protein is cytoplasmically localized with a granular pattern in all forms analyzed. Following puromycin treatment, EF-1α migrated with the heaviest gradient fractions in a sucrose polysome profile, indicating that its association with large protein complexes was independent of the translation machinery. We next characterized the EF-1α-associated mRNAs in unstressed and stressed epimastigotes. We observed that specific subsets of mRNAs were associated with EF-1α-mRNPs in unstressed or stressed epimastigotes. Some mRNAs were identified in both physiological conditions, whereas others were condition-specific. Gene ontology analysis identified enrichment of gene sets involved in single-organism metabolic processes, amino acid metabolic processes, ATP and metal ion binding, glycolysis, glutamine metabolic processes, and cobalt and iron ion binding.

These results indicate that in T. cruzi, as in other eukaryotes, EF-1α may play a non-canonical cellular role. We observed the enrichment of functionally related transcripts bound to EF-1α in normal growth conditions as well as in nutritionally stressed cell indicating a potential role of EF-1α mRNP in stress response.

The online version of this article (doi:10.1186/s12866-015-0436-2) contains supplementary material, which is available to authorized users.

• Cancer
• DNA damage
• Posttranscriptional regulation
• RNA-binding proteins
• Translational control
• p53

• Active Transport, Cell Nucleus
• Animals
• Cell Cycle
• Cell Proliferation
• DNA Damage
• Gene Expression
• Humans
• RNA Interference
• RNA Transport
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• Regulon

• R01 CA157268 NCI NIH HHS
• R01CA157268 NCI NIH HHS
• R01 ES005777 NIEHS NIH HHS
• P30 CA014236 NCI NIH HHS
• R01ES005777 NIEHS NIH HHS

• 14-3-3 protein
• kinetoplastid
• mRNA cap
• social motility
• translation initiation factor

• Amino Acid Sequence
• Eukaryotic Initiation Factor-4E/chemistry
• Eukaryotic Initiation Factor-4E/metabolism
• Eukaryotic Initiation Factor-4G/metabolism
• Gene Knockout Techniques
• Humans
• Molecular Sequence Data
• Protein Binding
• RNA Cap-Binding Proteins/metabolism
• RNA Caps/metabolism
• RNA Processing, Post-Transcriptional
• RNA, Protozoan/metabolism
• Sequence Alignment
• Trypanosoma brucei brucei/genetics
• Trypanosoma brucei brucei/metabolism

• R56 AI052348 NIAID NIH HHS
• R03 TW009035 FIC NIH HHS
• R21 AI073806 NIAID NIH HHS
• AI052348 NIAID NIH HHS
• TW009035 FIC NIH HHS
• R01 AI056034 NIAID NIH HHS
• GM089778 NIGMS NIH HHS
• R01 AI052348 NIAID NIH HHS
• AI073806 NIAID NIH HHS
• R01 GM089778 NIGMS NIH HHS
• AI056034 NIAID NIH HHS

Trypanosome gene expression is regulated almost exclusively at the posttranscriptional level, through mRNA stability, storage and degradation. Here, we characterize the ribonucleoprotein complex (mRNPs) corresponding to the zinc finger protein TcZC3H39 from T. cruzi comparing cells growing in normal conditions and under nutritional stress. The nutritional stress is a key step during T. cruzi differentiation from epimastigote form to human infective metacyclic trypomastigote form. The mechanisms by which the stress, altogether with other stimuli, triggers differentiation is not well understood. This work aims to characterize the TcZC3H39 protein during stress response. Using cells cultured in normal and stress conditions, we observed a dynamic change in TcZC3H39 granule distribution, which appeared broader in stressed epimastigotes. The protein core of the TcZC3H39-mRNP is composed of ribosomes, translation factors and RBPs. The TcZC3H39-mRNP could act sequestering highly expressed mRNAs and their associated ribosomes, potentially slowing translation in stress conditions. A shift were observed in the mRNAs associated with TcZC3H39: the number of targets in unstressed epimastigotes was smaller than that in stressed parasites, with no clear functional clustering in normal conditions. By contrast, in stressed parasites, the targets of TcZC3H39 were mRNAs encoding ribosomal proteins and a remarkable enrichment in mRNAs for the cytochrome c complex (COX), highly expressed mRNAs in the replicative form. This identification of a new component of RNA granules in T. cruzi, the TcZC3H39 protein, provides new insight into the mechanisms involved in parasite stress responses and the regulation of gene expression during T. cruzi differentiation.

• Trypanosoma cruzi
• gene expression
• mRNPs
• posttranscriptional regulation
• regulon
• stress response
• zinc finger

• Chagas Disease/parasitology
• Conserved Sequence
• DNA, Intergenic
• DNA, Protozoan/genetics
• Evolution, Molecular
• Genetic Variation
• Genome, Protozoan
• Genomics
• Trypanosoma cruzi/genetics

• D43 TW007888 FIC NIH HHS
• D43TW007888 FIC NIH HHS

The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6–48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.

Leishmania, the cause of a deadly spectrum of diseases in humans, surmounts a number of environmental challenges, including changes in the availability of salvageable nutrients, to successfully colonize its host. Adaptation to environmental stress is clearly of significance in parasite biology, but the underlying mechanisms are not well understood. To simulate the response to periodic nutrient scarcity in vivo, we have induced purine starvation in vitro. Purines are essential for growth and viability, and serve as the major energy currency of cells. Leishmania cannot synthesize purines and must salvage them from the surroundings. Extracellular purine depletion in culture induces a robust survival response in Leishmania, whereby growth arrests, but parasites persist for months. To profile the events that enable endurance of purine starvation, we used shotgun proteomics. Our data suggest that purine starvation induces extensive proteome remodeling, tailored to enhance purine capture and recycling, reduce energy expenditures, and maintain viability of the metabolically active, non-dividing population. Through global and targeted approaches, we reveal that proteome remodeling is multifaceted, and occurs through an array of responses at the mRNA, translational, and post-translational level. Our data provide one of the most inclusive views of adaptation to microenvironmental stress in Leishmania.

• Cis-element
• Post-transcriptional control
• RNA regulon
• RNA-binding protein
• Trypanosomes

• CAF1
• PAN2
• Trypanosoma
• deadenylation
• exosome
• mRNA decay
• mRNA degradation

• Cell Line
• Computational Biology
• Exoribonucleases/genetics
• Exoribonucleases/metabolism
• Exosome Multienzyme Ribonuclease Complex/genetics
• Exosome Multienzyme Ribonuclease Complex/metabolism
• Half-Life
• Multiprotein Complexes/genetics
• Multiprotein Complexes/metabolism
• Protozoan Proteins/genetics
• Protozoan Proteins/metabolism
• RNA Interference
• RNA Stability
• RNA Transport
• RNA, Messenger/genetics
• RNA, Messenger/metabolism
• RNA, Protozoan/genetics
• RNA, Protozoan/metabolism
• RNA, Small Nucleolar/genetics
• RNA, Small Nucleolar/metabolism
• Trypanosoma brucei brucei/enzymology
• Trypanosoma brucei brucei/genetics

• 3' Untranslated Regions/genetics
• 5' Untranslated Regions/genetics
• ELAV Proteins/genetics
• ELAV Proteins/metabolism
• Gene Expression Regulation
• Humans
• MicroRNAs/genetics
• MicroRNAs/metabolism
• RNA Splicing
• RNA-Binding Proteins/genetics
• RNA-Binding Proteins/metabolism
• Regulon
• Ribonucleoproteins/genetics
• Ribonucleoproteins/metabolism
• Transcription Factors/genetics
• Transcription Factors/metabolism

• R01 CA157268 NCI NIH HHS

Posttranscriptional gene regulation is a rapid and efficient process to adjust the proteome of a cell to a changing environment. RNA-binding proteins (RBPs) are the master regulators of mRNA processing and translation and are often aberrantly expressed in cancer. In addition to well-studied transcription factors, RBPs are emerging as fundamental players in tumor development. RBPs and their mRNA targets form a complex network that plays a crucial role in tumorigenesis. This paper describes mechanisms by which RBPs influence the expression of well-known oncogenes, focusing on precise examples that illustrate the versatility of RBPs in posttranscriptional control of cancer development. RBPs appeared very early in evolution, and new RNA-binding domains and combinations of them were generated in more complex organisms. The identification of RBPs, their mRNA targets, and their mechanism of action have provided novel potential targets for cancer therapy.

• rnaseq
• trypanosoma
• xrn1
• mrna decay
• polycistronic

• Exoribonucleases/metabolism
• Open Reading Frames
• RNA Stability
• RNA, Messenger/metabolism
• RNA, Protozoan/metabolism
• Transcriptome
• Trypanosoma brucei brucei/enzymology
• Trypanosoma brucei brucei/genetics

• 3' Untranslated Regions
• Antigens, Protozoan/genetics
• Blotting, Northern
• Cluster Analysis
• DNA, Protozoan/chemistry
• DNA, Protozoan/genetics
• Gene Expression Profiling
• Gene Library
• Humans
• Membrane Proteins/genetics
• Molecular Sequence Data
• Open Reading Frames
• Phylogeny
• Protozoan Proteins/genetics
• RNA, Messenger/biosynthesis
• Reverse Transcriptase Polymerase Chain Reaction
• Sequence Analysis, DNA
• Sequence Homology, Nucleic Acid
• Trypanosoma cruzi/genetics

We have previously shown that the Leishmania genome possess two widespread families of extinct retroposons termed Short Interspersed DEgenerated Retroposons (SIDER1/2) that play a role in post-transcriptional regulation. Moreover, we have demonstrated that SIDER2 retroposons promote mRNA degradation. Here we provide new insights into the mechanism by which unstable Leishmania mRNAs harboring a SIDER2 retroposon in their 3′-untranslated region are degraded. We show that, unlike most eukaryotic transcripts, SIDER2-bearing mRNAs do not undergo poly(A) tail shortening prior to rapid turnover, but instead, they are targeted for degradation by a site-specific endonucleolytic cleavage. The main cleavage site was mapped in two randomly selected SIDER2-containing mRNAs in vivo between an AU dinucleotide at the 5′-end of the second 79-nt signature (signature II), which represents the most conserved sequence amongst SIDER2 retroposons. Deletion of signature II abolished endonucleolytic cleavage and deadenylation-independent decay and increased mRNA stability. Interestingly, we show that overexpression of SIDER2 anti-sense RNA can increase sense transcript abundance and stability, and that complementarity to the cleavage region is required for protecting SIDER2-containing transcripts from degradation. These results establish a new paradigm for how unstable mRNAs are degraded in Leishmania and could serve as the basis for a better understanding of mRNA decay pathways in general.