RESUMEN
The nuclear envelope (NE) separates translation and transcription and is the location of multiple functions, including chromatin organization and nucleocytoplasmic transport. The molecular basis for many of these functions have diverged between eukaryotic lineages. Trypanosoma brucei, a member of the early branching eukaryotic lineage Discoba, highlights many of these, including a distinct lamina and kinetochore composition. Here, we describe a cohort of proteins interacting with both the lamina and NPC, which we term lamina-associated proteins (LAPs). LAPs represent a diverse group of proteins, including two candidate NPC-anchoring pore membrane proteins (POMs) with architecture conserved with S. cerevisiae and H. sapiens, and additional peripheral components of the NPC. While many of the LAPs are Kinetoplastid specific, we also identified broadly conserved proteins, indicating an amalgam of divergence and conservation within the trypanosome NE proteome, highlighting the diversity of nuclear biology across the eukaryotes, increasing our understanding of eukaryotic and NPC evolution.
Asunto(s)
Membrana Nuclear , Trypanosoma , Humanos , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Trypanosoma/metabolismoRESUMEN
The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.
Asunto(s)
Proteínas , Proteómica , Biotinilación , Poro Nuclear , Proteínas/química , Proteómica/métodos , Estreptavidina/químicaRESUMEN
Export of RNA from the nucleus is essential for all eukaryotic cells and has emerged as a major step in the control of gene expression. mRNA molecules are required to complete a complex series of processing events and pass a quality control system to protect the cytoplasm from the translation of aberrant proteins. Many of these events are highly conserved across eukaryotes, reflecting their ancient origin, but significant deviation from a canonical pathway as described from animals and fungi has emerged in the trypanosomatids. With significant implications for the mechanisms that control gene expression and hence differentiation, responses to altered environments and fitness as a parasite, these deviations may also reveal additional, previously unsuspected, mRNA export pathways.
Asunto(s)
ARN , Trypanosoma , Transporte Activo de Núcleo Celular/genética , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , ARN/genética , ARN/metabolismo , ARN Mensajero/genética , Trypanosoma/genética , Trypanosoma/metabolismoRESUMEN
BACKGROUND: Photosynthetic euglenids are major contributors to fresh water ecosystems. Euglena gracilis in particular has noted metabolic flexibility, reflected by an ability to thrive in a range of harsh environments. E. gracilis has been a popular model organism and of considerable biotechnological interest, but the absence of a gene catalogue has hampered both basic research and translational efforts. RESULTS: We report a detailed transcriptome and partial genome for E. gracilis Z1. The nuclear genome is estimated to be around 500 Mb in size, and the transcriptome encodes over 36,000 proteins and the genome possesses less than 1% coding sequence. Annotation of coding sequences indicates a highly sophisticated endomembrane system, RNA processing mechanisms and nuclear genome contributions from several photosynthetic lineages. Multiple gene families, including likely signal transduction components, have been massively expanded. Alterations in protein abundance are controlled post-transcriptionally between light and dark conditions, surprisingly similar to trypanosomatids. CONCLUSIONS: Our data provide evidence that a range of photosynthetic eukaryotes contributed to the Euglena nuclear genome, evidence in support of the 'shopping bag' hypothesis for plastid acquisition. We also suggest that euglenids possess unique regulatory mechanisms for achieving extreme adaptability, through mechanisms of paralog expansion and gene acquisition.
Asunto(s)
Euglena gracilis/genética , Genoma , Proteoma , Transcriptoma , Núcleo Celular , Euglena gracilis/metabolismo , PlastidiosRESUMEN
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.
Asunto(s)
Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/fisiología , Transporte Activo de Núcleo Celular , Antígenos de Superficie/inmunología , Núcleo Celular/metabolismo , Secuencia Conservada , Glicina , Poro Nuclear/metabolismo , Fenilalanina , Dominios Proteicos , Elementos Estructurales de las Proteínas , Alineación de Secuencia , Trypanosoma/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMEN
The core architecture of the eukaryotic cell was established well over one billion years ago, and is largely retained in all extant lineages. However, eukaryotic cells also possess lineage-specific features, frequently keyed to specific functional requirements. One quintessential core eukaryotic structure is the nuclear pore complex (NPC), responsible for regulating exchange of macromolecules between the nucleus and cytoplasm as well as acting as a nuclear organizational hub. NPC architecture has been best documented in one eukaryotic supergroup, the Opisthokonts (e.g. Saccharomyces cerevisiae and Homo sapiens), which although compositionally similar, have significant variations in certain NPC subcomplex structures. The variation of NPC structure across other taxa in the eukaryotic kingdom however, remains poorly understood. We explored trypanosomes, highly divergent organisms, and mapped and assigned their NPC proteins to specific substructures to reveal their NPC architecture. We showed that the NPC central structural scaffold is conserved, likely across all eukaryotes, but more peripheral elements can exhibit very significant lineage-specific losses, duplications or other alterations in their components. Amazingly, trypanosomes lack the major components of the mRNA export platform that are asymmetrically localized within yeast and vertebrate NPCs. Concomitant with this, the trypanosome NPC is ALMOST completely symmetric with the nuclear basket being the only major source of asymmetry. We suggest these features point toward a stepwise evolution of the NPC in which a coating scaffold first stabilized the pore after which selective gating emerged and expanded, leading to the addition of peripheral remodeling machineries on the nucleoplasmic and cytoplasmic sides of the pore.
Asunto(s)
Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Opisthorchis/química , Proteómica , Trypanosoma/metabolismo , Animales , Evolución Molecular , Humanos , Modelos Biológicos , Pliegue de Proteína , Trypanosoma/químicaAsunto(s)
Apicomplexa/fisiología , Apicomplexa/ultraestructura , Trypanosoma/fisiología , Trypanosoma/ultraestructura , Animales , Núcleo Celular/fisiología , Núcleo Celular/ultraestructura , Cromatina/fisiología , Cromatina/ultraestructura , Humanos , Poro Nuclear/fisiología , Poro Nuclear/ultraestructura , Infecciones por Protozoos/fisiopatologíaRESUMEN
Clathrin-mediated endocytosis (CME) is the most evolutionarily ancient endocytic mechanism known, and in many lineages the sole mechanism for internalisation. Significantly, in mammalian cells CME is responsible for the vast bulk of endocytic flux and has likely undergone multiple adaptations to accommodate specific requirements by individual species. In African trypanosomes, we previously demonstrated that CME is independent of the AP-2 adaptor protein complex, that orthologues to many of the animal and fungal CME protein cohort are absent, and that a novel, trypanosome-restricted protein cohort interacts with clathrin and drives CME. Here, we used a novel cryomilling affinity isolation strategy to preserve transient low-affinity interactions, giving the most comprehensive trypanosome clathrin interactome to date. We identified the trypanosome AP-1 complex, Trypanosoma brucei (Tb)EpsinR, several endosomal SNAREs plus orthologues of SMAP and the AP-2 associated kinase AAK1 as interacting with clathrin. Novel lineage-specific proteins were identified, which we designate TbCAP80 and TbCAP141. Their depletion produced extensive defects in endocytosis and endomembrane system organisation, revealing a novel molecular pathway subtending an early-branching and highly divergent form of CME, which is conserved and likely functionally important across the kinetoplastid parasites.
Asunto(s)
Endocitosis , Trypanosoma brucei brucei , Tripanosomiasis/metabolismo , Complejo 2 de Proteína Adaptadora/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Evolución Biológica , Clatrina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Humanos , Filogenia , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas SNARE/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Membrane transport is an essential component of pathogenesis for most infectious organisms. In African trypanosomes, transport to and from the plasma membrane is closely coupled to immune evasion and antigenic variation. In mammals and fungi an octameric exocyst complex mediates late steps in exocytosis, but comparative genomics suggested that trypanosomes retain only six canonical subunits, implying mechanistic divergence. We directly determined the composition of the Trypanosoma brucei exocyst by affinity isolation and demonstrate that the parasite complex is nonameric, retaining all eight canonical subunits (albeit highly divergent at the sequence level) plus a novel essential subunit, Exo99. Exo99 and Sec15 knockdowns have remarkably similar phenotypes in terms of viability and impact on morphology and trafficking pathways. Significantly, both Sec15 and Exo99 have a clear function in endocytosis, and global proteomic analysis indicates an important role in maintaining the surface proteome. Taken together these data indicate additional exocyst functions in trypanosomes, which likely include endocytosis, recycling and control of surface composition. Knockdowns in HeLa cells suggest that the role in endocytosis is shared with metazoan cells. We conclude that, whilst the trypanosome exocyst has novel components, overall functionality appears conserved, and suggest that the unique subunit may provide therapeutic opportunities.
Asunto(s)
Endocitosis/fisiología , Trypanosoma brucei brucei/patogenicidad , Evolución Biológica , Western Blotting , Membrana Celular/metabolismo , Células HeLa , Humanos , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , Transporte de Proteínas/fisiología , Proteómica , Proteínas Protozoarias/metabolismoRESUMEN
The nuclear lamina is a filamentous structure subtending the nuclear envelope and required for chromatin organization, transcriptional regulation and maintaining nuclear structure. The trypanosomatid coiled-coil NUP-1 protein is a lamina component functionally analogous to lamins, the major lamina proteins of metazoa. There is little evidence for shared ancestry, suggesting the presence of a distinct lamina system in trypanosomes. To find additional trypanosomatid lamina components we identified NUP-1 interacting proteins by affinity capture and mass-spectrometry. Multiple components of the nuclear pore complex (NPC) and a second coiled-coil protein, which we termed NUP-2, were found. NUP-2 has a punctate distribution at the nuclear periphery throughout the cell cycle and is in close proximity to NUP-1, the NPCs and telomeric chromosomal regions. RNAi-mediated silencing of NUP-2 leads to severe proliferation defects, gross alterations to nuclear structure, chromosomal organization and nuclear envelope architecture. Further, transcription is altered at telomere-proximal variant surface glycoprotein (VSG) expression sites (ESs), suggesting a role in controlling ES expression, although NUP-2 silencing does not increase VSG switching. Transcriptome analysis suggests specific alterations to Pol I-dependent transcription. NUP-1 is mislocalized in NUP-2 knockdown cells and vice versa, implying that NUP-1 and NUP-2 form a co-dependent network and identifying NUP-2 as a second trypanosomatid nuclear lamina component.
Asunto(s)
Lámina Nuclear/fisiología , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Cromosomas/genética , Cromosomas/metabolismo , Daño del ADN , Regulación de la Expresión Génica , Lámina Nuclear/ultraestructura , Poro Nuclear/metabolismo , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/genética , Transporte de Proteínas , Proteínas Protozoarias/genética , Transcriptoma , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/ultraestructuraRESUMEN
Nuclear pore proteins at the base of cilia were thought to regulate transport into cilia. In this issue of Developmental Cell, Del Viso et al. (2016) challenge this view, showing instead that pore proteins localize to ciliary basal bodies and that their perturbation leads to congenital heart disease.
Asunto(s)
Cilios/metabolismo , Cardiopatías Congénitas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Cilios/genética , Cilios/patología , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/patología , Humanos , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Transporte de Proteínas/genéticaRESUMEN
The parasitic protozoa Trypanosoma brucei and Plasmodium falciparum are lethal human parasites that have developed elegant strategies of immune evasion by antigenic variation. Despite the vast evolutionary distance between the two taxa, both parasites employ strict monoallelic expression of their membrane proteins, variant surface glycoproteins in Trypanosomes and the var, rif and stevor genes in Plasmodium, in order to evade their host's immune system. Additionally, both telomeric location and epigenetic controls are prominent features of these membrane proteins. As such, telomeres, chromatin structure and nuclear organization all contribute to control of gene expression and immune evasion. Here, we discuss the importance of epigenetics and sub-nuclear context for the survival of these disease-causing parasites.
Asunto(s)
Genes Protozoarios , Genómica , Membrana Nuclear/metabolismo , Parásitos/genética , Parásitos/metabolismo , Animales , Variación Antigénica , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Orden Génico , Genoma , Genómica/métodos , Interacciones Huésped-Parásitos/inmunología , Parásitos/inmunología , Parásitos/patogenicidadRESUMEN
Functional understanding of the nuclear envelope requires the identification of its component proteins and their interactions. Trypanosomes cause human and livestock diseases worldwide but are so divergent from animals and fungi that in silico searches for homologs of proteins are frequently of low value. Here we describe a strategy for the straightforward identification of nuclear envelope proteins from trypanosomes that classifies proteins and their interaction networks in the nuclear pore complex. Milling frozen whole cells into a powder and rapid screening of buffer conditions for optimization of complex isolation is described. The method is inexpensive and potentially applicable to many organisms, providing fast access to functional information.
Asunto(s)
Proteínas de la Membrana/aislamiento & purificación , Complejos Multiproteicos/aislamiento & purificación , Membrana Nuclear/metabolismo , Proteínas Protozoarias/aislamiento & purificación , Trypanosoma/metabolismo , Cromatografía de Afinidad/instrumentación , Cromatografía de Afinidad/métodos , Electroforesis en Gel de PoliacrilamidaRESUMEN
The nuclear pore complex (NPC) is responsible for nucleocytoplasmic transport and constitutes a hub for control of gene expression. The components of NPCs from several eukaryotic lineages have been determined, but only the yeast and vertebrate NPCs have been extensively characterized at the quaternary level. Significantly, recent evidence indicates that compositional similarity does not necessarily correspond to homologous architecture between NPCs from different taxa. To address this, we describe the interactome of the trypanosome NPC, a representative, highly divergent eukaryote. We identify numerous new NPC components and report an exhaustive interactome, allowing assignment of trypanosome nucleoporins to discrete NPC substructures. Remarkably, despite retaining similar protein composition, there are exceptional architectural dissimilarities between opisthokont (yeast and vertebrates) and excavate (trypanosomes) NPCs. Whilst elements of the inner core are conserved, numerous peripheral structures are highly divergent, perhaps reflecting requirements to interface with divergent nuclear and cytoplasmic functions. Moreover, the trypanosome NPC has almost complete nucleocytoplasmic symmetry, in contrast to the opisthokont NPC; this may reflect divergence in RNA export processes at the NPC cytoplasmic face, as we find evidence supporting Ran-dependent mRNA export in trypanosomes, similar to protein transport. We propose a model of stepwise acquisition of nucleocytoplasmic mechanistic complexity and demonstrate that detailed dissection of macromolecular complexes provides fuller understanding of evolutionary processes.
Asunto(s)
Evolución Molecular , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/genética , Microscopía Inmunoelectrónica , Poro Nuclear/química , Poro Nuclear/metabolismo , Estructura Cuaternaria de Proteína , Trypanosoma brucei bruceiRESUMEN
The nuclear pore complex (NPC) is the sole mediator of bidirectional nucleo-cytoplasmic transport and is also an important scaffold for chromatin organization and transcriptional regulation. Proteomic studies of numerous diverse eukaryotic species initially characterized the NPC as built with a number of remarkably similar structural features, suggesting its status as an ancient and conserved eukaryotic cell component. However, further detailed analyses now suggest that several key specific NPC features have a more convoluted evolutionary history than initially assumed. Recently we reported on TbNup92, a component in trypanosomes of one such conserved structural feature, a basket-like structure on the nuclear face of the NPC. We showed that TbNup92 has similar roles to nuclear basket proteins from yeasts and animals (Mlp and Tpr, respectively) in interacting with both the NPC and the mitotic spindle. However, comparative genomics suggests that TbNup92 and Mlp/Tpr may be products of distinct evolutionary histories, raising the possibility that these gene products are analogs rather than direct orthologs. Taken together with recent evidence for divergence in the nuclear lamina and kinetochores, it is apparent that the trypanosome nucleus functions by employing several novel or highly divergent protein complexes in parallel with conserved elements. These findings have major implications for how the trypanosomatid nucleus operates and the evolution of hierarchical nuclear organization.
Asunto(s)
Poro Nuclear/metabolismo , Animales , Núcleo Celular/metabolismo , Mitosis/fisiología , Membrana Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Protozoarias/metabolismo , Huso Acromático/metabolismo , Trypanosoma brucei brucei/metabolismoRESUMEN
The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle-dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina.
Asunto(s)
Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas Protozoarias/metabolismo , Transcripción Genética , Trypanosoma brucei brucei/metabolismo , Núcleo Celular/metabolismo , Segregación Cromosómica , Evolución Molecular , Puntos de Control de la Fase G2 del Ciclo Celular , Mitosis , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Filogenia , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/citologíaRESUMEN
Cilia and flagella are membrane-sheathed, microtubule-based protrusions that decorate the surface of many eukaryotic cells. At their base, they form a selective barrier that concentrates certain proteins within the cilia but excludes others. Kee et al. (2012) now propose that nuclear pore complex proteins form a fundamental part of this diffusion barrier.
RESUMEN
Trypanosomatids represent the causative agents of major diseases in humans, livestock and plants, with inevitable suffering and economic hardship as a result. They are also evolutionarily highly divergent organisms, and the many unique aspects of trypanosome biology provide opportunities in terms of identification of drug targets, the challenge of exploiting these putative targets and, at the same time, significant scope for exploration of novel and divergent cell biology. We can estimate from genome sequences that the degree of divergence of trypanosomes from animals and fungi is extreme, with perhaps one third to one half of predicted trypanosome proteins having no known function based on homology or recognizable protein domains/architecture. Two highly important aspects of trypanosome biology are the flagellar pocket and the nuclear envelope, where in silico analysis clearly suggests great potential divergence in the proteome. The flagellar pocket is the sole site of endo- and exocytosis in trypanosomes and plays important roles in immune evasion via variant surface glycoprotein (VSG) trafficking and providing a location for sequestration of various invariant receptors. The trypanosome nuclear envelope has been largely unexplored but, by analogy with higher eukaryotes, roles in the regulation of chromatin and most significantly, in controlling VSG gene expression are expected. Here we discuss recent successful proteomics-based approaches towards characterization of the nuclear envelope and the endocytic apparatus, the identification of conserved and novel trypanosomatid-specific features, and the implications of these findings.
Asunto(s)
Flagelos/metabolismo , Membrana Nuclear/metabolismo , Proteómica/métodos , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/fisiología , Tripanosomiasis Africana/parasitología , Animales , Evolución Molecular , Humanos , Transporte de Proteínas/genética , Proteoma , Proteínas Protozoarias/genética , Biología de Sistemas , Trypanosoma brucei brucei/genéticaRESUMEN
BACKGROUND: African trypanosomes belong to a eukaryotic lineage which displays many unusual genetic features. The mechanisms of chromosome segregation in these diploid protozoan parasites are poorly understood. Centromeres in Trypanosoma brucei have been localised to chromosomal regions that contain an array of ~147 bp AT-rich tandem repeats. Initial estimates from the genome sequencing project suggested that these arrays ranged from 2 - 8 kb. In this paper, we show that the centromeric repeat regions are much more extensive. RESULTS: We used a long-range restriction endonuclease mapping approach to more accurately define the sizes of the centromeric repeat arrays on the 8 T. brucei chromosomes where unambiguous assembly data were available. The results indicate that the sizes of the arrays on different chromosomes vary from 20 to 120 kb. In addition, we found instances of length heterogeneity between chromosome homologues. For example, values of 20 and 65 kb were obtained for the arrays on chromosome 1, and 50 and 75 kb for chromosome 5. CONCLUSIONS: Our results show that centromeric repeat arrays on T. brucei chromosomes are more similar in size to those of higher eukaryotes than previously suspected. This information provides a firmer framework for investigating aspects of chromosome segregation and will allow epigenetic features associated with the process to be more accurately mapped.
Asunto(s)
Centrómero/genética , Secuencias Repetidas en Tándem/genética , Trypanosoma brucei brucei/genética , Genoma de Protozoos , Análisis de Secuencia por Matrices de Oligonucleótidos , Mapeo RestrictivoRESUMEN
African trypanosomes are major pathogens of humans and livestock and represent a model for studies of unusual protozoal biology. We describe a high-throughput phenotyping approach termed RNA interference (RNAi) target sequencing, or RIT-seq that, using Illumina sequencing, maps fitness-costs associated with RNAi. We scored the abundance of >90,000 integrated RNAi targets recovered from trypanosome libraries before and after induction of RNAi. Data are presented for 7435 protein coding sequences, >99% of a non-redundant set in the Trypanosoma brucei genome. Analysis of bloodstream and insect life-cycle stages and differentiated libraries revealed genome-scale knockdown profiles of growth and development, linking thousands of previously uncharacterized and "hypothetical" genes to essential functions. Genes underlying prominent features of trypanosome biology are highlighted, including the constitutive emphasis on post-transcriptional gene expression control, the importance of flagellar motility and glycolysis in the bloodstream, and of carboxylic acid metabolism and phosphorylation during differentiation from the bloodstream to the insect stage. The current data set also provides much needed genetic validation to identify new drug targets. RIT-seq represents a versatile new tool for genome-scale functional analyses and for the exploitation of genome sequence data.