Divergent Eukaryotes Research Articles

The interaction of circRNAs and RNA binding proteins: An important part of circRNA maintenance and function.

The widespread expression of circular RNAs (circRNAs) is regarded as a feature of gene expression in highly diverged eukaryotes. Recent studies have shown that circRNAs can act as a miRNA sponge to repress miRNA function, participate in splicing of target genes, translate genes into protein and interact with RNA binding proteins (RBPs). RBPs are a broad class of proteins involved in gene transcription and translation, and interaction with RBPs is considered an important part of circRNA function, which can serve as an essential element underlying the functions of circRNAs, including genesis, translation, transcriptional regulation of target genes, and extracellular transport. In this mini-review, we attempt to explore in detail the relationship between circRNAs and RBPs, and the interactions between the two factors. The goal of this review is to investigate the emerging studies of RBPs and circRNAs to better understand how their interaction alters cellular function.

CDK Phosphorylation of Translation Initiation Factors Couples Protein Translation with Cell-Cycle Transition

SUMMARYProtein translation in eukaryotes is cell-cycle dependent, with translation rates more robust in G1 phase of the cell cycle than in mitosis. However, whether the fundamental cell-cycle control machinery directly activates protein translation during the G1/S cell-cycle transition remains unknown. Using the early divergent eukaryote Trypanosoma brucei as a model organism, we report that the G1 cyclin-dependent kinase CRK1 phosphorylates two translation initiation factors, eIF4E4 and PABP1, to promote the G1/S cell-cycle transition and global protein translation. Phosphorylation of eIF4E4 by CRK1 enhances binding to the m7G cap structure and interaction with eIF4E4 and eIF4G3, and phosphorylation of PABP1 by CRK1 promotes association with the poly(A) sequence, self-interaction, and interaction with eIF4E4. These findings demonstrate that cyclin-dependent kinase-mediated regulation of translation initiation factors couples global protein translation with the G1/S cell-cycle transition.

Selective human inhibitors of ATR and ATM render Leishmania major promastigotes sensitive to oxidative damage

All cellular processes, including those involved in normal cell metabolism to those responsible for cell proliferation or death, are finely controlled by cell signaling pathways, whose core proteins constitute the family of phosphatidylinositol 3-kinase-related kinases (PIKKs). Ataxia Telangiectasia Mutated (ATM) and Ataxia Telangiectasia and Rad3 related (ATR) are two important PIKK proteins that act in response to DNA damage, phosphorylating a large number of proteins to exert control over genomic integrity. The genus Leishmania belongs to a group of early divergent eukaryotes in evolution and has a highly plastic genome, probably owing to the existence of signaling pathways designed to maintain genomic integrity. The objective of this study was to evaluate the use of specific human inhibitors of ATR and ATM in Leishmania major. Bioinformatic analyses revealed the existence of the putative PIKK genes ATR and ATM, in addition to mTOR and DNA-PKcs in Leishmania spp. Moreover, it was possible to suggest that the inhibitors VE-821 and KU-55933 have binding affinity for the catalytic sites of putative L. major ATR and ATM, respectively. Promastigotes of L. major exposed to these inhibitors show slight growth impairment and minor changes in cell cycle and morphology. It is noteworthy that treatment of promastigotes with inhibitors VE-821 and KU-55933 enhanced the oxidative damage caused by hydrogen peroxide. These inhibitors could significantly reduce the number of surviving L. major cells following H2O2 exposure whilst also decreasing their evaluated IC50 to H2O2 to less than half of that observed for non-treated cells. These results suggest that the use of specific inhibitors of ATR and ATM in Leishmania interferes in the signaling pathways of this parasite, which can impair its tolerance to DNA damage and affect its genome integrity. ATR and ATM could constitute novel targets for drug development and/or repositioning for treatment of leishmaniases.

In silico analysis of the EF-hand proteins in the genome of Giardia intestinalis assembly A.

Giardia intestinalis is a parasite that inhabits the small intestine of humans and other mammals, causing a disease that can manifest itself with acute diarrhea. This parasite is an early divergent eukaryote with a compact genome and a life cycle composed of two distinct cell types: the trophozoite, the replicative form, and the cyst, the infectious form. Signal transduction pathways implicated in differentiation processes of G. intestinalis are largely unknown. Calcium, considered an essential messenger in cell signaling, has been shown to regulate a myriad of key cell processes including metabolism, motility, and exocytosis, among other important functions, through calcium-binding proteins (CaBPs). The most important and largest family of CaBPs is the EF-hand protein family. To investigate the nature of calcium signaling pathways present in this protozoan, an in silico analysis of the genome to identify genes encoding EF-hand proteins was undertaken. Twenty-eight sequences containing EF-hand domains were found; most of which have only a pair of domains, and half of the sequences were divergent or unique to Giardia. In addition, the transcription pattern for eight genes encoding EF-hand proteins was assessed during encystation. It was found that all the genes were differentially transcribed suggesting a different function in this process. The in silico results suggest that in G. intestinalis, calcium is involved in the regulation of protein phosphorylation through kinases and phosphatases.

DNA-damage inducible protein 1 is a conserved metacaspase substrate that is cleaved and further destabilized in yeast under specific metabolic conditions.

Metacaspases, distant relatives of metazoan caspases, have been shown to participate in programmed cell death in plants and in progression of the cell cycle and removal of protein aggregates in unicellular eukaryotes. However, since natural proteolytic substrates have scarcely been identified to date, their roles in these processes remain unclear. Here, we report that the DNA-damage inducible protein 1 (Ddi1) represents a conserved protein substrate for metacaspases belonging to divergent unicellular eukaryotes (trypanosomes and yeasts). We show that although the recognized cleavage sequence is not identical among the different model organisms tested, in all of them the proteolysis consequence is the removal of the ubiquitin-associated domain (UBA) present in the protein. We also demonstrate that Ddi1 cleavage is tightly regulated in vivo as it only takes place in yeast when calcium increases but under specific metabolic conditions. Finally, we show that metacaspase-mediated Ddi1 cleavage reduces the stability of this protein which can certainly impact on the many functions ascribed for it, including shuttle to the proteasome, cell cycle control, late secretory pathway regulation, among others.

Plasma Circular RNAs, Hsa_circRNA_025016, Predict Postoperative Atrial Fibrillation After Isolated Off‐Pump Coronary Artery Bypass Grafting

BackgroundCircular RNAs (circRNAs) are pervasively expressed in highly divergent eukaryotes and are stable in body fluids. However, the link between circRNAs and onset of atrial fibrillation (AF) has not previously been investigated. We aimed to identify plasma circRNAs that are able predict AF after cardiac surgery.Methods and ResultsPlasma circRNA expression profiles were investigated in a total of 769 patients with or without AF after isolated off‐pump coronary artery bypass grafting. First, a circRNA microarray was used to screen 13 617 human circRNAs in plasma samples from patients in the discovery cohort (n=30). A quantitative polymerase chain reaction assay was then applied to evaluate the expression of 9 selected circRNAs in the training cohort (n=365). This approach revealed that hsa_circRNA_025016 was upregulated in patients with new‐onset AF with a high diagnostic accuracy as assessed by the area under the receiver operating characteristic curve (=0.802). Additionally, a satisfactory diagnostic performance of hsa_circRNA_025016 was found in the validation cohort (n=284). Furthermore, Kyoto Encyclopedia of Genes and Genomes biological pathway analysis indicated that hsa_circ_025016 participated in melanogenesis, insulin secretion, and the thyroid hormone signaling pathway. A positive correlation between hsa_circ_025016 and fasting blood glucose was also identified in both cohorts.ConclusionsHsa_circ_025016 is a potential biomarker for predicting new‐onset AF after isolated off‐pump coronary artery bypass grafting.

Single-subunit oligosaccharyltransferases of Trypanosoma brucei display different and predictable peptide acceptor specificities

Trypanosoma brucei causes African trypanosomiasis and contains three full-length oligosaccharyltransferase (OST) genes; two of which, TbSTT3A and TbSTT3B, are expressed in the bloodstream form of the parasite. These OSTs have different peptide acceptor and lipid-linked oligosaccharide donor specificities, and trypanosomes do not follow many of the canonical rules developed for other eukaryotic N-glycosylation pathways, raising questions as to the basic architecture and detailed function of trypanosome OSTs. Here, we show by blue-native gel electrophoresis and stable isotope labeling in cell culture proteomics that the TbSTT3A and TbSTT3B proteins associate with each other in large complexes that contain no other detectable protein subunits. We probed the peptide acceptor specificities of the OSTs in vivo using a transgenic glycoprotein reporter system and performed glycoproteomics on endogenous parasite glycoproteins using sequential endoglycosidase H and peptide:N-glycosidase-F digestions. This allowed us to assess the relative occupancies of numerous N-glycosylation sites by endoglycosidase H-resistant N-glycans originating from Man5GlcNAc2-PP-dolichol transferred by TbSTT3A, and endoglycosidase H-sensitive N-glycans originating from Man9GlcNAc2-PP-dolichol transferred by TbSTT3B. Using machine learning, we assessed the features that best define TbSTT3A and TbSTT3B substrates in vivo and built an algorithm to predict the types of N-glycan most likely to predominate at all the putative N-glycosylation sites in the parasite proteome. Finally, molecular modeling was used to suggest why TbSTT3A has a distinct preference for sequons containing and/or flanked by acidic amino acid residues. Together, these studies provide insights into how a highly divergent eukaryote has re-wired protein N-glycosylation to provide protein sequence-specific N-glycan modifications. Data are available via ProteomeXchange with identifiers PXD007236, PXD007267, and PXD007268.

Sub-minute Phosphoregulation of Cell Cycle Systems during Plasmodium Gamete Formation

SummaryThe transmission of malaria parasites to mosquitoes relies on the rapid induction of sexual reproduction upon their ingestion into a blood meal. Haploid female and male gametocytes become activated and emerge from their host cells, and the males enter the cell cycle to produce eight microgametes. The synchronized nature of gametogenesis allowed us to investigate phosphorylation signaling during its first minute in Plasmodium berghei via a high-resolution time course of the phosphoproteome. This revealed an unexpectedly broad response, with proteins related to distinct cell cycle events undergoing simultaneous phosphoregulation. We implicate several protein kinases in the process, and we validate our analyses on the plant-like calcium-dependent protein kinase 4 (CDPK4) and a homolog of serine/arginine-rich protein kinases (SRPK1). Mutants in these kinases displayed distinct phosphoproteomic disruptions, consistent with differences in their phenotypes. The results reveal the central role of protein phosphorylation in the atypical cell cycle regulation of a divergent eukaryote.

Genome-wide and protein kinase-focused RNAi screens reveal conserved and novel damage response pathways in Trypanosoma brucei.

All cells are subject to structural damage that must be addressed for continued growth. A wide range of damage affects the genome, meaning multiple pathways have evolved to repair or bypass the resulting DNA lesions. Though many repair pathways are conserved, their presence or function can reflect the life style of individual organisms. To identify genome maintenance pathways in a divergent eukaryote and important parasite, Trypanosoma brucei, we performed RNAi screens to identify genes important for survival following exposure to the alkylating agent methyl methanesulphonate. Amongst a cohort of broadly conserved and, therefore, early evolved repair pathways, we reveal multiple activities not so far examined functionally in T. brucei, including DNA polymerases, DNA helicases and chromatin factors. In addition, the screens reveal Trypanosoma- or kinetoplastid-specific repair-associated activities. We also provide focused analyses of repair-associated protein kinases and show that loss of at least nine, and potentially as many as 30 protein kinases, including a nuclear aurora kinase, sensitises T. brucei to alkylation damage. Our results demonstrate the potential for synthetic lethal genome-wide screening of gene function in T. brucei and provide an evolutionary perspective on the repair pathways that underpin effective responses to damage, with particular relevance for related kinetoplastid pathogens. By revealing that a large number of diverse T. brucei protein kinases act in the response to damage, we expand the range of eukaryotic signalling factors implicated in genome maintenance activities.

Experimental and bioinformatic characterization of CaBP2933 an EF-Hand protein of Giardia intestinalis

Giardia intestinalis is a parasite that inhabits the small intestine of humans. This parasite is a divergent eukaryote with a compact genome. The calcium ion is an essential messenger in cell signaling. Calcium’s role as a messenger is mediated through calcium-binding proteins (CaBPs) that decode the message. The most important family of CaBPs is the EF-Hand protein family. In this study we have explored the role of EF-Hand protein CaBP2933. We analyzed its location, confirmed its ability to bind calcium and identified some of its interacting proteins. Take together our results suggest that CaBP2933 is involved in vesicular trafficking during encystation, via an interaction with kinesin-3 motor protein.

Proximity Interactions among Basal Body Components in Trypanosoma brucei Identify Novel Regulators of Basal Body Biogenesis and Inheritance.

ABSTRACTThe basal body shares similar architecture with centrioles in animals and is involved in nucleating flagellar axonemal microtubules in flagellated eukaryotes. The early-branching Trypanosoma brucei possesses a motile flagellum nucleated from the basal body that consists of a mature basal body and an adjacent pro-basal body. Little is known about the basal body proteome and its roles in basal body biogenesis and flagellar axoneme assembly in T. brucei. Here, we report the identification of 14 conserved centriole/basal body protein homologs and 25 trypanosome-specific basal body proteins. These proteins localize to distinct subdomains of the basal body, and several of them form a ring-like structure surrounding the basal body barrel. Functional characterization of representative basal body proteins revealed distinct roles in basal body duplication/separation and flagellar axoneme assembly. Overall, this work identified novel proteins required for basal body duplication and separation and uncovered new functions of conserved basal body proteins in basal body duplication and separation, highlighting an unusual mechanism of basal body biogenesis and inheritance in this early divergent eukaryote.

Detecting circular RNAs: bioinformatic and experimental challenges.

The pervasive expression of circular RNAs (circRNAs) is a recently discovered feature of gene expression in highly diverged eukaryotes. Numerous algorithms that are used to detect genome-wide circRNA expression from RNA sequencing (RNA-seq) data have been developed in the past few years, but there is little overlap in their predictions and no clear gold-standard method to assess the accuracy of these algorithms. We review sources of experimental and bioinformatic biases that complicate the accurate discovery of circRNAs and discuss statistical approaches to address these biases. We conclude with a discussion of the current experimental progress on the topic.

Phylogenetic Resolution of Deep Eukaryotic and Fungal Relationships Using Highly Conserved Low-Copy Nuclear Genes.

A comprehensive and reliable eukaryotic tree of life is important for many aspects of biological studies from comparative developmental and physiological analyses to translational medicine and agriculture. Both gene-rich and taxon-rich approaches are effective strategies to improve phylogenetic accuracy and are greatly facilitated by marker genes that are universally distributed, well conserved, and orthologous among divergent eukaryotes. In this article, we report the identification of 943 low-copy eukaryotic genes and we show that many of these genes are promising tools in resolving eukaryotic phylogenies, despite the challenges of determining deep eukaryotic relationships. As a case study, we demonstrate that smaller subsets of ∼20 and 52 genes could resolve controversial relationships among widely divergent taxa and provide strong support for deep relationships such as the monophyly and branching order of several eukaryotic supergroups. In addition, the use of these genes resulted in fungal phylogenies that are congruent with previous phylogenomic studies that used much larger datasets, and successfully resolved several difficult relationships (e.g., forming a highly supported clade with Microsporidia, Mitosporidium and Rozella sister to other fungi). We propose that these genes are excellent for both gene-rich and taxon-rich analyses and can be applied at multiple taxonomic levels and facilitate a more complete understanding of the eukaryotic tree of life.

Repurposing of conserved autophagy-related protein ATG8 in a divergent eukaryote

ABSTRACTToxoplasma gondii and other apicomplexan parasites contain a peculiar non-photosynthetic plastid called the apicoplast, which is essential for their survival. The localization of autophagy-related protein ATG8 to the apicoplast in several apicomplexan species and life stages has recently been described, and we have shown this protein is essential for proper inheritance of this complex plastid into daughter cells during cell division. Although the mechanism behind ATG8 association to the apicoplast in T. gondii is related to the canonical conjugation system leading to autophagosome formation, its singular role seems independent from the initial catabolic purpose of autophagy. Here we also discuss further the functional evolution and innovative adaptations of the autophagy machinery to maintain this organelle during parasite division.

Interactome Mapping Reveals the Evolutionary History of the Nuclear Pore Complex.

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.

Formation of interference-sensitive meiotic cross-overs requires sufficient DNA leading-strand elongation

Meiosis halves diploid genomes to haploid and is essential for sexual reproduction in eukaryotes. Meiotic recombination ensures physical association of homologs and their subsequent accurate segregation and results in the redistribution of genetic variations among progeny. Most organisms have two classes of cross-overs (COs): interference-sensitive (type I) and -insensitive (type II) COs. DNA synthesis is essential for meiotic recombination, but whether DNA synthesis has a role in differentiating meiotic CO pathways is unknown. Here, we show that Arabidopsis POL2A, the homolog of the yeast DNA polymerase-ε (a leading-strand DNA polymerase), is required for plant fertility and meiosis. Mutations in POL2A cause reduced fertility and meiotic defects, including abnormal chromosome association, improper chromosome segregation, and fragmentation. Observation of prophase I cell distribution suggests that pol2a mutants likely delay progression of meiotic recombination. In addition, the residual COs in pol2a have reduced CO interference, and the double mutant of pol2a with mus81, which affects type II COs, displayed more severe defects than either single mutant, indicating that POL2A functions in the type I pathway. We hypothesize that sufficient leading-strand DNA elongation promotes formation of some type I COs. Given that meiotic recombination and DNA synthesis are conserved in divergent eukaryotes, this study and our previous study suggest a novel role for DNA synthesis in the differentiation of meiotic recombination pathways.

Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development.

BackgroundThe pervasive expression of circular RNA is a recently discovered feature of gene expression in highly diverged eukaryotes, but the functions of most circular RNAs are still unknown. Computational methods to discover and quantify circular RNA are essential. Moreover, discovering biological contexts where circular RNAs are regulated will shed light on potential functional roles they may play.ResultsWe present a new algorithm that increases the sensitivity and specificity of circular RNA detection by discovering and quantifying circular and linear RNA splicing events at both annotated and un-annotated exon boundaries, including intergenic regions of the genome, with high statistical confidence. Unlike approaches that rely on read count and exon homology to determine confidence in prediction of circular RNA expression, our algorithm uses a statistical approach. Using our algorithm, we unveiled striking induction of general and tissue-specific circular RNAs, including in the heart and lung, during human fetal development. We discover regions of the human fetal brain, such as the frontal cortex, with marked enrichment for genes where circular RNA isoforms are dominant.ConclusionsThe vast majority of circular RNA production occurs at major spliceosome splice sites; however, we find the first examples of developmentally induced circular RNAs processed by the minor spliceosome, and an enriched propensity of minor spliceosome donors to splice into circular RNA at un-annotated, rather than annotated, exons. Together, these results suggest a potentially significant role for circular RNA in human development.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0690-5) contains supplementary material, which is available to authorized users.

Assembling the components of the quorum sensing pathway in African trypanosomes.

African trypanosomes, parasites that cause human sleeping sickness, undergo a density-dependent differentiation in the bloodstream of their mammalian hosts. This process is driven by a released parasite-derived factor that causes parasites to accumulate in G1 and become quiescent. This is accompanied by morphological transformation to ‘stumpy’ forms that are adapted to survival and further development when taken up in the blood meal of tsetse flies, the vector for trypanosomiasis. Although the soluble signal driving differentiation to stumpy forms is unidentified, a recent genome-wide RNAi screen identified many of the intracellular signalling and effector molecules required for the response to this signal. These resemble components of nutritional starvation and quiescence pathways in other eukaryotes, suggesting that parasite development shares similarities with the adaptive quiescence of organisms such as yeasts and Dictyostelium in response to nutritional starvation and stress. Here, the trypanosome signalling pathway is discussed in the context of these conserved pathways and the possible contributions of opposing ‘slender retainer’ and ‘stumpy inducer’ arms described. As evolutionarily highly divergent eukaryotes, the organisation and conservation of this developmental pathway can provide insight into the developmental cycle of other protozoan parasites, as well as the adaptive and programmed developmental responses of all eukaryotic cells.

Mad2, Bub3, and Mps1 regulate chromosome segregation and mitotic synchrony in Giardia intestinalis, a binucleate protist lacking an anaphase-promoting complex.

The binucleate pathogen Giardia intestinalis is a highly divergent eukaryote with a semiopen mitosis, lacking an anaphase-promoting complex/cyclosome (APC/C) and many of the mitotic checkpoint complex (MCC) proteins. However, Giardia has some MCC components (Bub3, Mad2, and Mps1) and proteins from the cohesin system (Smc1 and Smc3). Mad2 localizes to the cytoplasm, but Bub3 and Mps1 are either located on chromosomes or in the cytoplasm, depending on the cell cycle stage. Depletion of Bub3, Mad2, or Mps1 resulted in a lowered mitotic index, errors in chromosome segregation (including lagging chromosomes), and abnormalities in spindle morphology. During interphase, MCC knockdown cells have an abnormal number of nuclei, either one nucleus usually on the left-hand side of the cell or two nuclei with one mislocalized. These results suggest that the minimal set of MCC proteins in Giardia play a major role in regulating many aspects of mitosis, including chromosome segregation, coordination of mitosis between the two nuclei, and subsequent nuclear positioning. The critical importance of MCC proteins in an organism that lacks their canonical target, the APC/C, suggests a broader role for these proteins and hints at new pathways to be discovered.

Mikrocytids Are a Broadly Distributed and Divergent Radiation of Parasites in Aquatic Invertebrates

Microcell parasites have independently evolved in several eukaryotic lineages and are increasingly recognized as important and emerging pathogens of diverse hosts, including species of economic importance subject to international legislation concerning the trading of aquatic animals [1-3]. The microcell Mikrocytos mackini causes Denman Island disease of oysters and represents one of the most genetically divergent eukaryotes known. Mikrocytos has remained an isolated lineage with a limited distribution. We investigated two emerging diseases of juvenile crabs and oysters from the UK using massively parallel sequencing and targeted primer approaches to reveal that their causative agents are highly divergent lineages related to M. mackini (Paramikrocytos canceri n. gen. et n. sp. and M. mimicus sp. nov., respectively). We demonstrate a major new globally distributed parasite radiation (Mikrocytida ord. nov.) with phylogenetic affinities to the commercially important haplosporidian parasites of invertebrates. Mikrocytids have eluded detection because of their small size, intracellular habit, and extreme sequence divergence. P. canceri was frequently detected in a range of shoreline invertebrates, demonstrating that these newly recognized parasites are in fact common, diverse, and widespread and should be considered when assessing the risks of aquaculture activities, invasive species spread, and movements of ballast water and sediments with associated invertebrates.