Eukaryotic Ribosomal RNA Research Articles

Hmo1 Promotes Efficient Transcription Elongation by RNA Polymerase I in Saccharomyces cerevisiae.

RNA polymerase I (Pol I) is responsible for synthesizing the three largest eukaryotic ribosomal RNAs (rRNAs), which form the backbone of the ribosome. Transcription by Pol I is required for cell growth and, therefore, is subject to complex and intricate regulatory mechanisms. To accomplish this robust regulation, the cell engages a series of trans-acting transcription factors. One such factor, high mobility group protein 1 (Hmo1), has long been established as a trans-acting factor for Pol I in Saccharomyces cerevisiae; however, the mechanism by which Hmo1 promotes rRNA synthesis has not been defined. Here, we investigated the effect of the deletion of HMO1 on transcription elongation by Pol I in vivo. We determined that Hmo1 is an important activator of transcription elongation, and without this protein, Pol I accumulates across rDNA in a sequence-specific manner. Our results demonstrate that Hmo1 promotes efficient transcription elongation by rendering Pol I less sensitive to pausing in the G-rich regions of rDNA.

Promise and limitations of 18S genetic screening of extracted fecal DNA from wild capuchins

Genomic screening of fecal DNA provides insight into diet, parasite infection dynamics, and other aspects of the ecology and pathogens of wild populations. Here, we amplify and sequence the V4/V5 regions of the eukaryotic 18S ribosomal RNA gene from fecal DNA of wild capuchin monkeys (Cebus imitator). We collected 94 fecal samples from 26 individuals, each sampled 1-4 times across a 19-month period and examined the eukaryotic diversity in 63 of these samples which had sufficient numbers and quality of reads during downstream analyses. We found a total of 234 distinct amplicon sequence variants (ASVs) classified as Eukaryotes in our samples. Of these, 66 were assigned to the phylum Nematoda. 64 ASVs are from taxa that possibly parasitize monkeys or their food items: 33 were assigned to lungworms (Superfamily Metastrongyloidae; genus Angiostrongylus), two to the genus Strongyloides, and one to the genus Austrostrongylus. The remaining 28 ASVs were assigned to nematodes that likely parasitize plants and/or invertebrates that the monkeys consume. Taken together with past dietary and coprological study of the same primate population, our results suggest that invertebrate consumption and parasitic infection, especially by lungworms, is common and widespread among this population of wild monkeys. We also discuss limitations of our approach, including the amplification of off-target ASVs, and make suggestions for future research. Overall, 18S screening shows promise for identifying various components of the capuchin gastrointestinal eukaryotic ecosystem, including parasitic helminths, and its utility will increase with the improvement of genetic databases.

Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution.

NAT10 is an essential enzyme that catalyzes N4-acetylcytidine (ac4C) in eukaryotic transfer RNA and 18S ribosomal RNA. Recent studies suggested that rRNA acetylation is dependent on SNORD13, a box C/D small nucleolar RNA predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10’s essential function remain to be defined. Here, we demonstrate that SNORD13 is required for acetylation of a single cytidine of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for human cell growth, ribosome biogenesis, translation and development. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation ‘machinery’ that led to the characterization of many novel metazoan SNORD13 genes. This includes an atypical SNORD13-like RNA in Drosophila melanogaster which guides ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that Caenorhabditis elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across eukaryotic evolution and raise new questions regarding the biological and evolutionary relevance of this highly conserved rRNA modification.

Isomorphic Fluorescent Nucleosides Facilitate Real-Time Monitoring of RNA Depurination by Ribosome Inactivating Proteins.

Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with tz G and th G, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP-mediated depurination can be monitored in real time by steady-state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs' substrate recognition features.

METTL5 regulates cranial suture fusion via Wnt signaling

METTL5 is a methyltransferase that mediates eukaryotic 18S ribosomal RNA m6A modification, and its mutations lead to intellectual disability, microcephaly, and facial dysmorphism in patients. However, the role of METTL5 in craniofacial development remains poorly understood. This study demonstrates that Mettl5 knockout mice exhibit poor ossification, widened cranial sutures, and a cleidocranial dysplasia-like phenotype. Deletion of Mettl5 leads to increased proliferation and decreased osteogenic differentiation of suture mesenchymal stem cells. Mechanistically, we find that Wnt signaling is significantly downregulated after Mettl5 knockout. Overall, we reveal an essential role of METTL5 in craniofacial development and osteogenic differentiation of suture mesenchymal stem cells, making METTL5 a potential diagnostic and therapeutic target for craniofacial developmental diseases.

Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling.

Nucleotides in RNA and DNA are chemically modified by numerous enzymes that alter their function. Eukaryotic ribosomal RNA (rRNA) is modified at more than 100 locations, particularly at highly conserved and functionally important nucleotides. During ribosome biogenesis, modifications are added at various stages of assembly. The existence of differently modified classes of ribosomes in normal cells is unknown because no method exists to simultaneously evaluate the modification status at all sites within a single rRNA molecule. Using a combination of yeast genetics and nanopore direct RNA sequencing, we developed a reliable method to track the modification status of single rRNA molecules at 37 sites in 18 S rRNA and 73 sites in 25 S rRNA. We use our method to characterize patterns of modification heterogeneity and identify concerted modification of nucleotides found near functional centers of the ribosome. Distinct, undermodified subpopulations of rRNAs accumulate upon loss of Dbp3 or Prp43 RNA helicases, suggesting overlapping roles in ribosome biogenesis. Modification profiles are surprisingly resistant to change in response to many genetic and acute environmental conditions that affect translation, ribosome biogenesis, and pre-mRNA splicing. The ability to capture single-molecule RNA modification profiles provides new insights into the roles of nucleotide modifications in RNA function.

Regulation of antiviral innate immunity by chemical modification of viral RNA.

More than 100 chemical modifications of RNA, termed the epitranscriptome, have been described, most of which occur in prokaryotic and eukaryotic ribosomal, transfer, and noncoding RNA and eukaryotic messenger RNA. DNA and RNA viruses can modify their RNA either directly via genome-encoded enzymes or by hijacking the host enzymatic machinery. Among the many RNA modifications described to date, four play particularly important roles in promoting viral infection by facilitating viral gene expression and replication and by enabling escape from the host innate immune response. Here, we discuss our current understanding of the mechanisms by which the RNA modifications such as N6 -methyladenosine (m6A), N6 ,2'-O-dimethyladenosine (m6Am), 5-methylcytidine (m5C), N4-acetylcytidine (ac4C), and 2'-O-methylation (Nm) promote viral replication and/or suppress recognition by innate sensors and downstream activation of the host antiviral response. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution.

DNA Intercalators Inhibit Eukaryotic Ribosomal RNA Synthesis by Impairing the Initiation of Transcription.

In eukaryotes, ribosome biogenesis is driven by the synthesis of the ribosomal RNA (rRNA) by RNA polymerase I (Pol-I) and is tightly linked to cell growth and proliferation. The 3D-structure of the rDNA promoter plays an important, yet not fully understood role in regulating rRNA synthesis. We hypothesized that DNA intercalators/groove binders could affect this structure and disrupt rRNA transcription. To test this hypothesis, we investigated the effect of a number of compounds on Pol-I transcription in vitro and in cells. We find that intercalators/groove binders are potent inhibitors of Pol-I specific transcription both in vitro and in cells, regardless of their specificity and the strength of its interaction with DNA. Importantly, the synthetic ability of Pol-I is unaffected, suggesting that these compounds are not targeting post-initiating events. Notably, the tested compounds have limited effect on transcription by Pol-II and III, demonstrating the hypersensitivity of Pol-I transcription. We propose that stability of pre-initiation complex and initiation are affected as result of altered 3D architecture of the rDNA promoter, which is well in line with the recently reported importance of biophysical rDNA promoter properties on initiation complex formation in the yeast system.

Metabarcoding of zooplankton diversity within the Chukchi Borderland, Arctic Ocean: improved resolution from multi-gene markers and region-specific DNA databases

DNA-based analyses have become powerful tools for characterizing the metazoan biodiversity of diverse marine ecosystems. Metabarcoding (i.e., large-scale taxonomic identification of complex samples via high-throughput sequencing of a DNA barcode region) frequently uses hypervariable regions of the nuclear eukaryotic 18S ribosomal RNA (rRNA) gene. However, species-level taxonomic identification is hampered by the conservative nature of the 18S gene in comparison to the mitochondrial cytochrome oxidase I (COI) barcode gene. Additionally, metabarcoding relies on reference DNA sequence databases for classification of millions of unknown sequence reads and molecular operational taxonomic units (OTUs); databases that are at present depauperate for marine zooplankton taxa. Here, we characterized the mesozooplankton community for the Chukchi Borderland (CBL) region, western Arctic Ocean, through metabarcoding analysis of the V4 and V9 hypervariable regions of 18S rRNA and a portion of COI. Characterization of zooplankton diversity for the epipelagic and upper mesopelagic layers (0–500 m) was based upon 17 metazoan taxonomic categories encompassing 24 orders in 14 classes. Taxonomic classification using the V4 and V9 markers was most reliable for orders, with copepods dominating OTU counts. To increase taxonomic resolution and allow detection of species, V4, V9, and COI OTUs were classified against DNA sequence databases for the Arctic Ocean for the subclass Copepoda. The geographic region-specific databases for 18S rRNA and COI resulted in the detection and identification of 6 genera and 49 species of copepods representing 23 families, a marked increase in the taxonomic classification of the 18S rRNA markers. The greatest copepod species diversity was captured with V4 (34 species) followed by COI (28 species) with the least copepod diversity detected by V9 (5 species). Our results demonstrate the power of using multiple gene markers, with DNA reference databases that are specific to the geographic region of interest, providing more accurate metabarcoding biodiversity measures for the copepod assemblages compared to universal metazoan sequence reference databases. Results from this study highlight the need for continued DNA barcode sequencing to increase species representation in the reference sequence databases that are crucial for accurate characterization of mesozooplankton communities.

Interaction Networks of Ribosomal Expansion Segments in Kinetoplastids.

Expansion segments (ES) are insertions of a few to hundreds of nucleotides at discrete locations on eukaryotic ribosomal RNA (rRNA) chains. Some cluster around 'hot spots' involved in translation regulation and some may participate in biogenesis. Whether ES play the same roles in different organisms is currently unclear, especially since their size may vary dramatically from one species to another and very little is known about their functions. Most likely, ES variation is linked to adaptation to a particular environment. In this chapter, we compare the interaction networks of ES from four kinetoplastid parasites, which have evolved in diverse insect vectors and mammalian hosts: Trypanosoma cruzi, Trypanosoma brucei, Leishmania donovani and Leishmania major. Here, we comparatively analyze ribosome structures from these representative kinetoplastids and ascertain meaningful structural differences from mammalian ribosomes. We base our analysis on sequence alignments and three-dimensional structures of 80S ribosomes solved by cryo-electron microscopy (cryo-EM). Striking differences in size are observed between ribosomes of different parasites, indicating that not all ES are expanded equally. Larger ES are not always matched by large surrounding ES or proteins extensions in their vicinity, a particularity that may lead to clues about their biological function. ES display different species-specific patterns of conservation, which underscore the density of their interaction network at the surface of the ribosome. Making sense of the conservation patterns of ES is part of a global effort to lay the basis for functional studies aimed at discovering unique kinetoplastid-specific sites suitable for therapeutic applications against these human and often animal pathogens.

Catheter tips are a possible resource for biological study oncatheter failure.

Few studies have investigated the molecular mechanisms of catheter failure (CF). Herein, we performed histological and molecular biological analyses of the catheter tip to demonstrate its potential as a resource for biological investigation. Additionally, we searched for risk factors for the development of inflammation and coagulation, which are pathological conditions clarified by biological analysis. The CF group included 30 failed catheters involving thrombus and subcutaneous edema identified by ultrasonography. The No-CF group included 26 catheters with no complications. The removed catheter tips were fixed for hematoxylin-eosin (HE) staining with the application of a real-time reverse transcriptase polymerase chain reaction for eukaryotic 18S ribosomal RNA (rRNA), interleukin 1β, tumor necrosis factor α, tissue plasminogen activator, and plasminogen activator inhibitor 1 (SERPINE1). HE staining identified attached nuclear cells on the inner surfaces of both CF and No-CF catheters. The 18S rRNA was amplified in all samples. The expression level of SERPINE1 was significantly higher in the CF group than in the No-CF group (p = 0.01), whereas the expression levels of other genes did not differ between the groups. Symptoms of CF associated with the expression of SERPINE1 were analyzed. The catheter being in contact with blood vessels during placement was a suggested factor related to the high expression of SERPINE1 (p = 0.04). Catheter tips are a potential resource for biological investigation, and expression analysis of the attached cells can reflect the pathological condition of the catheterized tissue. Further studies using catheter tips are required to elucidate the molecular mechanisms of CF.

Transcriptional Mutagenesis Prevents Ribosomal DNA Deterioration: The Role of Duplications and Deletions.

Clashes between transcription and replication complexes can cause point mutations and chromosome rearrangements on heavily transcribed genes. In eukaryotic ribosomal RNA genes, the system that prevents transcription–replication conflicts also causes frequent copy number variation. Such fast mutational dynamics do not alter growth rates in yeast and are thus selectively near neutral. It was recently found that yeast regulates these mutations by means of a signaling cascade that depends on the availability of nutrients.Here, we investigate the long-term evolutionary effect of the mutational dynamics observed in yeast. We developed an in silico model of single-cell organisms whose genomes mutate more frequently when transcriptional load is larger. We show that mutations induced by high transcriptional load are beneficial when biased toward gene duplications and deletions: they decrease mutational load even though they increase the overall mutation rates. In contrast, genome stability is compromised when mutations are not biased toward gene duplications and deletions, even when mutations occur much less frequently.Taken together, our results show that the mutational dynamics observed in yeast are beneficial for the long-term stability of the genome and pave the way for a theory of evolution where genetic operators are themselves cause and outcome of the evolutionary dynamics.

Small Non-Coding RNAs Derived From Eukaryotic Ribosomal RNA.

The advent of RNA-sequencing (RNA-Seq) technologies has markedly improved our knowledge and expanded the compendium of small non-coding RNAs, most of which derive from the processing of longer RNA precursors. In this review article, we will present a nonexhaustive list of referenced small non-coding RNAs (ncRNAs) derived from eukaryotic ribosomal RNA (rRNA), called rRNA fragments (rRFs). We will focus on the rRFs that are experimentally verified, and discuss their origin, length, structure, biogenesis, association with known regulatory proteins, and potential role(s) as regulator of gene expression. This relatively new class of ncRNAs remained poorly investigated and underappreciated until recently, due mainly to the a priori exclusion of rRNA sequences—because of their overabundance—from RNA-Seq datasets. The situation surrounding rRFs resembles that of microRNAs (miRNAs), which used to be readily discarded from further analyses, for more than five decades, because no one could believe that RNA of such a short length could bear biological significance. As if we had not yet learned our lesson not to restrain our investigative, scientific mind from challenging widely accepted beliefs or dogmas, and from looking for the hidden treasures in the most unexpected places.

Landscape of the complete RNA chemical modifications in the human 80S ribosome.

During ribosome biogenesis, ribosomal RNAs acquire various chemical modifications that ensure the fidelity of translation, and dysregulation of the modification processes can cause proteome changes as observed in cancer and inherited human disorders. Here, we report the complete chemical modifications of all RNAs of the human 80S ribosome as determined with quantitative mass spectrometry. We assigned 228 sites with 14 different post-transcriptional modifications, most of which are located in functional regions of the ribosome. All modifications detected are typical of eukaryotic ribosomal RNAs, and no human-specific modifications were observed, in contrast to a recently reported cryo-electron microscopy analysis. While human ribosomal RNAs appeared to have little polymorphism regarding the post-transcriptional modifications, we found that pseudouridylation at two specific sites in 28S ribosomal RNA are significantly reduced in ribosomes of patients with familial dyskeratosis congenita, a genetic disease caused by a point mutation in the pseudouridine synthase gene DKC1. The landscape of the entire epitranscriptomic ribosomal RNA modifications provides a firm basis for understanding ribosome function and dysfunction associated with human disease.

The internal transcribed spacer 2 of Jenufa (Chlorophyta, Chlorophyceae) is extraordinarily long: A hypothesis

Aided by a host of bioinformatics tools, primary and secondary structural analyses of the internal transcribed spacer 2 (ITS2) from the eukaryotic ribosomal RNA repeat have a long and enviable record of service to diversity studies of fungi, plants and protists. Automation of annotation, secondary structure estimation and sequence alignment have become routine for the vast majority of ITS2 sequences. Challenges to the bioinformatics pipeline for ITS2 analysis generally arise in cases where the sequence length lies well outside the norm. These sequences generally defy protocols for annotation and secondary structure prediction. The long ITS2 sequences (ca. 600 nucleotides) from the green alga, Jenufa, offered an opportunity to explore this problem. Custom BLAST parameters revealed the presence of 4-helix structures (200–250 nucleotides) embedded in the 5′ portion of several long ITS2 sequences of Jenufa. Of special note is the ITS2 sequence of J. lobulosa where a 4-helix structure was obtained for both the embedded ITS2 and for the complete sequence. Phylogenetic analysis of these typically-sized sequences resolved Golenkinia longispicula as the sister to Jenufa. Our observations indicate that other long ITS2 sequences should be examined for evidence of expansion or duplication. In addition, if the embedded ITS2 sequences are functional, then ribogenesis is almost certainly more diverse than is already apparent from studies of humans and yeast.

ITSoneDB: a comprehensive collection of eukaryotic ribosomal RNA Internal Transcribed Spacer 1 (ITS1) sequences.

A holistic understanding of environmental communities is the new challenge of metagenomics. Accordingly, the amplicon-based or metabarcoding approach, largely applied to investigate bacterial microbiomes, is moving to the eukaryotic world too. Indeed, the analysis of metabarcoding data may provide a comprehensive assessment of both bacterial and eukaryotic composition in a variety of environments, including human body. In this respect, whereas hypervariable regions of the 16S rRNA are the de facto standard barcode for bacteria, the Internal Transcribed Spacer 1 (ITS1) of ribosomal RNA gene cluster has shown a high potential in discriminating eukaryotes at deep taxonomic levels. As metabarcoding data analysis rely on the availability of a well-curated barcode reference resource, a comprehensive collection of ITS1 sequences supplied with robust taxonomies, is highly needed. To address this issue, we created ITSoneDB (available at http://itsonedb.cloud.ba.infn.it/) which in its current version hosts 985 240 ITS1 sequences spanning over 134 000 eukaryotic species. Each ITS1 is mapped on the NCBI reference taxonomy with its start and end positions precisely annotated. ITSoneDB has been developed in agreement to the FAIR guidelines by enabling the users to query and download its content through a simple web-interface and access relevant metadata by cross-linking to European Nucleotide Archive.

Synthesis, Function, and Heterogeneity of snoRNA-Guided Posttranscriptional Nucleoside Modifications in Eukaryotic Ribosomal RNAs.

Ribosomal RNAs contain numerous 2'-O-methylated nucleosides and pseudouridines. Methylation of the 2' oxygen of ribose moieties and isomerization of uridines into pseudouridines are catalyzed by C/D and H/ACA small nucleolar ribonucleoprotein particles, respectively. We review the composition, structure, and mode of action of archaeal and eukaryotic C/D and H/ACA particles. Most rRNA modifications cluster in functionally crucial regions of the rRNAs, suggesting they play important roles in translation. Some of these modifications promote global translation efficiency or modulate translation fidelity. Strikingly, recent quantitative nucleoside modification profiling methods have revealed that a subset of modification sites is not always fully modified. The finding of such ribosome heterogeneity is in line with the concept of specialized ribosomes that could preferentially translate specific mRNAs. This emerging concept is supported by findings that some human diseases are caused by defects in the rRNA modification machinery correlated with a significant alteration of IRES-dependent translation.

Mineral Type Structures Soil Microbial Communities

ABSTRACTSoil microorganisms living in close contact with minerals play key roles in the biogeochemical cycling of elements, soil formation, and plant nutrition. Yet, the composition of microbial communities inhabiting the mineralosphere (i.e., the soil surrounding minerals) is poorly understood. Here, we explored the composition of soil microbial communities associated with different types of minerals in various soil horizons. To this effect, a field experiment was set up in which mineral specimens of apatite, biotite, and oligoclase were buried in the organic, eluvial, and upper illuvial horizons of a podzol soil. After an incubation period of two years, the soil attached to the mineral surfaces was collected, and microbial communities were analyzed by means of Illumina MiSeq sequencing of the 16S (prokaryotic) and 18S (eukaryotic) ribosomal RNA genes. We found that both composition and diversity of bacterial, archaeal, and fungal communities varied across the different mineral surfaces, and that mineral type had a greater influence on structuring microbial assemblages than soil horizon. Thus, our findings emphasize the importance of mineral surfaces as ecological niches in soils.

Yeast rRNA Expansion Segments: Folding and Function

Divergence between prokaryotic and eukaryotic ribosomal RNA (rRNA) and among eukaryotic ribosomal RNAs is focused in expansion segments (ESs). Eukaryotic ribosomes are significantly larger than prokaryotic ribosomes partly because of their ESs. We hypothesize that larger rRNAs of complex organisms could confer increased functionality to the ribosome. Here, we characterize the binding partners of Saccharomyces cerevisiae expansion segment 7 (ES7), which is the largest and most variable ES of the eukaryotic large ribosomal subunit and is located at the surface of the ribosome. In vitro RNA‐protein pull-down experiments using ES7 as a bait indicate that ES7 is a binding hub for a variety of non-ribosomal proteins essential to ribosomal function in eukaryotes. ES7-associated proteins observed here cluster into four groups based on biological process, (i) response to abiotic stimulus (e.g., response to external changes in temperature, pH, oxygen level, etc.), (ii) ribosomal large subunit biogenesis, (iii) protein transport and localization, and (iv) transcription elongation. Seven synthetases, Ala-, Arg-, Asp-, Asn-, Leu-, Lys- and TyrRS, appear to associate with ES7. Affinities of AspRS, TyrRS and LysRS for ES7 were confirmed by in vitro binding assays. The results suggest that ES7 in S. cerevisiae could play a role analogous to the multi-synthetase complex present in higher order organisms and could be important for the appropriate function of the ribosome. Thermal denaturation studies and footprinting experiments confirm that isolated ES7 is stable and maintains a near-native secondary and tertiary structure.

Next-Generation Sequencing Assessment of Eukaryotic Diversity in Oil Sands Tailings Ponds Sediments and SurfaceWater.

Tailings ponds in the Athabasca oil sands (Canada) contain fluid wastes, generated by the extraction of bitumen from oil sands ores. Although the autochthonous prokaryotic communities have been relatively well characterized, almost nothing is known about microbial eukaryotes living in the anoxic soft sediments of tailings ponds or in the thin oxic layer of water that covers them. We carried out the first next-generation sequencing study of microbial eukaryotic diversity in oil sands tailings ponds. In metagenomes prepared from tailings sediment and surface water, we detected very low numbers of sequences encoding eukaryotic small subunit ribosomal RNA representing seven major taxonomic groups of protists. We also produced and analysed three amplicon-based 18S rRNA libraries prepared from sediment samples. These revealed a more diverse set of taxa, 169 different OTUs encompassing up to eleven higher order groups of eukaryotes, according to detailed classification using homology searching and phylogenetic methods. The 10 most abundant OTUs accounted for >90% of the total of reads, vs. large numbers of rare OTUs (<1% abundance). Despite the anoxic and hydrocarbon-enriched nature of the environment, the tailings ponds harbour complex communities of microbial eukaryotes indicating that these organisms should be taken into account when studying the microbiology of the oil sands.