Tetrahymena Thermophila Genome Annotation
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PROVIDER: PRJEB75540 | ENA |
REPOSITORIES: ENA
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Project description:The ciliate Tetrahymena thermophila is a well-established unicellular model eukaryote, contributing significantly to foundational biological discoveries. Despite its acknowledged importance, current studies on Tetrahymena biology face challenges due to gene annotation inaccuracy, particularly the notable absence of untranslated regions (UTRs). To comprehensively annotate the Tetrahymena macronuclear genome, we collected extensive transcriptomic data spanning various cell stages. To ascertain transcript orientation and transcription start/end sites, we incorporated data on epigenetic marks displaying enrichment towards the 5' end of gene bodies, including H3 lysine 4 tri-methylation (H3K4me3), histone variant H2A.Z, nucleosome positioning and N6-methyldeoxyadenine (6mA). Cap-seq data was subsequently applied to validate the accuracy of identified transcription start sites. Additionally, we integrated Nanopore direct RNA sequencing (DRS), strand-specific RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) data. Using a newly developed bioinformatic pipeline, coupled with manual curation and experimental validation, our work yielded substantial improvements to the current gene models, including the addition of 2,481 new genes, updates to 23,936 existing genes, and the incorporation of 8,339 alternatively spliced isoforms. Furthermore, novel UTR information was annotated for 26,687 high-confidence genes. Intriguingly, 20% of protein-coding genes were identified to have natural antisense transcripts characterized by high diversity in alternative splicing, thus offering insights into understanding transcriptional regulation. Our work will enhance the utility of Tetrahymena as a robust genetic toolkit for advancing biological research, and provides a promising framework for genome annotation in other eukaryotes.
| S-EPMC11754650 | biostudies-literature
Project description:Tetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.We addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified.We report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.
| S-EPMC2612030 | biostudies-literature
Project description:Genome architecture varies greatly among eukaryotes. This diversity may profoundly affect the origin and maintenance of genetic variation within a population. Ciliates are microbial eukaryotes with unusual genome features, such as the separation of germline and somatic genomes within a single cell and amitotic division. These features have previously been proposed to increase the rate of molecular evolution in these species. Here, we assessed the fitness effects of genetic variation in the two genomes of natural isolates of the ciliate Tetrahymena thermophila. We find more extensive genetic variation in fitness in the transcriptionally silent germline genome than in the expressed somatic genome. Surprisingly, this variation is not primarily deleterious, but has both beneficial and deleterious effects. We conclude that Tetrahymena genome architecture allows for the maintenance of genetic variation that would otherwise be eliminated by selection. We consider the effect of selection on the two genomes and the impacts of reproductive strategies and the mechanism of sex determination on the structure of this variation.
| S-EPMC4899097 | biostudies-literature
Project description:The growth, survival, and life cycle progression of the freshwater ciliated protozoan Tetrahymena thermophila are responsive to protein signals thought to be released by constitutive secretion. In addition to providing insights about ciliate communication, studies of constitutive secretion are of interest for evaluating the utility of T. thermophila as a platform for the expression of secreted protein therapeutics. For these reasons, we undertook an unbiased investigation of T. thermophila secreted proteins using wild-type and secretion mutant strains. Extensive tandem mass spectrometry analyses of secretome samples were performed. We identified a total of 207 secretome proteins, most of which were not detected in a set of abundant whole-cell protein identifications. Numerous proteases and other hydrolases were secreted from cells grown in rich medium but not cells transferred to a nutrient starvation condition. On the other hand, we detected the starvation-enhanced secretion of a small number of cytosolic proteins, suggestive of an exosome-like pathway in T. thermophila. Subsets of proteins from the T. thermophila regulated secretion pathway were detected with differential representation across strains and culture conditions. Finally, many secretome proteins had a predicted N-terminal signal sequence but no other annotated characteristic or functional classification. Our work provides the first comprehensive analysis of secreted proteins in T. thermophila and establishes the groundwork for future studies of constitutive protein secretion biology and biotechnology in ciliates.
| S-EPMC2863955 | biostudies-literature
Project description:Multifunctional acyltransferases are able to catalyze the esterification of various acyl-acceptors with activated fatty acids. Here we describe the identification of four proteins from Tetrahymena thermophila that share certain properties with mammalian acyltransferases regarding their predicted transmembrane structure, their molecular mass and the typical acyltransferase motif. Expression of the Tetrahymena sequences results in production of triacylglycerols and wax esters in recombinant yeast when appropriate substrates are provided. The in vitro characterization shows, that these enzymes are capable of esterifying different acyl-acceptors including fatty alcohols, diols, diacylglycerols and isoprenols with acyl-CoA thioesters. Based on these catalytic activities and the sequence similarities of the Tetrahymena proteins with acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) family members, we conclude that we identified a new group of DGAT2-related multifunctional acyltransferases from protozoan organisms.
| S-EPMC3311841 | biostudies-literature
Project description:Genetically programmed DNA rearrangements can regulate mRNA expression at an individual locus or, for some organisms, on a genome-wide scale. Ciliates rely on a remarkable process of whole-genome remodeling by DNA elimination to differentiate an expressed macronucleus (MAC) from a copy of the germline micronucleus (MIC) in each cycle of sexual reproduction. Here we describe results from the first high-throughput sequencing effort to investigate ciliate genome restructuring, comparing Sanger long-read sequences from a Tetrahymena thermophila MIC genome library to the MAC genome assembly. With almost 25% coverage of the unique-sequence MAC genome by MIC genome sequence reads, we created a resource for positional analysis of MIC-specific DNA removal that pinpoints MAC genome sites of DNA elimination at nucleotide resolution. The widespread distribution of internal eliminated sequences (IES) in promoter regions and introns suggests that MAC genome restructuring is essential not only for what it removes (for example, active transposons) but also for what it creates (for example, splicing-competent introns). Consistent with the heterogeneous boundaries and epigenetically modulated efficiency of individual IES deletions studied to date, we find that IES sites are dramatically under-represented in the ∼25% of the MAC genome encoding exons. As an exception to this general rule, we discovered a previously unknown class of small (<500 bp) IES with precise elimination boundaries that can contribute the 3' exon of an mRNA expressed during genome restructuring, providing a new mechanism for expanding mRNA complexity in a developmentally regulated manner.
| S-EPMC3276166 | biostudies-literature
Project description:The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.
| S-EPMC1557398 | biostudies-literature
Project description:We describe phylogenetic and functional studies of three septins in the free-living ciliate Tetrahymena thermophila. Both deletion and overproduction of septins led to vacuolization of mitochondria, destabilization of the nuclear envelope, and increased autophagy. All three green fluorescent protein-tagged septins localized to mitochondria. Specific septins localized to the outer mitochondrial membrane, to septa formed during mitochondrial scission, or to the mitochondrion-associated endoplasmic reticulum. The only other septins known to localize to mitochondria are human ARTS and murine M-septin, both alternatively spliced forms of Sep4 (S. Larisch, Cell Cycle 3:1021-1023, 2004; S. Takahashi, R. Inatome, H. Yamamura, and S. Yanagi, Genes Cells 8:81-93, 2003). It therefore appears that septins have been recruited to mitochondrial functions independently in at least two eukaryotic lineages and in both cases are involved in apoptotic events.
| S-EPMC2519767 | biostudies-literature
Project description:The maintenance of chromosome integrity is crucial for genetic stability. However, programmed chromosome fragmentations are known to occur in many organisms, and in the ciliate Tetrahymena the five germline chromosomes are fragmented into hundreds of minichromosomes during somatic nuclear differentiation. Here, we showed that there are different fates of these minichromosomes after chromosome breakage. Among the 326 somatic minichromosomes identified using genomic data, 50 are selectively eliminated from the mature somatic genome. Interestingly, many and probably most of these minichromosomes are eliminated during the growth period between 6 and 20 doublings right after conjugation. Genes with potential conjugation-specific functions are found in these minichromosomes. This study revealed a new mode of programmed DNA elimination in ciliates similar to those observed in parasitic nematodes, which could play a role in developmental gene regulation.
| S-EPMC5091840 | biostudies-literature
Project description:In conjugating Tetrahymena thermophila, massive DNA elimination occurs upon the development of the new somatic genome from the germ line genome. Small, approximately 28-nucleotide scan RNAs (scnRNAs) and Twi1p, an Argonaute family member, mediate H3K27me3 and H3K9me3 histone H3 modifications, which lead to heterochromatin formation and the excision of the heterochromatinized germ line-limited sequences. In our search for new factors involved in developmental DNA rearrangement, we identified two Twi1p-interacting proteins, Wag1p and CnjBp. Both proteins contain GW (glycine and tryptophan) repeats, which are characteristic of several Argonaute-interacting proteins in other organisms. Wag1p and CnjBp colocalize with Twi1p in the parental macronucleus early in conjugation and in the new developing macronucleus during later developmental stages. Around the time DNA elimination occurs, Wag1p forms multiple nuclear bodies in the developing macronuclei that do not colocalize with heterochromatic DNA elimination structures. Analyses of DeltaWAG1, DeltaCnjB, and double DeltaWAG1 DeltaCnjB knockout strains revealed that WAG1 and CnjB genes need to be deleted together to inhibit the downregulation of specific scnRNAs, the formation of DNA elimination structures, and DNA excision. Thus, Wag1p and CnjBp are two novel players with overlapping functions in RNA interference-mediated genome rearrangement in Tetrahymena.
| S-EPMC2738283 | biostudies-literature