Miao Tian

Transcriptome Analysis of the Model Protozoan, Tetrahymena thermophila, Using Deep RNA Sequencing

Background The ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST) database limited the quality of the original genome annotation. Methodology/Principal Findings This RNA-seq study describes the first deep sequencing analysis of the T. thermophila transcriptome during the three major stages of the life cycle: growth, starvation and conjugation. Uniquely mapped reads covered more than 96% of the 24,725 predicted gene models in the somatic genome. More than 1,000 new transcribed regions were identified. The great dynamic range of RNA-seq allowed detection of a nearly six order-of-magnitude range of measurable gene expression orchestrated by this cell. RNA-seq also allowed the first prediction of transcript untranslated regions (UTRs) and an updated (larger) size estimate of the T. thermophila transcriptome: 57 Mb, or about 55% of the somatic genome. Our study identified nearly 1,500 alternative splicing (AS) events distributed over 5.2% of T. thermophila genes. This percentage represents a two order-of-magnitude increase over previous EST-based estimates in Tetrahymena. Evidence of stage-specific regulation of alternative splicing was also obtained. Finally, our study allowed us to completely confirm about 26.8% of the genes originally predicted by the gene finder, to correct coding sequence boundaries and intron-exon junctions for about a third, and to reassign microarray probes and correct earlier microarray data. Conclusions/Significance RNA-seq data significantly improve the genome annotation and provide a fully comprehensive view of the global transcriptome of T. thermophila. To our knowledge, 5.2% of T. thermophila genes with AS is the highest percentage of genes showing AS reported in a unicellular eukaryote. Tetrahymena thus becomes an excellent unicellular model eukaryote in which to investigate mechanisms of alternative splicing.

Tetrahymena Functional Genomics Database (TetraFGD): an integrated resource for Tetrahymena functional genomics

The ciliated protozoan Tetrahymena thermophila is a useful unicellular model organism for studies of eukaryotic cellular and molecular biology. Researches on T. thermophila have contributed to a series of remarkable basic biological principles. After the macronuclear genome was sequenced, substantial progress has been made in functional genomics research on T. thermophila, including genome-wide microarray analysis of the T. thermophila life cycle, a T. thermophila gene network analysis based on the microarray data and transcriptome analysis by deep RNA sequencing. To meet the growing demands for the Tetrahymena research community, we integrated these data to provide a public access database: Tetrahymena functional genomics database (TetraFGD). TetraFGD contains three major resources, including the RNA-Seq transcriptome, microarray and gene networks. The RNA-Seq data define gene structures and transcriptome, with special emphasis on exon–intron boundaries; the microarray data describe gene expression of 20 time points during three major stages of the T. thermophila life cycle; the gene network data identify potential gene–gene interactions of 15 049 genes. The TetraFGD provides user-friendly search functions that assist researchers in accessing gene models, transcripts, gene expression data and gene–gene relationships. In conclusion, the TetraFGD is an important functional genomic resource for researchers who focus on the Tetrahymena or other ciliates. Database URL: http://tfgd.ihb.ac.cn/

Phosphoproteomic Analysis of Protein Phosphorylation Networks in Tetrahymena thermophila, a Model Single-celled Organism

Tetrahymena thermophila is a widely used unicellular eukaryotic model organism in biological research and contains more than 1000 protein kinases and phosphatases with specificity for Ser/Thr/Tyr residues. However, only a few dozen phosphorylation sites in T. thermophila are known, presenting a major obstacle to further understanding of the regulatory roles of reversible phosphorylation in this organism. In this study, we used high-accuracy mass-spectrometry-based proteomics to conduct global and site-specific phosphoproteome profiling of T. thermophila. In total, 1384 phosphopeptides and 2238 phosphorylation sites from 1008 T. thermophila proteins were identified through the combined use of peptide prefractionation, TiO2 enrichment, and two-dimensional LC-MS/MS analysis. The identified phosphoproteins are implicated in the regulation of various biological processes such as transport, gene expression, and mRNA metabolic process. Moreover, integrated analysis of the T. thermophila phosphoproteome and gene network revealed the potential biological functions of many previously unannotated proteins and predicted some putative kinase–substrate pairs. Our data provide the first global survey of phosphorylation in T. thermophila using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided dataset is a valuable resource for the future understanding of signaling pathways in this important model organism.

Genome of the facultative scuticociliatosis pathogen Pseudocohnilembus persalinus provides insight into its virulence through horizontal gene transfer

Certain ciliates of the subclass Scuticociliatia (scuticociliates) are facultative parasites of fishes in which they cause a suite of diseases collectively termed scuticociliatosis. Hitherto, comparatively little was known about genetics and genomics of scuticociliates or the mechanism of scuticociliatosis. In this study, a laboratory culture of the facultatively pathogenic scuticociliate Pseudocohnilembus persalinus was established and its genome sequenced, giving the first genome of a marine ciliate. Genome-wide horizontal gene transfer (HGT) analysis showed P. persalinus has acquired many unique prokaryote-derived genes that potentially contribute to the virulence of this organism, including cell adhesion, hemolysis and heme utilization genes. These findings give new insights into our understanding of the pathology of scuticociliates.

Phylogenomic analyses reveal subclass Scuticociliatia as the sister group of subclass Hymenostomatia within class Oligohymenophorea

Scuticociliates and hymenostomes are two groups of the ciliate class Oligohymenophorea, a diverse clade that includes two model genera, Tetrahymena and Paramecium, which have been intensively studied due to their ease of culture and their amenability to a wide range of biochemical and genetic investigations. However, phylogenetic relationships among the subclasses of the Oligohymenophorea, and especially between the Scuticociliatia and Hymenostomatia, are not clearly resolved. Here, we investigate the phylogenetic relationship between the subclasses Scuticociliatia and Hymenostomatia based on omics data. The transcriptomes of five species, comprising four oligohymenophoreans and one colpodean, were sequenced. A supermatrix was constructed for phylogenomic analyses based on 113 genes encoding 43,528 amino acid residues from 26 taxa, including ten representatives of the class Oligohymenophorea. Our phylogenomic analyses revealed that the monophyletic Scuticociliatia is sister to the monophyletic Hymenostomatia, which together form the terminal branch within the monophyletic class Oligohymenophorea. Competing hypotheses for this relationship were rejected by topological tests. Our results provide corroborative evidence for the close relationship between the subclasses Scuticociliatia and Hymenostomatia, justifying the possible use of the model hymenostome T. thermophila as an effective experimental system to study the molecular and cellular biology of the scuticociliates.

Nonsense-mediated mRNA decay in Tetrahymena is EJC independent and requires a protozoa-specific nuclease

Abstract Nonsense-mediated mRNA decay (NMD) is essential for removing premature termination codon-containing transcripts from cells. Studying the NMD pathway in model organisms can help to elucidate the NMD mechanism in humans and improve our understanding of how this biologically important process has evolved. Ciliates are among the earliest branching eukaryotes; their NMD mechanism is poorly understood and may be primordial. We demonstrate that highly conserved Upf proteins (Upf1a, Upf2 and Upf3) are involved in the NMD pathway of the ciliate, Tetrahymena thermophila. We further show that a novel protozoa-specific nuclease, Smg6L, is responsible for destroying many NMD-targeted transcripts. Transcriptome-wide identification and characterization of NMD-targeted transcripts in vegetative Tetrahymena cells showed that many have exon–exon junctions downstream of the termination codon. However, Tetrahymena may lack a functional exon junction complex (EJC), and the Tetrahymena ortholog of an EJC core component, Mago nashi (Mag1), is dispensable for NMD. Therefore, NMD is EJC independent in this early branching eukaryote.

Cdk3, a conjugation-specific cyclin-dependent kinase, is essential for the initiation of meiosis in Tetrahymena thermophila

ABSTRACT Meiosis is an important process in sexual reproduction. Meiosis initiation has been found to be highly diverse among species. In yeast, it has been established that cyclin-dependent kinases (Cdks) and cyclins are essential components in the meiosis initiation pathway. In this study, we identified 4 Cdks in the model ciliate, Tetrahymena thermophila, and we found one of them, Cdk3, which is specifically expressed during early conjugation, to be essential for meiosis initiation. Cdk3 deletion led to arrest at the pair formation stage of conjugation. We then confirmed that Cdk3 acts upstream of double-strand break (DSB) formation. Moreover, we detected that Cdk3 is necessary for the expression of many genes involved in early meiotic events. Through proteomic quantification of phosphorylation, co-expression analysis and RNA-Seq analyses, we identified a conjugation-specific cyclin, Cyc2, which most likely partners with Cdk3 to initiate meiosis.

Cyc17, a meiosis-specific cyclin, is essential for anaphase initiation and chromosome segregation in Tetrahymena thermophila

ABSTRACT Although the role of cyclins in controlling nuclear division is well established, their function in ciliate meiosis remains unknown. In ciliates, the cyclin family has undergone massive expansion which suggests that diverse cell cycle systems exist, and this warrants further investigation. A screen for cyclins in the model ciliate Tetrahymena thermophila showed that there are 34 cyclins in this organism. Only 1 cyclin, Cyc17, contains the complete cyclin core and is specifically expressed during meiosis. Deletion of CYC17 led to meiotic arrest at the diakinesis-like metaphase I stage. Expression of genes involved in DNA metabolism and chromosome organization (chromatin remodeling and basic chromosomal structure) was repressed in cyc17 knockout matings. Further investigation suggested that Cyc17 is involved in regulating spindle pole attachment, and is thus essential for chromosome segregation at meiosis. These findings suggest a simple model in which chromosome segregation is influenced by Cyc17.

E2fl1 is a meiosis-specific transcription factor in the protist Tetrahymena thermophila

ABSTRACT Members of the E2F family of transcription factors have been reported to regulate the expression of genes involved in cell cycle control, DNA replication, and DNA repair in multicellular eukaryotes. Here, E2FL1, a meiosis-specific E2F transcription factor gene, was identified in the model ciliate Tetrahymena thermophila. Loss of this gene resulted in meiotic arrest prior to anaphase I. The cytological experiments revealed that the meiotic homologous pairing was not affected in the absence of E2FL1, but the paired homologous chromosomes did not separate and assumed a peculiar tandem arrangement. This is the first time that an E2F family member has been shown to regulate meiotic events. Moreover, BrdU incorporation showed that DSB processing during meiosis was abnormal upon the deletion of E2FL1. Transcriptome sequencing analysis revealed that E2FL1 knockout decreased the expression of genes involved in DNA replication and DNA repair in T. thermophila, suggesting that the function of E2F is highly conserved in eukaryotes. In addition, E2FL1 deletion inhibited the expression of related homologous chromosome segregation genes in T. thermophila. The result may explain the meiotic arrest phenotype at anaphase I. Finally, by searching for E2F DNA-binding motifs in the entire T. thermophila genome, we identified 714 genes containing at least one E2F DNA-binding motif; of these, 235 downregulated represent putative E2FL1 target genes.

A DP-like transcription factor protein interacts with E2fl1 to regulate meiosis in Tetrahymena thermophila

ABSTRACT Evolutionarily conserved E2F family transcription factors regulate the cell cycle via controlling gene expression in a wide range of eukaryotes. We previously demonstrated that the meiosis-specific transcription factor E2fl1 had an important role in meiosis in the model ciliate Tetrahymena thermophila. Here, we report that expression of another E2F family transcription factor gene DPL2 correlates highly with that of E2FL1. Similar to e2fl1Δ cells, dpl2Δ cells undergo meiotic arrest prior to anaphase I, with the five chromosomes adopting an abnormal tandem arrangement. Immunofluorescence staining and immunoprecipitation experiments demonstrate that Dpl2 and E2fl1 form a complex during meiosis. We previously identified several meiotic regulatory proteins in T. thermophila. Cyc2 and Tcdk3 may cooperate to initiate meiosis and Cyc17 is essential for initiating meiotic anaphase. We investigate the relationship of these regulators with Dpl2 and E2fl1, and then construct a meiotic regulatory network by measuring changes in meiotic genes expression in knockout cells. We conclude that the E2fl1/Dpl2 complex plays a central role in meiosis in T. thermophila.