Using digital PCR to predict ciliate abundance from ribosomal RNA gene copy numbers.

Ciliates play a key role in most ecosystems. Their abundance in natural samples is crucial for answering many ecological questions. Traditional methods of quantifying individual species, which rely on microscopy, are often labour-intensive, time-consuming and can be highly biassed. As a result, we investigated the potential of digital polymerase chain reaction (dPCR) for quantifying ciliates. A significant challenge in this process is the high variation in the copy number of the taxonomic marker gene (ribosomal RNA [rRNA]). We first quantified the rRNA gene copy numbers (GCN) of the model ciliate, Paramecium tetraurelia, during different stages of the cell cycle and growth phases. The per-cell rRNA GCN varied between approximately 11,000 and 130,000, averaging around 50,000 copies per cell. Despite these variations in per-cell rRNA GCN, we found a highly significant correlation between GCN and cell numbers. This is likely due to the coexistence of different cellular stages in an uncontrolled (environmental) ciliate population. Thanks to the high sensitivity of dPCR, we were able to detect the target gene in a sample that contained only a single cell. The dPCR approach presented here is a valuable addition to the molecular toolbox in protistan ecology. It may guide future studies in quantifying and monitoring the abundance of targeted (even rare) ciliates in natural samples.

Inter-generational nuclear crosstalk links the control of gene expression to programmed genome rearrangement during the Paramecium sexual cycle.

Multinucleate cells are found in many eukaryotes, but how multiple nuclei coordinate their functions is still poorly understood. In the cytoplasm of the ciliate Paramecium tetraurelia, two micronuclei (MIC) serving sexual reproduction coexist with a somatic macronucleus (MAC) dedicated to gene expression. During sexual processes, the MAC is progressively destroyed while still ensuring transcription, and new MACs develop from copies of the zygotic MIC. Several gene clusters are successively induced and switched off before vegetative growth resumes. Concomitantly, programmed genome rearrangement (PGR) removes transposons and their relics from the new MACs. Development of the new MACs is controlled by the old MAC, since the latter expresses genes involved in PGR, including the PGM gene encoding the essential PiggyMac endonuclease that cleaves the ends of eliminated sequences. Using RNA deep sequencing and transcriptome analysis, we show that impairing PGR upregulates key known PGR genes, together with ∼600 other genes possibly also involved in PGR. Among these genes, 42% are no longer induced when no new MACs are formed, including 180 genes that are co-expressed with PGM under all tested conditions. We propose that bi-directional crosstalk between the two coexisting generations of MACs links gene expression to the progression of MAC development.

Methods for Paramecium tetraurelia ciliary membrane protein identification and function.

In this chapter we provide some tools to study the ciliary proteins that make it possible for Paramecium cells to swim by beating their cilia. These proteins include many ion channels, accessory proteins, peripheral proteins, structural proteins, rootlets of cilia, and enzymes. Some of these proteins are also found in the soma membrane, but their distinct and critical functions are in the cilia. Paramecium has 4000 or more cilia per cell, giving it an advantage for biochemical studies over cells that have one primarily cilium per cell. Nonetheless, a challenge for studies of many ciliary proteins in Paramecium is their low abundance. We discuss here several strategies to overcome this challenge and other challenges such as working with very large channel proteins. We also include for completeness other techniques that are critical to the study of swimming behavior, such as genetic crosses, recording of swimming patterns, electrical recordings, expression of very large channel proteins, RNA Interference, among others.

Developmental timing of programmed DNA elimination in Paramecium tetraurelia recapitulates germline transposon evolutionary dynamics.

With its nuclear dualism, the ciliate Paramecium constitutes a unique model to study how host genomes cope with transposable elements (TEs). P. tetraurelia harbors two germline micronuclei (MICs) and a polyploid somatic macronucleus (MAC) that develops from one MIC at each sexual cycle. Throughout evolution, the MIC genome has been continuously colonized by TEs and related sequences that are removed from the somatic genome during MAC development. Whereas TE elimination is generally imprecise, excision of approximately 45,000 TE-derived internal eliminated sequences (IESs) is precise, allowing for functional gene assembly. Programmed DNA elimination is concomitant with genome amplification. It is guided by noncoding RNAs and repressive chromatin marks. A subset of IESs is excised independently of this epigenetic control, raising the question of how IESs are targeted for elimination. To gain insight into the determinants of IES excision, we established the developmental timing of DNA elimination genome-wide by combining fluorescence-assisted nuclear sorting with high-throughput sequencing. Essentially all IESs are excised within only one endoreplication round (32C to 64C), whereas TEs are eliminated at a later stage. We show that DNA elimination proceeds independently of replication. We defined four IES classes according to excision timing. The earliest excised IESs tend to be independent of epigenetic factors, display strong sequence signals at their ends, and originate from the most ancient integration events. We conclude that old IESs have been optimized during evolution for early and accurate excision by acquiring stronger sequence determinants and escaping epigenetic control.

Identification of novel, functional, long noncoding RNAs involved in programmed, large-scale genome rearrangements.

Noncoding RNAs (ncRNAs) make up to ∼98% percent of the transcriptome of a given organism. In recent years, one relatively new class of ncRNAs, long noncoding RNAs (lncRNAs), were shown to be more than mere by-products of gene expression and regulation. The unicellular eukaryote Paramecium tetraurelia is a member of the ciliate phylum, an extremely heterogeneous group of organisms found in most bodies of water across the globe. A hallmark of ciliate genetics is nuclear dimorphism and programmed elimination of transposons and transposon-derived DNA elements, the latter of which is essential for the maintenance of the somatic genome. Paramecium and ciliates in general harbor a plethora of different ncRNA species, some of which drive the process of large-scale genome rearrangements, including DNA elimination, during sexual development. Here, we identify and validate the first known functional lncRNAs in ciliates to date. Using deep-sequencing and subsequent bioinformatic processing and experimental validation, we show that Paramecium expresses at least 15 lncRNAs. These candidates were predicted by a highly conservative pipeline, and informatic analyses hint at differential expression during development. Depletion of two lncRNAs, lnc1 and lnc15, resulted in clear phenotypes, decreased survival, morphological impairment, and a global effect on DNA elimination.

Micromanipulation in Paramecium: From non-mendelian inheritance to the outlook for versatile micromachines.

This review addresses nine areas of knowledge revealed by micromanipulations performed with Paramecium. Microinjection has shown that sexual maturation and senescence of Paramecium caudatum is a programmed process conducted by a specific gene and its product protein. In Paramecium tetraurelia, autogamy was revealed to depend on the number of DNA syntheses rather than the number of cell divisions in clonal aging. The cytoplasmic complementarity test established that microinjection of wild-type cytoplasm can correct genetic defects of mutants. The concept of complementarity together with protein chemistry revealed compounds that control membrane excitability. In non-Mendelian inheritance, noncoding small RNAs made from the parental micronucleus regulate the rearrangement of the progeny's macronuclear DNA. The macronucleus has the potential to be used as a factory for genetic engineering. The development and differentiation of progeny's nuclei in mating pairs are controlled by the parental macronucleus. The chemical reaction processes associated with exocytosis have been revealed by microinjection of various enzymes and antibodies. Using the fusion gene of histone H2B and yellow-fluorescence protein, it was revealed that the fusion gene-mRNA is transferred between cells during mating. Experiments with endosymbiotic bacteria and the host shed light on the conditions needed to establish sustainable symbiotic relationships.

The transient Spt4-Spt5 complex as an upstream regulator of non-coding RNAs during development.

The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline and somatic nuclei, massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs - 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.

Early developmental, meiosis-specific proteins - Spo11, Msh4-1, and Msh5 - Affect subsequent genome reorganization in Paramecium tetraurelia.

Developmental DNA elimination in Paramecium tetraurelia occurs through a trans-nuclear comparison of the genomes of two distinct types of nuclei: the germline micronucleus (MIC) and the somatic macronucleus (MAC). During sexual reproduction, which starts with meiosis of the germline nuclei, MIC-limited sequences including Internal Eliminated Sequences (IESs) and transposons are eliminated from the developing MAC in a process guided by noncoding RNAs (scnRNAs and iesRNAs). However, our current understanding of this mechanism is still very limited. Therefore, studying both genetic and epigenetic aspects of these processes is a crucial step to understand this phenomenon in more detail. Here, we describe the involvement of homologs of classical meiotic proteins, Spo11, Msh4-1, and Msh5 in this phenomenon. Based on our analyses, we propose that proper functioning of Spo11, Msh4-1, and Msh5 during Paramecium sexual reproduction are necessary for genome reorganization and viable progeny. Also, we show that double-strand breaks (DSBs) in DNA induced during meiosis by Spo11 are crucial for proper IESs excision. In summary, our investigations show that early sexual reproduction processes may significantly influence later somatic genome integrity.

One Cell, Two Gears: Extensive Somatic Genome Plasticity Accompanies High Germline Genome Stability in Paramecium.

Mutation accumulation (MA) experiments are conventionally employed to study spontaneous germline mutations. However, MA experiments can also shed light on somatic genome plasticity in a habitual and genetic drift-maximizing environment. Here, we revisit an MA experiment that uncovered extraordinary germline genome stability in Paramecium tetraurelia, a single-celled eukaryote with nuclear dimorphism. Our re-examination of isogenic P. tetraurelia MA lines propagated in nutrient-rich medium for >40 sexual cycles reveals that their polyploid somatic genome accrued hundreds of intervening DNA segments (IESs), which are normally eliminated during germline-soma differentiation. These IESs frequently occupy a fraction of the somatic DNA copies of a given locus, producing IES excision/retention polymorphisms, and preferentially fall into a class of epigenetically controlled sequences. Relative to control lines, retained IESs are flanked by stronger cis-acting signals and interrupt an excess of highly expressed coding exons. These findings suggest that P. tetraurelia's elevated germline DNA replication fidelity is associated with pervasive somatic genome plasticity. They show that MA regimes are powerful tools for investigating the role that developmental plasticity, somatic mutations, and epimutations have in ecology and evolution.

Massive colonization of protein-coding exons by selfish genetic elements in Paramecium germline genomes.

Ciliates are unicellular eukaryotes with both a germline genome and a somatic genome in the same cytoplasm. The somatic macronucleus (MAC), responsible for gene expression, is not sexually transmitted but develops from a copy of the germline micronucleus (MIC) at each sexual generation. In the MIC genome of Paramecium tetraurelia, genes are interrupted by tens of thousands of unique intervening sequences called internal eliminated sequences (IESs), which have to be precisely excised during the development of the new MAC to restore functional genes. To understand the evolutionary origin of this peculiar genomic architecture, we sequenced the MIC genomes of 9 Paramecium species (from approximately 100 Mb in Paramecium aurelia species to >1.5 Gb in Paramecium caudatum). We detected several waves of IES gains, both in ancestral and in more recent lineages. While the vast majority of IESs are single copy in present-day genomes, we identified several families of mobile IESs, including nonautonomous elements acquired via horizontal transfer, which generated tens to thousands of new copies. These observations provide the first direct evidence that transposable elements can account for the massive proliferation of IESs in Paramecium. The comparison of IESs of different evolutionary ages indicates that, over time, IESs shorten and diverge rapidly in sequence while they acquire features that allow them to be more efficiently excised. We nevertheless identified rare cases of IESs that are under strong purifying selection across the aurelia clade. The cases examined contain or overlap cellular genes that are inactivated by excision during development, suggesting conserved regulatory mechanisms. Similar to the evolution of introns in eukaryotes, the evolution of Paramecium IESs highlights the major role played by selfish genetic elements in shaping the complexity of genome architecture and gene expression.

Primary ciliary dyskinesia relative protein ZMYND10 is involved in regulating ciliary function and intraflagellar transport in Paramecium tetraurelia.

Cilia are highly conserved in most eukaryotes and are regarded as an important organelle for motility and sensation in various species. Cilia are microscopic, hair-like cytoskeletal structures that protrude from the cell surface. The major focus in studies of cilia has been concentrated on the ciliary dysfunction in vertebrates that causes multisymptomatic diseases, which together are referred to as ciliopathies. To date, the understanding of ciliopathies has largely depended on the study of ciliary structure and function in different animal models. Zinc finger MYND-type containing 10 (ZMYND10) is a ciliary protein that was recently found to be mutated in patients with primary ciliary dyskinesia (PCD). In Paramecium tetraurelia, we identified two ZMYND10 genes, arising from a whole-genome duplication. Using RNAi, we found that the depletion of ZMYND10 in P. tetraurelia causes severe ciliary defects, thus provoking swimming dysfunction and lethality. Moreover, we found that the absence of ZMYND10 caused the abnormal localization of the intraflagellar transport (IFT) protein IFT43 along cilia. These results suggest that ZMYND10 is involved in the regulation of ciliary function and IFT, which may contribute to the study of PCD pathogenesis.

Intraflagellar Transport 80 Is Required for Cilia Construction and Maintenance in Paramecium tetraurelia.

Intraflagellar transport (IFT) represents a bidirectional dynamic process that carries cargo essential for cilia building and the maintenance of ciliary function, which is important for the locomotion of single cells, intracellular and intercellular signalling transduction. Accumulated evidence has revealed that defects in IFT cause several clinical disorders. Here, we determined the role of IFT80, an IFT-B protein that is mutated in Jeune asphyxiating thoracic dystrophy. Using the RNAi method in the ciliate Paramecium as model, we found that loss of IFT80 prevents cilia biogenesis and causes strong cell lethality. A specific antibody against IFT80 was also prepared in our study, which labelled IFT80 in cilia of Paramecium. GFP fusion experiments were performed to illustrate the dynamic movement of IFT-A and IFT-B proteins in cilia of Paramecium; then, we found that the depletion of IFT80 in cells prevents IFT-A and IFT-B proteins from entering the cilia. Our results showed the distribution change of other IFT proteins in cells that were depleted of IFT80, and we discuss the possible roles of IFT80 in Paramecium.

MKS-NPHP module proteins control ciliary shedding at the transition zone.

Ciliary shedding occurs from unicellular organisms to metazoans. Although required during the cell cycle and during neurogenesis, the process remains poorly understood. In all cellular models, this phenomenon occurs distal to the transition zone (TZ), suggesting conserved molecular mechanisms. The TZ module proteins (Meckel Gruber syndrome [MKS]/Nephronophtysis [NPHP]/Centrosomal protein of 290 kDa [CEP290]/Retinitis pigmentosa GTPase regulator-Interacting Protein 1-Like Protein [RPGRIP1L]) are known to cooperate to establish TZ formation and function. To determine whether they control deciliation, we studied the function of 5 of them (Transmembrane protein 107 [TMEM107], Transmembrane protein 216 [TMEM216], CEP290, RPGRIP1L, and NPHP4) in Paramecium. All proteins are recruited to the TZ of growing cilia and localize with 9-fold symmetry at the level of the most distal part of the TZ. We demonstrate that depletion of the MKS2/TMEM216 and TMEM107 proteins induces constant deciliation of some cilia, while depletion of either NPHP4, CEP290, or RPGRIP1L prevents Ca2+/EtOH deciliation. Our results constitute the first evidence for a role of conserved TZ proteins in deciliation and open new directions for understanding motile cilia physiology.

Environmentally induced plasticity of programmed DNA elimination boosts somatic variability in Paramecium tetraurelia.

Can ecological changes impact somatic genome development? Efforts to resolve this question could reveal a direct link between environmental changes and somatic variability, potentially illuminating our understanding of how variation can surface from a single genotype under stress. Here, we tackle this question by leveraging the biological properties of ciliates. When Paramecium tetraurelia reproduces sexually, its polyploid somatic genome regenerates from the germline genome through a developmental process that involves the removal of thousands of ORF-interrupting sequences known as internal eliminated sequences (IESs). We show that exposure to nonstandard culture temperatures impacts the efficiency of this process of programmed DNA elimination, prompting the emergence of hundreds of incompletely excised IESs in the newly developed somatic genome. These alternative DNA isoforms display a patterned genomic topography, impact gene expression, and might be inherited transgenerationally. On this basis, we conclude that environmentally induced developmental thermoplasticity contributes to genotypic diversification in Paramecium.

Loss of a Fragile Chromosome Region leads to the Screwy Phenotype in Paramecium tetraurelia.

A conspicuous cell-shape phenotype known as "screwy" was reported to result from mutations at two or three uncharacterized loci in the ciliate Paramecium tetraurelia. Here, we describe a new screwy mutation, Spinning Top, which appeared spontaneously in the cross of an unrelated mutant with reference strain 51. The macronuclear (MAC) genome of the Spinning Top mutant is shown to lack a ~28.5-kb segment containing 18 genes at the end of one chromosome, which appears to result from a collinear deletion in the micronuclear (MIC) genome. We tested several candidate genes from the deleted locus by dsRNA-induced silencing in wild-type cells, and identified a single gene responsible for the phenotype. This gene, named Spade, encodes a 566-aa glutamine-rich protein with a C2HC zinc finger. Its silencing leads to a fast phenotype switch during vegetative growth, but cells recover a wild-type phenotype only 5-6 divisions after silencing is stopped. We analyzed 5 independently-obtained mutant alleles of the Sc1 locus, and concluded that all of them also lack the Spade gene and a number of neighboring genes in the MAC and MIC genomes. Mapping of the MAC deletion breakpoints revealed two different positions among the 5 alleles, both of which differ from the Spinning Top breakpoint. These results suggest that this MIC chromosome region is intrinsically unstable in strain 51.

The Polycomb protein Ezl1 mediates H3K9 and H3K27 methylation to repress transposable elements in Paramecium.

In animals and plants, the H3K9me3 and H3K27me3 chromatin silencing marks are deposited by different protein machineries. H3K9me3 is catalyzed by the SET-domain SU(VAR)3-9 enzymes, while H3K27me3 is catalyzed by the SET-domain Enhancer-of-zeste enzymes, which are the catalytic subunits of Polycomb Repressive Complex 2 (PRC2). Here, we show that the Enhancer-of-zeste-like protein Ezl1 from the unicellular eukaryote Paramecium tetraurelia, which exhibits significant sequence and structural similarities with human EZH2, catalyzes methylation of histone H3 in vitro and in vivo with an apparent specificity toward K9 and K27. We find that H3K9me3 and H3K27me3 co-occur at multiple families of transposable elements in an Ezl1-dependent manner. We demonstrate that loss of these histone marks results in global transcriptional hyperactivation of transposable elements with modest effects on protein-coding gene expression. Our study suggests that although often considered functionally distinct, H3K9me3 and H3K27me3 may share a common evolutionary history as well as a common ancestral role in silencing transposable elements.

A Novel Role for Polycystin-2 (Pkd2) in P. tetraurelia as a Probable Mg2+ Channel Necessary for Mg2+-Induced Behavior.

A human ciliopathy gene codes for Polycystin-2 (Pkd2), a non-selective cation channel. Here, the Pkd2 channel was explored in the ciliate Paramecium tetraurelia using combinations of RNA interference, over-expression, and epitope-tagging, in a search for function and novel interacting partners. Upon depletion of Pkd2, cells exhibited a phenotype similar to eccentric (XntA1), a Paramecium mutant lacking the inward Ca2+-dependent Mg2+ conductance. Further investigation showed both Pkd2 and XntA localize to the cilia and cell membrane, but do not require one another for trafficking. The XntA-myc protein co-immunoprecipitates Pkd2-FLAG, but not vice versa, suggesting two populations of Pkd2-FLAG, one of which interacts with XntA. Electrophysiology data showed that depletion and over-expression of Pkd2 led to smaller and larger depolarizations in Mg2+ solutions, respectively. Over-expression of Pkd2-FLAG in the XntA1 mutant caused slower swimming, supporting an increase in Mg2+ permeability, in agreement with the electrophysiology data. We propose that Pkd2 in P. tetraurelia collaborates with XntA for Mg2+-induced behavior. Our data suggest Pkd2 is sufficient and necessary for Mg2+ conductance and membrane permeability to Mg2+, and that Pkd2 is potentially a Mg2+-permeable channel.

Identification of a miniature Sae2/Ctp1/CtIP ortholog from Paramecium tetraurelia required for sexual reproduction and DNA double-strand break repair.

DNA double-strand breaks (DSBs) induced by genotoxic agents can cause cell death or contribute to chromosomal instability, a major driving force of cancer. By contrast, Spo11-dependent DSBs formed during meiosis are aimed at generating genetic diversity. In eukaryotes, CtIP and the Mre11 nuclease complex are essential for accurate processing and repair of both unscheduled and programmed DSBs by homologous recombination (HR). Here, we applied bioinformatics and genetic analysis to identify Paramecium tetraurelia CtIP (PtCtIP), the smallest known Sae2/Ctp1/CtIP ortholog, as a key factor for the completion of meiosis and the recovery of viable sexual progeny. Using in vitro assays, we find that purified recombinant PtCtIP preferentially binds to double-stranded DNA substrates but does not contain intrinsic nuclease activity. Moreover, mutation of the evolutionarily conserved C-terminal 'RHR' motif abrogates DNA binding of PtCtIP but not its ability to functionally interact with Mre11. Translating our findings into mammalian cells, we provide evidence that disruption of the 'RHR' motif abrogates accumulation of human CtIP at sites of DSBs. Consequently, cells expressing the DNA binding mutant CtIPR837A/R839A are defective in DSB resection and HR. Collectively, our work highlights minimal structural requirements for CtIP protein family members to facilitate the processing of DSBs, thereby maintaining genome stability as well as enabling sexual reproduction.

A mating-type mutagenesis screen identifies a zinc-finger protein required for specific DNA excision events in Paramecium.

In the ciliate Paramecium tetraurelia, functional genes are reconstituted during development of the somatic macronucleus through the precise excision of ∼45 000 single-copy Internal Eliminated Sequences (IESs), thought to be the degenerate remnants of ancient transposon insertions. Like introns, IESs are marked only by a weak consensus at their ends. How such a diverse set of sequences is faithfully recognized and precisely excised remains unclear: specialized small RNAs have been implicated, but in their absence up to ∼60% of IESs are still correctly excised. To get further insight, we designed a mutagenesis screen based on the hypersensitivity of a specific excision event in the mtA gene, which determines mating types. Unlike most IES-containing genes, the active form of mtA is the unexcised one, allowing the recovery of hypomorphic alleles of essential IES recognition/excision factors. Such is the case of one mutation recovered in the Piwi gene PTIWI09, a key player in small RNA-mediated IES recognition. Another mutation identified a novel protein with a C2H2 zinc finger, mtGa, which is required for excision of a small subset of IESs characterized by enrichment in a 5-bp motif. The unexpected implication of a sequence-specific factor establishes a new paradigm for IES recognition and/or excision.

The splicing of tiny introns of Paramecium is controlled by MAGO.

The exon junction complex (EJC) is a key element of the splicing machinery. The EJC core is composed of eIF4A3, MAGO, Y14 and MLN51. Few accessory proteins, such as CWC22 or UPF3, bind transiently to the EJC. The EJC has been implicated in the control of the splicing of long introns. To ascertain whether the EJC controls the splicing of short introns, we used Paramecium tetraurelia as a model organism, since it has thousands of very tiny introns. To elucidate whether EJC affects intron splicing in P. tetraurelia, we searched for EJC protein-coding genes, and silenced those genes coding for eIF4A3, MAGO and CWC22. We found that P. tetraurelia likely assembles an active EJC with only three of the core proteins, since MLN51 is lacking. Silencing of eIF4A3 or CWC22 genes, but not that of MAGO, caused lethality. Silencing of the MAGO gene caused either an increase, decrease, or no change in intron retention levels of some intron-containing mRNAs used as reporters. We suggest that a fine-tuning expression of EJC genes is required for steady intron removal in P. tetraurelia. Taking into consideration our results and those published by others, we conclude that the EJC controls splicing independently of the intron size.