Dicer-like Enzymes with Sequence Cleavage Preferences.

Dicer proteins are known to produce small RNAs (sRNAs) from long double-stranded RNA (dsRNA) templates. These sRNAs are bound by Argonaute proteins, which select the guide strand, often with a 5' end sequence bias. However, Dicer proteins have never been shown to have sequence cleavage preferences. In Paramecium development, two classes of sRNAs that are required for DNA elimination are produced by three Dicer-like enzymes: Dcl2, Dcl3, and Dcl5. Through in vitro cleavage assays, we demonstrate that Dcl2 has a strict size preference for 25 nt and a sequence preference for 5' U and 5' AGA, while Dcl3 has a sequence preference for 5' UNG. Dcl5, however, has cleavage preferences for 5' UAG and 3' CUAC/UN, which leads to the production of RNAs precisely matching short excised DNA elements with corresponding end base preferences. Thus, we characterize three Dicer-like enzymes that are involved in Paramecium development and propose a biological role for their sequence-biased cleavage products.

Paramecium Genetics, Genomics, and Evolution.

The ciliate genus Paramecium served as one of the first model systems in microbial eukaryotic genetics, contributing much to the early understanding of phenomena as diverse as genome rearrangement, cryptic speciation, cytoplasmic inheritance, and endosymbiosis, as well as more recently to the evolution of mating types, introns, and roles of small RNAs in DNA processing. Substantial progress has recently been made in the area of comparative and population genomics. Paramecium species combine some of the lowest known mutation rates with some of the largest known effective populations, along with likely very high recombination rates, thereby harboring a population-genetic environment that promotes an exceptionally efficient capacity for selection. As a consequence, the genomes are extraordinarily streamlined, with very small intergenic regions combined with small numbers of tiny introns. The subject of the bulk of Paramecium research, the ancient Paramecium aurelia species complex, is descended from two whole-genome duplication events that retain high degrees of synteny, thereby providing an exceptional platform for studying the fates of duplicate genes. Despite having a common ancestor dating to several hundred million years ago, the known descendant species are morphologically indistinguishable, raising significant questions about the common view that gene duplications lead to the origins of evolutionary novelties.

Chromatin remodeling is required for sRNA-guided DNA elimination in Paramecium.

Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The unicellular ciliate Paramecium is a model to study dynamic genome organization in eukaryotic cells, given its unique feature of nuclear dimorphism. Here, the formation of the somatic macronucleus during sexual reproduction requires eliminating thousands of transposon remnants (IESs) and transposable elements scattered throughout the germline micronuclear genome. The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries. Here we show that IES recognition and precise excision are facilitated by recruiting ISWI1, a Paramecium homolog of the chromatin remodeler ISWI. ISWI1 knockdown substantially inhibits DNA elimination, quantitatively similar to development-specific sRNA gene knockdowns but with much greater aberrant IES excision at alternative boundaries. We also identify key development-specific sRNA biogenesis and transport proteins, Ptiwi01 and Ptiwi09, as ISWI1 cofactors in our co-immunoprecipitation studies. Nucleosome profiling indicates that increased nucleosome density correlates with the requirement for ISWI1 and other proteins necessary for IES excision. We propose that chromatin remodeling together with small RNAs is essential for efficient and precise DNA elimination in Paramecium.

GC content, but not nucleosome positioning, directly contributes to intron splicing efficiency in Paramecium.

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur cotranscriptionally, and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We show that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, among the smallest periodicities reported. Our analysis has revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint favoring introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for nonsense-mediated decay effectors, we show that introns located at the edge of nucleosomes display higher splicing efficiency than those at the center. However, multiple regression analysis indicates that the low GC content of introns, rather than nucleosome positioning, is associated with high splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning, and intron evolution in Paramecium.

Broad domains of histone marks in the highly compact Paramecium macronuclear genome.

The unicellular ciliate Paramecium contains a large vegetative macronucleus with several unusual characteristics, including an extremely high coding density and high polyploidy. As macronculear chromatin is devoid of heterochromatin, our study characterizes the functional epigenomic organization necessary for gene regulation and proper Pol II activity. Histone marks (H3K4me3, H3K9ac, H3K27me3) reveal no narrow peaks but broad domains along gene bodies, whereas intergenic regions are devoid of nucleosomes. Our data implicate H3K4me3 levels inside ORFs to be the main factor associated with gene expression, and H3K27me3 appears in association with H3K4me3 in plastic genes. Silent and lowly expressed genes show low nucleosome occupancy, suggesting that gene inactivation does not involve increased nucleosome occupancy and chromatin condensation. Because of a high occupancy of Pol II along highly expressed ORFs, transcriptional elongation appears to be quite different from that of other species. This is supported by missing heptameric repeats in the C-terminal domain of Pol II and a divergent elongation system. Our data imply that unoccupied DNA is the default state, whereas gene activation requires nucleosome recruitment together with broad domains of H3K4me3. In summary, gene activation and silencing in Paramecium run counter to the current understanding of chromatin biology.

The genome and comparative transcriptome of the euryhaline model ciliate Paramecium duboscqui reveal adaptations to environmental salinity.

BACKGROUND: As a potential model organism for studies of environmental and cell biology, Paramecium duboscqui is a special euryhaline species of Paramecium that can be found in fresh, brackish, or marine water in natural salinity ranges between 0‰ and 33‰. However, the genome information as well as molecular mechanisms that account for its remarkable halotolerant traits remain extremely unknown. To characterize its genome feature, we combined PacBio and Illumina sequencing to assemble the first high-quality and near-complete macronuclear genome of P. duboscqui. Meanwhile, comparative transcriptomic profiles under different salinities gave underlying insight into the molecular mechanism of its adaptations to environmental salinity. RESULTS: The results showed that the MAC genome of P. duboscqui comprises 160 contigs, with 113 of them possessing telomere (~ 28.82 Mb haploid genome size). Through comparative genomic analyses with the other ciliate, we found that gene families encoding transmembrane transporter proteins have been expanded in P. duboscqui, showing enormous potential in salinity adaptation. Like other Paramecium, P. duboscqui utilizes TGA as its only termination codon and has reassigned TAA and TAG to encode glutamine. P. duboscqui showed different growth rates under different salinities, with an optimum growth rate in 5‰ salinity. A comparison of the transcriptomic profiles among P. duboscqui grown under different concentrations showed that genes involved in protein folding, oxygen respiration, and glutathione-dependent detoxification were upregulated in the high-salt group, whereas genes encoding DNA-binding proteins and transcription factors were upregulated in the low-salt group, suggesting distinct mechanisms for responding to low and high salinity. Weighted gene coexpression network analysis (WGCNA) linked the hub genes expressed at 30‰ salinity to cysteine-type peptidase activity, lipid transfer, sodium hydrogen exchange, and cell division, with the hub genes expressed at 0‰ salinity involved in transmembrane transport and protein localization. CONCLUSIONS: This study characterizes a new euryhaline model Paramecium, provides novel insights into Paramecium evolution, and describes the molecular mechanisms that have allow P. duboscqui to adapt to different osmotic environments.

Dynamics of Gene Loss following Ancient Whole-Genome Duplication in the Cryptic Paramecium Complex.

Whole-genome duplications (WGDs) have shaped the gene repertoire of many eukaryotic lineages. The redundancy created by WGDs typically results in a phase of massive gene loss. However, some WGD-derived paralogs are maintained over long evolutionary periods, and the relative contributions of different selective pressures to their maintenance are still debated. Previous studies have revealed a history of three successive WGDs in the lineage of the ciliate Paramecium tetraurelia and two of its sister species from the Paramecium aurelia complex. Here, we report the genome sequence and analysis of 10 additional P. aurelia species and 1 additional out group, revealing aspects of post-WGD evolution in 13 species sharing a common ancestral WGD. Contrary to the morphological radiation of vertebrates that putatively followed two WGD events, members of the cryptic P. aurelia complex have remained morphologically indistinguishable after hundreds of millions of years. Biases in gene retention compatible with dosage constraints appear to play a major role opposing post-WGD gene loss across all 13 species. In addition, post-WGD gene loss has been slower in Paramecium than in other species having experienced genome duplication, suggesting that the selective pressures against post-WGD gene loss are especially strong in Paramecium. A near complete lack of recent single-gene duplications in Paramecium provides additional evidence for strong selective pressures against gene dosage changes. This exceptional data set of 13 species sharing an ancestral WGD and 2 closely related out group species will be a useful resource for future studies on Paramecium as a major model organism in the evolutionary cell biology.

Paramecium (Oligohymenophorea, Ciliophora) diversity in Thailand sheds light on the genus biogeography and reveals new phylogenetic lineages.

Paramecium (Ciliophora, Oligohymenophorea) is a good model to study ciliate biogeography. Extensive sampling mainly in northern hemisphere has led to 16 valid morphological species description thus far. However, a majority of hard-to-reach regions, including South East Asia, are underinvestigated. Our study combined traditional morphological and molecular approaches to reveal the biodiversity of Paramecium in Thailand from more than 110 samples collected in 10 provinces. Representatives of seven morphological species were identified from our collection, including the rare species, such as P. gigas and P. jenningsi. Additionally, we detected five different sibling species of the P. aurelia complex, described a new cryptic species P. hiwatashii n. sp. phylogenetically related to P. caudatum, and discovered a potentially new genetic species of the P. bursaria species complex. We also documented a variety of bacterial cytoplasmic symbionts from at least nine monoclonal cultures of Paramecium.

Anoctamin-like protein 1 regulates repolarization in Paramecium behavioral responses.

Paramecium exhibits responsive behavior to environmental changes, moving either closer to or further away from stimuli. Electrophysiological experiments have revealed that these behavioral responses are controlled by membrane potentials. Anoctamin, a Ca2+-activated Cl- channel, is involved in the regulation of membrane potential in mammals. However, it remains uncertain whether Cl- channels like anoctamin regulate Paramecium behavior. Herein, replacement of external Cl- ions with acetate ion and application of Cl- channel blocker niflumic acid (NFA, 0.1 µM) increased spontaneous avoiding reactions (sARs). Hence, we hypothesized that anoctamin is involved in the stabilization of membrane potential fluctuation. Paramecium cells in which the anoctamin-like protein 1 gene was knocked down displayed frequent sARs in the culture medium without external stimulation. Treatment of anoctamin-like protein 1-knockdown cells with the Ca2+ chelator BAPTA or Ca-channel blocker nicardipine reversed the increase in sARs. Electrophysiological experiments revealed extension of membrane depolarization when positive currents were applied to anoctamin-like protein 1-knockdown cells. We concluded that anoctamin-like protein 1 works as a Cl-channel and stabilizes the membrane potential oscillation, reducing sARs.

Emergent RNA-RNA interactions can promote stability in a facultative phototrophic endosymbiosis.

Eukaryote-eukaryote endosymbiosis was responsible for the spread of chloroplast (plastid) organelles. Stability is required for the metabolic and genetic integration that drives the establishment of new organelles, yet the mechanisms that act to stabilize emergent endosymbioses-between two fundamentally selfish biological organisms-are unclear. Theory suggests that enforcement mechanisms, which punish misbehavior, may act to stabilize such interactions by resolving conflict. However, how such mechanisms can emerge in a facultative endosymbiosis has yet to be explored. Here, we propose that endosymbiont-host RNA-RNA interactions, arising from digestion of the endosymbiont population, can result in a cost to host growth for breakdown of the endosymbiosis. Using the model facultative endosymbiosis between Paramecium bursaria and Chlorella spp., we demonstrate that this mechanism is dependent on the host RNA-interference (RNAi) system. We reveal through small RNA (sRNA) sequencing that endosymbiont-derived messenger RNA (mRNA) released upon endosymbiont digestion can be processed by the host RNAi system into 23-nt sRNA. We predict multiple regions of shared sequence identity between endosymbiont and host mRNA, and demonstrate through delivery of synthetic endosymbiont sRNA that exposure to these regions can knock down expression of complementary host genes, resulting in a cost to host growth. This process of host gene knockdown in response to endosymbiont-derived RNA processing by host RNAi factors, which we term "RNAi collisions," represents a mechanism that can promote stability in a facultative eukaryote-eukaryote endosymbiosis. Specifically, by imposing a cost for breakdown of the endosymbiosis, endosymbiont-host RNA-RNA interactions may drive maintenance of the symbiosis across fluctuating ecological conditions.

Paramecium: RNA sequence-structure phylogenetics.

Organisms classified as members of the genus Paramecium belong to the best-known group of single-celled eukaryotes. Nevertheless, the phylogeny within the genus Paramecium has been discussed and revisited in recent decades and remains partly unresolved. By applying an RNA sequence-structure approach, we attempt to increase accuracy and robustness of phylogenetic trees. For each individual 18S and internal transcribed spacer 2 (ITS2) sequence, a putative secondary structure was predicted through homology modelling. While searching for a structural template, we found, in contrast to the available literature, that the ITS2 molecule consists of three helices in members of the genus Paramecium and four helices in members of the genus Tetrahymena. Two sequencestructure neighbor-joining overall trees were reconstructed with (1) more than 400 taxa (ITS2) and (2) more than 200 taxa (18S). For smaller subsets, neighbor-joining, maximum-parsimony, and maximum-likelihood analyses were executed using sequence-structure information simultaneously. Based on a combined data set (ITS2+18S rDNA) a well-supported tree was reconstructed with bootstrap values over 50 in at least one of the applied analyses. Our results are in general agreement with those published in the available literature based on multi-gene analyses. Our study supports the simultaneous use of sequence-structure data to reconstruct accurate and robust phylogenetic trees.

The Paramecium histone chaperone Spt16-1 is required for Pgm endonuclease function in programmed genome rearrangements.

In Paramecium tetraurelia, a large proportion of the germline genome is reproducibly removed from the somatic genome after sexual events via a process involving small (s)RNA-directed heterochromatin formation and DNA excision and repair. How germline limited DNA sequences are specifically recognized in the context of chromatin remains elusive. Here, we use a reverse genetics approach to identify factors involved in programmed genome rearrangements. We have identified a P. tetraurelia homolog of the highly conserved histone chaperone Spt16 subunit of the FACT complex, Spt16-1, and show its expression is developmentally regulated. A functional GFP-Spt16-1 fusion protein localized exclusively in the nuclei where genome rearrangements take place. Gene silencing of Spt16-1 showed it is required for the elimination of all germline-limited sequences, for the survival of sexual progeny, and for the accumulation of internal eliminated sequence (ies)RNAs, an sRNA population produced when elimination occurs. Normal accumulation of 25 nt scanRNAs and deposition of silent histone marks H3K9me3 and H3K27me3 indicated that Spt16-1 does not regulate the scanRNA-directed heterochromatin pathway involved in the early steps of DNA elimination. We further show that Spt16-1 is required for the correct nuclear localization of the PiggyMac (Pgm) endonuclease, which generates the DNA double-strand breaks required for DNA elimination. Thus, Spt16-1 is essential for Pgm function during programmed genome rearrangements. We propose a model in which Spt16-1 mediates interactions between the excision machinery and chromatin, facilitating endonuclease access to DNA cleavage sites during genome rearrangements.

Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination.

Gene duplication and diversification drive the emergence of novel functions during evolution. Because of whole genome duplications, ciliates from the Paramecium aurelia group constitute a remarkable system to study the evolutionary fate of duplicated genes. Paramecium species harbor two types of nuclei: a germline micronucleus (MIC) and a somatic macronucleus (MAC) that forms from the MIC at each sexual cycle. During MAC development, ~45,000 germline Internal Eliminated Sequences (IES) are excised precisely from the genome through a 'cut-and-close' mechanism. Here, we have studied the P. tetraurelia paralogs of KU80, which encode a key DNA double-strand break repair factor involved in non-homologous end joining. The three KU80 genes have different transcription patterns, KU80a and KU80b being constitutively expressed, while KU80c is specifically induced during MAC development. Immunofluorescence microscopy and high-throughput DNA sequencing revealed that Ku80c stably anchors the PiggyMac (Pgm) endonuclease in the developing MAC and is essential for IES excision genome-wide, providing a molecular explanation for the previously reported Ku-dependent licensing of DNA cleavage at IES ends. Expressing Ku80a under KU80c transcription signals failed to complement a depletion of endogenous Ku80c, indicating that the two paralogous proteins have distinct properties. Domain-swap experiments identified the α/ß domain of Ku80c as the major determinant for its specialized function, while its C-terminal part is required for excision of only a small subset of IESs located in IES-dense regions. We conclude that Ku80c has acquired the ability to license Pgm-dependent DNA cleavage, securing precise DNA elimination during programmed rearrangements. The present study thus provides novel evidence for functional diversification of genes issued from a whole-genome duplication.

N-glycans from Paramecium bursaria chlorella virus MA-1D: Re-evaluation of the oligosaccharide common core structure.

Paramecium bursaria chlorella virus MA-1D is a chlorovirus that infects Chlorella variabilis strain NC64A, a symbiont of the protozoan Paramecium bursaria. MA-1D has a 339-kb genome encoding ca. 366 proteins and 11 tRNAs. Like other chloroviruses, its major capsid protein (MCP) is decorated with N-glycans, whose structures have been solved in this work by using nuclear magnetic spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry along with MS/MS experiments. This analysis identified three N-linked oligosaccharides that differ in the nonstoichiometric presence of three monosaccharides, with the largest oligosaccharide composed of eight residues organized in a highly branched fashion. The N-glycans described here share several features with those of the other chloroviruses except that they lack a distal xylose unit that was believed to be part of a conserved core region for all the chloroviruses. Examination of the MA-1D genome detected a gene with strong homology to the putative xylosyltransferase in the reference chlorovirus PBCV-1 and in virus NY-2A, albeit mutated with a premature stop codon. This discovery means that we need to reconsider the essential features of the common core glycan region in the chloroviruses.

Genetics and Epigenetics of Mating Type Determination in Paramecium and Tetrahymena.

While sex is an ancient and highly conserved eukaryotic invention, self-incompatibility systems such as mating types or sexes appear to be derived limitations that show considerable evolutionary plasticity. Within a single class of ciliates, Paramecium and Tetrahymena species have long been known to present a wide variety of mating type numbers and modes of inheritance, but only recently have the genes involved been identified. Although similar transmembrane proteins mediate self/nonself recognition in both ciliates, the mechanisms of mating type determination differ widely, ranging from Mendelian systems to developmental nuclear differentiation, either stochastic or maternally inherited. The non-Mendelian systems rely on programmed editing of the germline genome that occurs during differentiation of the somatic nucleus, and they have co-opted different DNA recombination mechanisms-some previously unknown. Here we review the recent molecular advances and some remaining unsolved questions and discuss the possible implications of these diverse mechanisms for inbreeding/outbreeding balance regulation.

The conformational cycle of a prototypical voltage-gated sodium channel.

Electrical signaling was a dramatic development in evolution, allowing complex single-cell organisms like Paramecium to coordinate movement and early metazoans like worms and jellyfish to send regulatory signals rapidly over increasing distances. But how are electrical signals generated in biology? In fact, voltage-gated sodium channels conduct sodium currents that initiate electrical signals in all kingdoms of life, from bacteria to man. They are responsible for generating the action potential in vertebrate nerve and muscle, neuroendocrine cells, and other cell types1,2. Because of the high level of conservation of their core structure, it is likely that their fundamental mechanisms of action are conserved as well. Here we describe the complete cycle of conformational changes that a bacterial sodium channel undergoes as it transitions from resting to activated/open and inactivated/closed states, based on high-resolution structural studies of a single sodium channel. We further relate this conformational cycle of the ancestral sodium channel to the function of its vertebrate orthologs. The strong conservation of amino acid sequence and three-dimensional structure suggests that this model, at a fundamental level, is relevant for both prokaryotic and eukaryotic sodium channels, as well as voltage-gated calcium and potassium channels.

Paramecium epigenetics in development and proliferation.

The term epigenetics is used for any layer of genetic information aside from the DNA base-sequence information. Mammalian epigenetic research increased our understanding of chromatin dynamics in terms of cytosine methylation and histone modification during differentiation, aging, and disease. Instead, ciliate epigenetics focused more on small RNA-mediated effects. On the one hand, these do concern the transport of RNA from parental to daughter nuclei, representing a regulated transfer of epigenetic information across generations. On the other hand, studies of Paramecium, Tetrahymena, Oxytricha, and Stylonychia revealed an almost unique function of transgenerational RNA. Rather than solely controlling chromatin dynamics, they control sexual progeny's DNA content quantitatively and qualitatively. Thus epigenetics seems to control genetics, at least genetics of the vegetative macronucleus. This combination offers ciliates, in particular, an epigenetically controlled genetic variability. This review summarizes the epigenetic mechanisms that contribute to macronuclear heterogeneity and relates these to nuclear dimorphism. This system's adaptive and evolutionary possibilities raise the critical question of whether such a system is limited to unicellular organisms or binuclear cells. We discuss here the relevance of ciliate genetics and epigenetics to multicellular organisms.

Comparative Analysis Between Paramecium Strains with Different Syngens Using the RAPD Method.

Paramecium spp. are a genus of free-living protists that live mainly in freshwater environments. They are ciliates with high motility and phagocytosis and have been used to analyze cell motility and as a host model for pathogens. Besides such biological characteristics, apart from the usual morphological and genetic classification of species, the existence of taxonomies (such as syngens) and mating types related to Paramecium's unique reproduction is known. In this study, we attempted to develop a simple method to identify Paramecium strains, which are difficult to distinguish morphologically, using random amplified polymorphic DNA (RAPD) analysis. Consequently, we can observe strain-specific band patterns. We also confirm that the presence of endosymbiotic Chlorella cells affects the band pattern of P. bursaria. Furthermore, the results of the RAPD analysis using several P. caudatum strains with different syngens show that it is possible to detect a band specific to a certain syngen. By improving the reaction conditions and random primers, based on the results of this study, RAPD analysis can be applied to the identification of Paramecium strains and their syngen confirmation tests.

ParameciumDB 2019: integrating genomic data across the genus for functional and evolutionary biology.

ParameciumDB (https://paramecium.i2bc.paris-saclay.fr) is a community model organism database for the genome and genetics of the ciliate Paramecium. ParameciumDB development relies on the GMOD (www.gmod.org) toolkit. The ParameciumDB web site has been publicly available since 2006 when the P. tetraurelia somatic genome sequence was released, revealing that a series of whole genome duplications punctuated the evolutionary history of the species. The genome is linked to available genetic data and stocks. ParameciumDB has undergone major changes in its content and website since the last update published in 2011. Genomes from multiple Paramecium species, especially from the P. aurelia complex, are now included in ParameciumDB. A new modern web interface accompanies this transition to a database for the whole Paramecium genus. Gene pages have been enriched with orthology relationships, among the Paramecium species and with a panel of model organisms across the eukaryotic tree. This update also presents expert curation of Paramecium mitochondrial genomes.

Diversification of small RNA amplification mechanisms for targeting transposon-related sequences in ciliates.

The silencing of repetitive transposable elements (TEs) is ensured by signal amplification of the initial small RNA trigger, which occurs at distinct steps of TE silencing in different eukaryotes. How such a variety of secondary small RNA biogenesis mechanisms has evolved has not been thoroughly elucidated. Ciliated protozoa perform small RNA-directed programmed DNA elimination of thousands of TE-related internal eliminated sequences (IESs) in the newly developed somatic nucleus. In the ciliate Paramecium, secondary small RNAs are produced after the excision of IESs. In this study, we show that in another ciliate, Tetrahymena, secondary small RNAs accumulate at least a few hours before their derived IESs are excised. We also demonstrate that DNA excision is dispensable for their biogenesis in this ciliate. Therefore, unlike in Paramecium, small RNA amplification occurs before IES excision in Tetrahymena This study reveals the remarkable diversity of secondary small RNA biogenesis mechanisms, even among ciliates with similar DNA elimination processes, and thus raises the possibility that the evolution of TE-targeting small RNA amplification can be traced by investigating the DNA elimination mechanisms of ciliates.