Small RNAs target their complementary chromatin regions for gene silencing through nascent long non-coding RNAs (lncRNAs). In the ciliated protozoan
, the interaction between Piwi-associated small ...RNAs (scnRNAs) and the nascent lncRNA transcripts from the somatic genome has been proposed to induce target-directed small RNA degradation (TDSD), and scnRNAs not targeted for TDSD later target the germline-limited sequences for programmed DNA elimination. In this study, we show that the SUMO E3 ligase Ema2 is required for the accumulation of lncRNAs from the somatic genome and thus for TDSD and completing DNA elimination to make viable sexual progeny. Ema2 interacts with the SUMO E2 conjugating enzyme Ubc9 and enhances SUMOylation of the transcription regulator Spt6. We further show that Ema2 promotes the association of Spt6 and RNA polymerase II with chromatin. These results suggest that Ema2-directed SUMOylation actively promotes lncRNA transcription, which is a prerequisite for communication between the genome and small RNAs.
Programmed genome rearrangements in ciliates provide fascinating examples of flexible epigenetic genome regulations and important insights into the interaction between transposable elements (TEs) and ...host genomes. DNA elimination in Tetrahymena thermophila removes approximately 12 000 internal eliminated sequences (IESs), which correspond to one-third of the genome, when the somatic macronucleus (MAC) differentiates from the germline micronucleus (MIC). More than half of the IESs, many of which show high similarity to TEs, are targeted for elimination in cis by the small RNA-mediated genome comparison of the MIC to the MAC. Other IESs are targeted for elimination in trans by the same small RNAs through repetitive sequences. Furthermore, the small RNA–heterochromatin feedback loop ensures robust DNA elimination. Here, we review an updated picture of the DNA elimination mechanism, discuss the physiological and evolutionary roles of DNA elimination, and outline the key questions that remain unanswered.
The ciliated protozoan Tetrahymena undergoes extensive programmed DNA elimination when the germline micronucleus produces the new macronucleus during sexual reproduction. DNA elimination is ...epigenetically controlled by DNA sequences of the parental macronuclear genome, and this epigenetic regulation is mediated by small RNAs (scan RNAs scnRNAs) of ∼28-30 nucleotides that are produced and function by an RNAi-related mechanism. Here, we examine scnRNA production and turnover by deep sequencing. scnRNAs are produced exclusively from the micronucleus and nonhomogeneously from a variety of chromosomal locations. scnRNAs are preferentially derived from the eliminated sequences, and this preference is mainly determined at the level of transcription. Despite this bias, a significant fraction of scnRNAs is also derived from the macronuclear-destined sequences, and these scnRNAs are degraded during the course of sexual reproduction. These results indicate that the pattern of DNA elimination in the new macronucleus is shaped by the biased transcription in the micronucleus and the selective degradation of scnRNAs in the parental macronucleus.
The germline genome of the binucleated ciliate
undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of ...the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped
chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare
's germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.
Small RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the ...germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production.
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•Two types of siRNAs (scnRNAs) are expressed in Tetrahymena sexual reproduction•Early-scnRNAs are produced from Type-A IESs containing potentially active transposons•Early-scnRNAs trans-recognize Type-B IESs and trigger late-scnRNA production in cis•Late-scnRNA production forms trans-recognition network for robust IES elimination
Noto et al. show that a class of Tetrahymena small RNAs from internal eliminated sequences (IESs) containing potentially active transposons recognize not only the IESs from which they are derived but also other IESs in trans, triggering the expression of another class of small RNAs and ensuring robust DNA elimination.
Tetrahymena eliminates micronuclear-limited sequences from the developing macronucleus during sexual reproduction. Homology between the sequences to be eliminated and approximately 28-nucleotide ...small RNAs (scnRNAs) associated with an Argonaute family protein Twi1p likely underlies this elimination process. However, the mechanism by which Twi1p-scnRNA complexes identify micronuclear-limited sequences is not well understood. We show that a Twi1p-associated putative RNA helicase Ema1p is required for the interaction between Twi1p and chromatin. This requirement explains the phenotypes of EMA1 KO strains, including loss of selective down-regulation of scnRNAs homologous to macronuclear-destined sequences, loss of H3K9 and K27 methylation in the developing new macronucleus, and failure to eliminate DNA. We further demonstrate that Twi1p interacts with noncoding transcripts derived from parental and developing macronuclei and this interaction is greatly reduced in the absence of Ema1p. We propose that Ema1p functions in DNA elimination by stimulating base-pairing interactions between scnRNAs and noncoding transcripts in both parental and developing new macronuclei.
RNAi-mediated positive feedback loops are pivotal for the maintenance of heterochromatin, but how they are downregulated at heterochromatin-euchromatin borders is not well understood. In the ciliated ...protozoan Tetrahymena, heterochromatin is formed exclusively on the sequences that are removed from the somatic genome by programmed DNA elimination, and an RNAi-mediated feedback loop is important for assembling heterochromatin on the eliminated sequences. In this study, we show that the heterochromatin protein 1 (HP1)-like protein Coi6p, its interaction partners Coi7p and Lia5p, and the histone demethylase Jmj1p are crucial for confining the production of small RNAs and the formation of heterochromatin to the eliminated sequences. The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. The results provide direct evidence for the existence of a dedicated mechanism that counteracts a positive feedback loop between RNAi and heterochromatin at heterochromatin-euchromatin borders to maintain the integrity of the somatic genome.
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•The HP1-like protein Coi6p confines small RNA and heterochromatin formation•Two Coi6p-binding proteins and the histone demethylase Jmj1p likely act with Coi6p•Coi6p and Jmj1p are important for preventing ectopic DNA elimination•Suppression of RNAi-heterochromatin feedback loop maintains somatic genome integrity
Regulation of a small RNA-mediated positive feedback loop is pivotal for confining heterochromatin to proper locations. Suhren et al. show the existence of a dedicated mechanism that counteracts a small RNA-heterochromatin positive feedback loop at heterochromatin-euchromatin borders to maintain the integrity of the somatic genome in Tetrahymena.
Epigenetic inheritance of acquired traits is widespread among eukaryotes, but how and to what extent such information is transgenerationally inherited is still unclear. The patterns of programmed DNA ...elimination in ciliates are epigenetically and transgenerationally inherited, and it has been proposed that small RNAs, which shuttle between the germline and the soma, regulate this epigenetic inheritance. In this study, we test the existence and role of such small-RNA-mediated communication by epigenetically disturbing the pattern of DNA elimination in Tetrahymena. We show that the pattern of DNA elimination is, indeed, determined by the selective turnover of small RNAs, which is induced by the interaction between germline-derived small RNAs and the somatic genome. In addition, we show that DNA elimination of an element is regulated by small-RNA-mediated communication with other eliminated elements. By contrast, no evidence obtained thus far supports the notion that transfer of epigenetic information from the soma to the germline, if any, regulates DNA elimination. Our results indicate that small-RNA-mediated trans-nuclear and trans-element communication, in addition to unknown information in the germline genome, contributes to determining the pattern of DNA elimination.
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•Small RNA turnover mediates germline-soma communication to regulate DNA elimination•Small-RNA-mediated inter-element communication also regulates DNA elimination•The initial small RNA production pattern is likely determined genetically
Noto and Mochizuki report that the pattern of DNA elimination in Tetrahymena is determined epigenetically by germline-soma and trans-element communications, which are mediated by the selective turnover and secondary production of small RNAs, respectively, and likely also genetically by unknown information in the germline genome.
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.
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