Masatoshi Mutazono

Tomoko Noto

Kazufumi Mochizuki
email: kazufumi.mochizuki@igh.cnrs.fr

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

Keywords: small RNA, transposon silencing, DNA elimination, ciliate

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 RNAdirected 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 prior to IES excision in Tetrahymena. This study reveals the remarkable diversity of secondary small RNA biogenesis mechanisms, even among ciliates showing 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.

Significance statement

Secondary amplification of small RNA triggers is necessary to silence repetitive transposable elements (TEs) and thus to maintain genome integrity. Despite their importance, these secondary small RNAs are produced by various mechanisms in different eukaryotes. How such a variety of mechanisms has evolved has not been thoroughly elucidated. In this report, we show that in the ciliate Tetrahymena secondary small RNA production occurs from the eliminated sequences prior to their excision during the small RNA-directed programmed DNA elimination of TE-related sequences.

This situation contrast with the excision-dependent secondary small RNA production observed in another ciliate, Paramecium. This study therefore shows the possibility of tracing the evolution of TE-targeting small RNA amplification by investigating DNA elimination mechanisms of ciliates. \body

Introduction

Signal amplification of the initial small RNA trigger is important to ensure the silencing of repetitive transposable elements (TEs). Curiously, secondary small RNA biogenesis occurs by various mechanisms that are coupled with distinct steps of TE silencing in different eukaryotes, such as nucleolytic cleavage of TE transcripts, recruitment of RNAdependent RNA polymerase, and heterochromatin-directed transcription [START_REF] Czech | piRNA-Guided Genome Defense: From Biogenesis to Silencing[END_REF][START_REF] Gebert | RNA-based regulation of transposon expression[END_REF][START_REF] Castel | RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond[END_REF][START_REF] Zhou | RNA Pol IV and V in gene silencing: Rebel polymerases evolving away from Pol II's rules[END_REF][START_REF] Luteijn | PIWI-interacting RNAs: from generation to transgenerational epigenetics[END_REF]. To understand how such a variety of small RNA amplification mechanisms have evolved, it is important to compare evolutionarily closely related TE-silencing processes that have distinct secondary small RNA biogenesis mechanisms. This study raises the possibility that the programmed DNA elimination mechanisms of ciliated protozoa can be used for such a comparative evolutionary biology approach.

Like most other ciliates, each cell of Tetrahymena thermophila (hereafter referred to as Tetrahymena) contains two types of nuclei: the transcriptionally inactive germline micronucleus (MIC) and the transcriptionally active somatic macronucleus (MAC).

During the sexual process of conjugation, the MIC produces new MICs and MACs, while the parental MAC is discarded. The MIC genome includes ~12,000 IESs, which are removed from the newly formed MAC. IESs contain many potentially active TEs and their remnants [START_REF] Hamilton | Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome[END_REF]. Each IES can be excised in linear or circular form. Because excised IESs in linear form are very short lived [START_REF] Austerberry | Specific DNA rearrangements in synchronously developing nuclei of Tetrahymena[END_REF][START_REF] Saveliev | Product analysis illuminates the final steps of IES deletion in Tetrahymena thermophila[END_REF] while circularized IESs are stable for more than several hours [START_REF] Saveliev | The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila[END_REF], circularized IESs should be produced either directly during the excision process or from liner IESs right after their excision.

Small RNAs play a key role in the DNA elimination in Tetrahymena [START_REF] Chalker | Epigenetics of ciliates[END_REF][START_REF] Noto | Whats, hows and whys of programmed DNA elimination in Tetrahymena[END_REF][START_REF] Yao | Programmed DNA deletion as an RNA-guided system of genome defense[END_REF]. At early conjugation stages (~2-3 hours post mixing [hpm]), ~60% of IESs, called Type-A IESs, and their genomic surroundings are bi-directionally transcribed in the MIC [START_REF] Chalker | Nongenic, bidirectional transcription precedes and may promote developmental DNA deletion in Tetrahymena thermophila[END_REF][START_REF] Schoeberl | Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena[END_REF], and the transcripts are processed to ~26-32-nt primary small RNAs called early-scnRNAs [START_REF] Mochizuki | A Dicer-like protein in Tetrahymena has distinct functions in genome rearrangement, chromosome segregation, and meiotic prophase[END_REF][START_REF] Malone | Germ line transcripts are processed by a Dicer-like protein that is essential for developmentally programmed genome rearrangements of Tetrahymena thermophila[END_REF]. Early-scnRNAs are loaded into the Piwi-clade Argonaute protein Twi1p [START_REF] Mochizuki | Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in tetrahymena[END_REF][START_REF] Noto | The Tetrahymena argonaute-binding protein Giw1p directs a mature argonaute-siRNA complex to the nucleus[END_REF] and move into the parental MAC, where those complementary to the MAC genome (i.e., non-IES sequences) are degraded by the process called scnRNA selection [START_REF] Schoeberl | Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena[END_REF][START_REF] Noto | Small RNA-Mediated trans-Nuclear and trans-Element Communications in Tetrahymena DNA Elimination[END_REF]. The remaining IES-specific early-scnRNAs are then shuttled into the developing new MACs (~7-8 hpm) and are believed to base pair with nascent transcripts from Type-A and Type-B IESs, the latter of which constitute ~40% of all IESs and share repetitive sequences with Type-A IESs [START_REF] Noto | Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination[END_REF]. These interactions recruit the Enhancer of zeste [E(z)]-like methyltransferase Ezl1p, which mediates the accumulation of tri-methylations of histone H3 at lysine 9 (H3K9me3) and lysine 27 (H3K27me3) and the HP1-like protein Pdd1p to IESs [START_REF] Liu | RNAi-dependent H3K27 methylation is required for heterochromatin formation and DNA elimination in Tetrahymena[END_REF][START_REF] Taverna | Methylation of histone h3 at lysine 9 targets programmed DNA elimination in tetrahymena[END_REF]. This heterochromatin nucleation induces the production of ~26-32-nt secondary small RNAs, called late-scnRNAs [START_REF] Noto | Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination[END_REF], and the excision of IESs by the domesticated piggyBac transposase Tpb2p [START_REF] Cheng | A domesticated piggyBac transposase plays key roles in heterochromatin dynamics and DNA cleavage during programmed DNA deletion in Tetrahymena thermophila[END_REF][START_REF] Vogt | A domesticated PiggyBac transposase interacts with heterochromatin and catalyzes reproducible DNA elimination in Tetrahymena[END_REF]. The flanking sequences are religated by non-homologous end joining (NHEJ) [START_REF] Lin | An essential role for the DNA breakage-repair protein Ku80 in programmed DNA rearrangements in Tetrahymena thermophila[END_REF]. Besides Type-A and Type-B IESs, there are ~20 IESs in the genome that produce few scnRNAs and are removed by the other piggyBac transposases, Tpb1p and Tpb6p [START_REF] Cheng | The piggyBac transposon-derived genes TPB1 and TPB6 mediate essential transposon-like excision during the developmental rearrangement of key genes in Tetrahymena thermophila[END_REF][START_REF] Feng | A germline-limited piggyBac transposase gene is required for precise excision in Tetrahymena genome rearrangement[END_REF].

The other ciliate Paramecium tetraurelia (hereafter referred to as Paramecium) exhibits a very similar DNA elimination process, including Dicer-dependent scnRNA production [START_REF] Sandoval | Functional diversification of Dicer-like proteins and small RNAs required for genome sculpting[END_REF]; loading of the small RNAs into Piwi-clade Argonaute proteins [START_REF] Bouhouche | Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling[END_REF]; scnRNA selection [START_REF] Sandoval | Functional diversification of Dicer-like proteins and small RNAs required for genome sculpting[END_REF][START_REF] Lepère | Maternal noncoding transcripts antagonize the targeting of DNA elimination by scanRNAs in Paramecium tetraurelia[END_REF]; scnRNA-and an E(z)-like protein-dependent accumulation of H3K9me3 and H3K27me3 on IESs [START_REF] Lhuillier-Akakpo | Local effect of enhancer of zeste-like reveals cooperation of epigenetic and cis-acting determinants for zygotic genome rearrangements[END_REF]; IES excision by a piggyBac transposase [START_REF] Baudry | PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia[END_REF][START_REF] Bischerour | Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium[END_REF]; and repair of the excision sites by NHEJ [START_REF] Kapusta | Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining[END_REF]. Recently, it has been reported that the secondary small RNAs, called iesRNAs in Paramecium, are produced after the excision of IESs [START_REF] Allen | Circular Concatemers of Ultra-Short DNA Segments Produce Regulatory RNAs[END_REF]. The excised IESs are concatenated and circularized and proposed to serve as templates for the production of precursors for iesRNAs in this ciliate [START_REF] Allen | Circular Concatemers of Ultra-Short DNA Segments Produce Regulatory RNAs[END_REF][START_REF] Hoehener | Dicer-like Enzymes with Sequence Cleavage Preferences[END_REF]. On the other hand, although heterochromatin formation has been shown to be essential for the biogenesis of late-scnRNAs in Tetrahymena [START_REF] Noto | Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination[END_REF], it remains to be determined if the late-scnRNA production is dependent on IES excision. To examine whether the post-excision production of secondary small RNAs is conserved, we investigated the causality between the excision of IESs and the biogenesis of secondary small RNAs in Tetrahymena.

Results

Highly sensitive assay for determining the timing of de novo IES excision

To understand the temporal relationship between the excision of IESs and the production of late-scnRNAs in Tetrahymena, we first aimed to determine the timing of IES excision.

It has been reported that de novo IES excision can be detected by PCR amplification of the ligation junctions of excised IES circles (jPCR) [START_REF] Saveliev | The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila[END_REF][START_REF] Yao | Detection of circular excised DNA deletion elements in Tetrahymena thermophila during development[END_REF]. However, the sensitivity of their detection has not been examined. To detect de novo DNA elimination events with high sensitivity, we have established a method combining jPCR with the digestion of linear DNA (DLD) and rolling circular amplification (RCA), referred to as DLR-jPCR (Figure 1A). Two wild-type Tetrahymena cultures possessing different mating types were mixed to initiate conjugation, and total genomic DNA was extracted at the onset of mixing and subsequently at 2-hr intervals from 4 to 20 hr post mixing (hpm). The genomic DNA was then used in DLR-jPCR assays. We chose 9 representative IESs, including 3 each of the major IES types, Type-A, Type-B and TPB1/6-dependent IESs, to investigate the appearances of IES circles. As shown in Figure 1B, IES circles were specifically detected in the late stages of conjugation (Figure 1B, RCA+), consistent with the fact that DNA elimination occurs during the development of the new MAC, which is formed at ~7 hpm. IES circles were detected for all 9 tested IES loci, indicating that DLR-jPCR can detect de novo IES excision events, regardless of the type of IES.

The timing of the appearance of IES circles varied among different IESs. For example, circles of IES737 and IES1147 were detected at 10 hpm, whereas those of the other IESs only appeared after 12 hpm (Figure 1B). Although we have failed to find any obvious rule defining the timing of DNA excision for different IESs to date, these results are consistent with the previous notion that some IESs are eliminated earlier than others [START_REF] Austerberry | Specific DNA rearrangements in synchronously developing nuclei of Tetrahymena[END_REF]. IES circles were detected continuously for at least several hours. A previous report [START_REF] Saveliev | The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila[END_REF] suggested that this situation is caused, at least in part, by the persistence of each IES circle for several hours before it is degraded, although cell-to-cell variability of the timing of IES excision may also contribute to the continuous detection of IES circles.

We failed to detect any IES circles when we performed similar reactions without RCA (Figure 1B, RCA-), indicating that the RCA step significantly sensitizes the detection of IES circles. To further assess the sensitivity of DLR-jPCR, we prepared serial dilutions of genomic DNAs after the RCA reaction and performed jPCR. As shown in Figure 1C, we found that DNA circles from IES737 could be detected even when genomic DNA from 12 hpm was diluted 2,000 times. This result indicates that DLR-jPCR is probably >2,000 times more sensitive than the previous IES circles detection methods, which do not include the RCA step, and the dynamic range of the detection sensitivity of the DLR-jPCR assay spans more than three orders of magnitude.

Late-scnRNA production is initiated before IES excision becomes detectable

We next sought to compare the timing of the appearance of IES circles with that of late-scnRNAs. When late-scnRNAs are produced in the new MAC, early-scnRNAs expressed from the MIC at earlier stages of conjugation persist. Early-and late-scnRNAs cannot be distinguished by simple biochemical properties, such as length and base composition [START_REF] Noto | Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination[END_REF]. Therefore, to specifically investigate late-scnRNAs, we focused on Type-B IESderived small RNAs, which are predominantly, if not exclusively, late-scnRNAs [START_REF] Noto | Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination[END_REF].

Small RNAs extracted from mating wild-type cells at 3, 6, 8, 10 or 12 hpm were sequenced (small RNA-Seq), and those that uniquely mapped to the three studied Type-B IESs indicated above were counted. As shown in Figure 2A-C, small RNAs derived from these Type-B IESs remained at a low level till 6 hpm and clearly increased after 8 hpm, indicating that late-scnRNAs began to be produced from these IESs at 8 hpm at the latest.

A similar result was obtained when small RNAs were mapped to each of the 3,293 Type-B IESs (Figure 2D). The earliest time point at which IES circles were detected for these IESs was 10 hpm (IES737) or later (IES5, IES4092) (Figure 1B). Therefore, late-scnRNAs expressed from an IES can be detected by small RNA-Seq before the excision of that IES becomes detectable by DLR-jPCR.

Although these results most likely indicate that the production of late-scnRNAs occurs prior to the excision of IESs, the above data do not exclude the possibility that late-scnRNAs are produced after IES excision, but the detection of late-scnRNAs by small RNA-Seq is more sensitive than that of IES circles by DLR-jPCR. Therefore, we next aimed to use a genetic mutant that is defective in IES excision to further investigate the relationship between the production of late-scnRNAs and the IES excision.

TPB2-null mutants block the accumulation of the Tpb2p excisase

Tpb2p is important for the DNA elimination of Type-A and Type-B IESs in vivo [START_REF] Cheng | A domesticated piggyBac transposase plays key roles in heterochromatin dynamics and DNA cleavage during programmed DNA deletion in Tetrahymena thermophila[END_REF] and catalyzes DNA double-strand breaks to form a 4-base 5' overhang in vitro [START_REF] Vogt | A domesticated PiggyBac transposase interacts with heterochromatin and catalyzes reproducible DNA elimination in Tetrahymena[END_REF], which is identical to the intermediate structure observed in DNA elimination in vivo [START_REF] Saveliev | The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila[END_REF]. Therefore, Tpb2p is believed to be the enzyme catalyzing the final excision step of Type-A and Type-B IESs. Previous loss-of-function studies of TPB2 were conducted using RNAi [START_REF] Cheng | A domesticated piggyBac transposase plays key roles in heterochromatin dynamics and DNA cleavage during programmed DNA deletion in Tetrahymena thermophila[END_REF] or conditional [START_REF] Vogt | A domesticated PiggyBac transposase interacts with heterochromatin and catalyzes reproducible DNA elimination in Tetrahymena[END_REF] knockdown. In these studies, IES elimination was not completely blocked, probably due to incomplete loss of TPB2 expression. Therefore, the conditions applied in these previous genetic studies cannot be used to explicitly determine the requirement for TPB2 in late-scnRNA biogenesis.

To produce TPB2-null mutants, we employed CRISPR-based mutagenesis technology.

Although CRISPR technology was used for mutagenesis of Tetrahymena in two previous reports [START_REF] Suhren | Negative Regulators of an RNAi-Heterochromatin Positive Feedback Loop Safeguard Somatic Genome Integrity in Tetrahymena[END_REF][START_REF] Howard-Till | A specialized condensin complex participates in somatic nuclear maturation in Tetrahymena thermophila[END_REF], we have made several modifications to the previous protocols to efficiently obtain Tetrahymena mutants (see SI Materials and Methods). We introduced a construct expressing Cas9 and gRNAs into the MAC of vegetative cells of two wild-type strains (B2086 and CU428) with different mating types. We expected the Cas9-gRNA complex to introduce a mutation in the MIC (Figure 3A). We used two gRNAs that target slightly different positions of the first exon of TPB2 (Figure 3B). After the induction of Cas9 expression, the cells were cloned, and the TPB2 locus in the MIC was amplified by PCR using primers "a" and "c" shown in Figure 3A. The products were then sequenced.

The PCR amplicon contains a chromosome breakage sequence (Cbs) that remains intact in the MIC but is severed to form the ends of two separate chromosomes in the MAC (Figure 3A). This PCR assay therefore specifically amplifies the TPB2 MIC locus.

Approximately half of the clones examined contained insertions or deletions around the targeted region. We choose two clones each from the B2086 and CU428 strains that contained one of the three frameshift mutations in TPB2, which were referred to as tpb2-1fs, tpb2-2fs and tpb2-3fs (Figure 3B). We did not detect any copies of wild-type TPB2 in the MIC in these cell lines, indicating that both copies of TPB2 in the diploid MIC were disrupted in these TPB2 mutants.

The TPB2 MAC locus of the TPB2 mutants was also analyzed by PCR using primers "b" and "c" shown in Figure 3A. Although this PCR assay was designed to amplify both the MAC and MIC loci, the MIC locus was underrepresented because of the difference in ploidy between the MAC (~50n) and the MIC (2n). We detected only the wild-type TPB2 locus in these PCR products, indicating that the CRISPR system used here did not efficiently induce mutations in the MAC (Figure 3B). This result might have occurred because Cas9 was fused to part of the MIC-localized protein MLH1 in our experiment to enhance the MIC localization of Cas9 and/or because DNA double-strand breaks might be repaired predominantly by homologous recombination with the sister copies in the polyploid MAC. Nevertheless, Tpb2p detected by western blot analysis was reduced to background levels in the TPB2 mutants (Figure 3C), indicating that TPB2 was exclusively expressed zygotically from the new MAC in the wild-type cells. We concluded that the mutants were TPB2-null mutants.

TPB2 mutants block DNA elimination of Type-A and Type-B IESs

H3K9me3 and H3K27me3, scnRNA-dependent heterochromatic modifications, specifically occur on IESs in wild-type cells [START_REF] Liu | RNAi-dependent H3K27 methylation is required for heterochromatin formation and DNA elimination in Tetrahymena[END_REF]. Consistent with a previous report describing RNAi knockdown of TPB2 [START_REF] Cheng | A domesticated piggyBac transposase plays key roles in heterochromatin dynamics and DNA cleavage during programmed DNA deletion in Tetrahymena thermophila[END_REF], these modifications accumulated in the new MAC in the TPB2 mutants at 8 hpm (SI Appendix, Figure S1). In the wild-type cells at 12 hpm, these heterochromatic marks accumulated in nuclear foci referred to as heterochromatin bodies (HBs). The formation of HBs is believed to be triggered by Tpb2p-induced DNA double-strand breaks [START_REF] Shieh | LIA5 is required for nuclear reorganization and programmed DNA rearrangements occurring during tetrahymena macronuclear differentiation[END_REF]. Consistently, HBs were not detected in the TPB2 mutants at 12 hpm (SI Appendix, Figure S1). The presence of H3K9/K27 me3 and the absence of HBs in the TPB2 mutants therefore indicate that heterochromatin is formed, but IES excision is generally blocked in these mutants.

We next directly investigated DNA elimination in the TPB2 mutants by DLR-jPCR. As shown in Figure 4, we could not detect IES circles from any of the tested Type-A and Type-B IESs at 12 and 18 hpm in the absence of Tpb2p (Figure 4A, TPB2 mut), whereas IES circles from all of these IESs were detected at one or both of those time points in wildtype cells (Figure 4A, WT). In contrast, IES circles from the three tested TPB1/6dependent IES loci were detected in the TPB2 mutants, although their appearances were delayed as compared to those in wild-type cells (Figure 4A), Therefore, DNA elimination of all studied Type-A and Type-B IESs was reduced to an undetectable level in the TPB2 mutants. According to the results of sensitivity testing of DLR-jPCR shown in Figure 1C, we estimate that the frequency of the excision of these IESs in the TPB2 mutants was reduced to less than ~1/2,000 of that in the wild-type cells.

Blocking DNA elimination does not result in loss of late-scnRNAs

Finally, to investigate the accumulation of late-scnRNAs in the absence of IES excision, the TPB2 mutants were mated, and their 26-to 32-nt small RNAs were analyzed. Total amount of small RNAs detected by denaturing gel electrophoresis was similar between the wild-type cells and the TPB2 mutants at both early-(3 hpm) and late-(10 and 12 hpm) conjugation stages (SI Appendix, Figure S2A). Small RNAs that uniquely mapped to the 3,293 Type-B IESs were increased between 3 and 10 hpm in the TPB2 mutants (Figure 4B, top) and small RNAs derived from the three representative Type-B IESs were detected in the TPB2 mutants at 10 hpm (Figure 4B, bottom) and 12 hpm. In contrast, the levels of Type-B IES-mapping small RNAs remained low at 10 and 12 hpm in PDD1 KO cells, which lacked the core heterochromatin component (41) (Figure 4B). Because early-scnRNAs are produced only at early conjugation stage (~3 hpm) [START_REF] Noto | Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination[END_REF], any increase of small RNAs at later stages should be attributed to the production of late-scnRNAs.

Therefore, the results above indicate that IES excision, but not heterochromatin formation, is dispensable for the production of late-scnRNAs.

The amounts of Type-B IES-mapping small RNAs at 10 and 12 hpm in the TPB2 mutants were lower compared to those in wild-type cells (Figure 4B, top). In contrast, Type-A IESmapping small RNAs were not obviously affected in the TPB2 mutants (SI Appendix, Figure S2B). It is highly likely that the reduction of Type-B IES-mapping small RNAs, as well as the delayed elimination of the TPB1/6-dependent IESs (Figure 4A) in the TPB2 mutants is indirectly caused by the genome-wide retention of IESs [START_REF] Cheng | A domesticated piggyBac transposase plays key roles in heterochromatin dynamics and DNA cleavage during programmed DNA deletion in Tetrahymena thermophila[END_REF], which should disturb the expression of numerous genes. Alternatively, because we noticed that small RNAs, which are normally produced predominantly from IESs, were also produced from non-IES sequences in the TPB2 mutants (Figure 4B, bottom), we suggest that Tpb2p may have a non-catalytic function to confine late-scnRNA production to IESs and the reduction of late-scnRNAs that map Type-B IESs might be due to the gain of late-scnRNA production out of IESs. Nevertheless, the reduction of late-scnRNAs from Type-B IESs in the TPB2 mutants was approximately two-fold, in contrast to the more than 2,000-fold reduction of IES excision in these mutant cells.

The results obtained with the TPB2 mutants do not exclude the possibilities that some late-scnRNA biogenesis occurs after IES excision in wild-type cells and/or late-scnRNA biogenesis normally occurs exclusively after IES excision in wild-type cells but some compensatory mechanism allows the TPB2 mutants to produce late-scnRNAs without IES excision. However, together with the fact that late-scnRNAs were detected even at the time when elimination of their derived IESs was not detectable in wild-type cells (Figure 1), the results shown in this study led us to conclude that the biogenesis of late-scnRNAs occurs prior to the excision of IESs.

Discussion

Two technical advances made in this study, the sensitive detection of de novo DNA elimination (Figure 1) and the production of TPB2-null mutants (Figure 3), enabled us to show that late-scnRNAs accumulate at least a few hours before the elimination of their derived IESs becomes detectable (Figure 2) and that IES excision is dispensable for their biogenesis (Figure 4). Therefore, secondary small RNAs are produced from IESs prior to their excision in Tetrahymena, at least in part. This situation contrast considerably with the excision-dependent secondary small RNA production observed in another ciliate, Paramecium, in which concatenated circles formed from excised IESs are suggested to be the templates for the transcription of the precursors of iesRNAs (secondary small RNAs in this ciliate) [START_REF] Sandoval | Functional diversification of Dicer-like proteins and small RNAs required for genome sculpting[END_REF][START_REF] Allen | Circular Concatemers of Ultra-Short DNA Segments Produce Regulatory RNAs[END_REF] (Figure 4C). The post-excision production of secondary small RNAs in Paramecium is further supported by the fact that injection of DNA fragments into cells during sexual reproduction can induce ectopic DNA elimination at complementary genomic loci [START_REF] Allen | Circular Concatemers of Ultra-Short DNA Segments Produce Regulatory RNAs[END_REF]. In contrast, our attempts to induce ectopic DNA elimination in Tetrahymena by introducing linear and circular DNAs have failed. Considering all of these findings together, we conclude that the modes of secondary small RNA biogenesis are fundamentally different between Paramecium and Tetrahymena. This difference was unexpected because these two ciliates use otherwise very similar molecular mechanisms for DNA elimination.

The post-excision production of iesRNAs from concatenated IES circles in Paramecium might allow "rolling circle transcription" to produce a large amount of small RNAs that is sufficient to target all copies of IESs in the highly polyploid (~800 copies) somatic MAC genome. Such massive amplification of the small RNA signal is probably not necessary in Tetrahymena, which exhibits only ~50 copies in the MAC in vegetative stage and 4 to 8 copies when IES elimination occurs. Instead, because the IESs of Tetrahymena are much longer (median = 2.8 kb) than those of Paramecium (median = 51 bp) [START_REF] Hamilton | Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome[END_REF][START_REF] Guérin | Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements[END_REF], secondary small RNA production in Tetrahymena is necessary to cover IES regions that are not complementary to the primary small RNAs. The fact that heterochromatin formation, but not the downstream IES excision, is necessary for the secondary small RNA biogenesis in Tetrahymena (Figure 4B) strongly indicates that the secondary small RNA production mechanism is directly linked to a heterochromatin machinery. Indeed, the cis-spreading of late-scnRNA biogenesis via an RNAi-heterochromatin feedback loop plays an important role in IES elimination in Tetrahymena [START_REF] Noto | Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination[END_REF][START_REF] Suhren | Negative Regulators of an RNAi-Heterochromatin Positive Feedback Loop Safeguard Somatic Genome Integrity in Tetrahymena[END_REF], which is reminiscent of the heterochromatin-driven production of small RNAs observed in fungi [START_REF] Yamanaka | RNAi triggered by specialized machinery silences developmental genes and retrotransposons[END_REF], plants [START_REF] Law | Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1[END_REF] and animals [START_REF] Klattenhoff | The Drosophila HP1 homolog Rhino is required for transposon silencing and piRNA production by dual-strand clusters[END_REF][START_REF] Andersen | A heterochromatin-dependent transcription machinery drives piRNA expression[END_REF] and thus likely originated from an ancient TE silencing mechanism. If the above speculations are correct, the remarkable difference in secondary small RNA biogenesis reflects the evolutionary history of genome organization after the two ciliate linages diverged several million years ago. In this context, it would be interesting to explore the genomes and DNA elimination mechanisms of other ciliates to trace the evolution of TE-targeting small RNA amplification. Such observations would further provide unique views regarding how TEs and their silencing mechanisms drive the evolution of genome structures and vice versa. Figure 3. Production of TPB2 mutants (A) A construct expressing Cas9 from the cadmium-inducible MTT1 promoter and TPB2targeting gRNAs from the constitutive U6 promoter was introduced into the MAC. Some of the Cas9-gRNA complexes are expected to enter the MIC and introduce a mutation at the TPB2 locus. Immediately next to the TPB2 locus, there is a chromosome breakage sequence (Cbs), which remains intact in the MIC but is severed to form the ends of two separate chromosomes in the MAC. PCR with primers "a" and "c" specifically amplifies the TPB2 MIC locus, while PCR with "b" and "c" amplifies both the TPB2 MAC and MIC loci. (B) Two gRNAs (T1 and T2) were used to target the sequences in the first exon of TPB2 (marked with yellow). The TPB2 MIC and MAC loci were amplified, respectively, with the primers "a" and "c" and the primers "b" and "c" shown in (A). Although primers "b" and "c" could amplify both the MAC and MIC loci, polyploid MAC copies were predominantly represented in this PCR assay. The detected insertions and deletions are indicated in red. (C) Total cell proteins were extracted at the indicated time points from conjugation cultures of either wild-type (WT) cells or TPB2 mutants (TPB2 mut, cross 1 = #6 x #38, cross 2 = #41 x #44) and analyzed by western blotting using an anti-Tpb2p antibody (top) and an anti-α-tubulin antibody (bottom). In Tetrahymena (left), primary (1˚) small RNAs fully or partially cover IESs and induce heterochromatin formation, which triggers the production of secondary (2˚) small RNAs. Secondary small RNAs further induce heterochromatin formation and small RNA production. Eventually, heterochromatinized IESs are excised. In Paramecium (right), primary small RNAs cover IESs homogeneously. The excised IESs form concatenated circles, which are transcribed to produce secondary small RNAs. Secondary small RNAs further induce IES elimination and small RNA production.

Figure 1 .

 1 Figure 1. Detection of de novo IES excision events(A) Linear DNAs in total genomic DNA were digested with exonucleases (digestion of linear DNA [DLD]). Then, the remaining circular DNAs, including IES circles produced by de novo IES excision in the new MAC, were amplified by rolling circle amplification (RCA), and the junction of the DNA circle from an IES locus was detected by PCR (jPCR). The whole procedure is referred to as DLR-jPCR. (B) Wild-type strains were mated, and genomic DNA was extracted at the indicated hour post-mixing (hpm). The circles excised from the indicated IES loci were analyzed by DLR-jPCR with (+) or without (-) RCA. (C) Genomic DNA from 12 hpm was treated as in (B), but the sample was diluted by the indicated factors before jPCR to detect the IES737 locus.

Figure 2 .

 2 Figure 2. Accumulation of late-scnRNAs in wild-type cells (A-C) Normalized numbers (RPM [reads per million]) of sequenced 26-to 32-nt small RNAs from wild-type cells at indicated time points uniquely mapping to the representative Type-B IES loci are shown as histograms with 50-nt bins. Sky-blue boxes indicate IESs. The fraction of unique sequences that can be mapped by small RNAs in each 50-nt bin is shown at the bottom. (D) Normalized numbers (RPKM [reads per kilobase of unique sequences per million]) of 26-to 32-nt small RNAs from wild-type cells at indicated time points that uniquely matched the 3,293 Type-B IESs are shown as box plots. The horizontal bar in the box indicates the median value. The bars on the top and bottom of the box indicate the minimum and maximum values, respectively, within 1.5x IQR.Figure3. Production of TPB2 mutants (A) A construct expressing Cas9 from the cadmium-inducible MTT1 promoter and TPB2targeting gRNAs from the constitutive U6 promoter was introduced into the MAC. Some of the Cas9-gRNA complexes are expected to enter the MIC and introduce a mutation at the TPB2 locus. Immediately next to the TPB2 locus, there is a chromosome breakage

Figure 4 .

 4 Figure 4. Analyses of DNA elimination and late-scnRNA accumulation in TPB2 mutants (A) Wild-type (WT) and TPB2 mutant (TPB2 mut, mating of #41 and #44) strains were mated and their genomic DNA was extracted at 0, 12 and 18 hr post-mixing (hpm). The excised circles from the indicated IES loci were detected by DLR-jPCR. (B) Wild-type (WT), TPB2 mutant (TPB2 mut, cross 1 = #6 x #38, cross 2 = #41 x #44) and PDD1 KO (∆PDD1) strains were mated and their small RNAs were sequenced. (Top) Normalized numbers (RPKM [reads per kilobase of unique sequences per million]) of 26-to 32-nt small RNAs at 3, 10 and 12 hr post-mixing (hpm) that uniquely matched the 3,293 Type-B IESs are shown as box plots as in Figure 2D. (Bottom) Normalized numbers (RPM [reads per million]) of sequenced 26-to 32-nt small RNAs at 10 hpm uniquely mapping to the indicated Type-B IES loci are shown as histograms with 50-nt bins. Sky-blue boxes indicate IESs. (C) Comparison of the timing of secondary small RNA biogenesis.In Tetrahymena (left), primary (1˚) small RNAs fully or partially cover IESs and induce heterochromatin formation, which triggers the production of secondary (2˚) small RNAs. Secondary small RNAs further induce heterochromatin formation and small RNA production. Eventually, heterochromatinized IESs are excised. In Paramecium (right), primary small RNAs cover IESs homogeneously. The excised IESs form concatenated circles, which are transcribed to produce secondary small RNAs. Secondary small RNAs further induce IES elimination and small RNA production.

Acknowledgments. We acknowledge the MRI facility, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04), and the NGS unit of Vienna BioCenter Core Facilities. This work was supported by an Advanced Grant from the "Investissements d'avenir" program Labex EpiGenMed (ANR-10-LABX-12-01), an "Accueil de Chercheurs de Haut Niveau [ACHN]" grant (ANR-16-ACHN-0017) from the French National Research Agency to KM.

Materials and Methods

Strains. The wild-type Tetrahymena thermophila strains B2086 and CU428 were obtained from the Tetrahymena Stock Center. The PDD1 KO strains (W3-3 and W39-1) were described previously [START_REF] Schwope | Mutations in Pdd1 reveal distinct requirements for its chromodomain and chromoshadow domain in directing histone methylation and heterochromatin elimination[END_REF]. The construction of the TPB2 mutant strains is described in SI Materials and Methods. DLR-jPCR assay. Total genomic DNA was extracted using the NucleoSpin Tissue kit (Macherey-Nagel). 5 µg of the genomic DNA was treated with exonuclease V (NEB) for 1 h at 37°C, purified using the QIAquick Gel Extraction kit (QIAGEN), and then treated with Plasmid-Safe DNase (Epicentre) for 16 h at 37°C. The remaining circular DNAs were ethanol precipitated and amplified by rolling circle amplification (RCA) using the Illustra TempliPhi Amprification kit (GE Healthcare) according to the manufacturer's instructions, except that the 30°C incubation was performed for more than 18 h. The junctions of circularized IESs were amplified by PCR using the primers listed in SI Appendix Table S1. The positions of the Type-A and Type-B IESs investigated in this study were described previously [START_REF] Noto | Small-RNA-Mediated Genome-wide trans-Recognition Network in Tetrahymena DNA Elimination[END_REF]. The positions of TPB1/6-dependent IESs were described in Cheng et al. 2016 (26).

Small RNA analyses. High-throughput sequencing and analyses of small RNAs were performed as described [START_REF] Schoeberl | Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena[END_REF], except that radiolabeled 16 nt and 32 nt RNA oligos (5'-GAAUAGUUUAAACUGU-3'; 5'-AUCUUGGUCGUACGCGGAAUAGUUUAAACUGU-3') were used to excise small RNAs and their intermediate products from gels. The raw sequencing data have been deposited under accession no. GSE125297.

Western blotting and