A SUMO E3 ligase promotes long non-coding RNA transcription to regulate small RNA-directed DNA elimination

  1. Salman Shehzada
  2. Tomoko Noto
  3. Julie Saksouk
  4. Kazufumi Mochizuki  Is a corresponding author
  1. Institute of Human Genetics (IGH), CNRS, University of Montpellier, France

Abstract

Small RNAs target their complementary chromatin regions for gene silencing through nascent long non-coding RNAs (lncRNAs). In the ciliated protozoan Tetrahymena, 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.

Editor's evaluation

This important study demonstrates that protein SUMOylation is essential for programmed DNA elimination guided by small RNAs during conjugation in Tetrahymena ciliates. The authors present convincing evidence that the E3 SUMO ligase Ema2 is necessary for the production of long non-coding RNAs from the somatic nucleus, targeted small RNA degradation, and DNA elimination. The authors also show that the transcription regulator Spt6 is a SUMOylation target of Ema2, though the relevance of this is not completely established. This paper is of broad significance and will appeal to those interested in non-coding RNA biology, the control of programmed genome rearrangements, or ciliate biology.

https://doi.org/10.7554/eLife.95337.sa0

Introduction

Small RNAs of approximately 20–30 nucleotides that are complexed with Argonaute family proteins target either mRNAs for post-transcriptional silencing or chromatin regions for transcriptional gene silencing (Holoch and Moazed, 2015; Wilson and Doudna, 2013). For the latter process, small RNAs are generally considered to recognize their genomic targets via nascent lncRNAs to induce heterochromatin formation. Therefore, chromatin regions that are targeted for silencing by small RNAs must be paradoxically transcribed to provide nascent lncRNAs.

In fission yeast, small interfering RNAs (siRNAs) mediate the deposition of histone 3 lysine 9 di- and trimethylation (H3K9me2/3) for heterochromatin assembly at centromeric repeats (Hall et al., 2002; Volpe et al., 2002). While the HP1 protein Swi6 binds to H3K9me2/3 for transcriptional silencing, phosphorylation of histone H3 serine 10 at the M phase of the cell cycle evicts Swi6, passively allowing lncRNA transcription from the centromeric repeats at G1 and S phase and thus promoting H3K9me2/3 deposition to newly assembled nucleosomes (Chen et al., 2008; Kloc et al., 2008). In contrast, the HP1 paralog Rhino in fruit flies, which is specifically enriched at PIWI-interacting RNA (piRNA) clusters by binding to H3K9me2/3 (Le Thomas et al., 2014), actively recruits dedicated variants of basal transcription factors, allowing lncRNA transcription from heterochromatin while preventing mRNA transcription from transposons in the same loci (Andersen et al., 2017). A similar active heterochromatin-dependent lncRNA transcription has been reported in plants, where SHH1, a reader of H3K9me2/3 as well as mono-methylated H3K9, recruits the plant-specific RNA polymerase IV (Law et al., 2013; Law et al., 2011).

Because small RNA-producing loci are also small RNA targets in most of the studied small RNA-directed heterochromatin formation processes, it poses a challenge to separately investigate lncRNA transcription for small RNA biogenesis and that for small RNA-dependent recruitment of downstream effectors in these processes. In contrast, the source and target loci of small RNAs reside in different nuclei during programmed DNA elimination in some ciliated protozoans such as Tetrahymena thermophila and Paramecium tetraurelia, which provides a unique system to study the mechanism and roles of lncRNA transcription in small RNA-directed chromatin regulation.

In most ciliates, each cell contains two types of nuclei, the diploid germline micronucleus (MIC) and the polyploid somatic macronucleus (MAC). During conjugation, a sexual reproduction process in ciliates, the MIC undergoes meiosis and fertilization, followed by the formation of the new MIC and MAC, while the parental MAC is degraded (Chalker et al., 2013). Programmed DNA elimination occurs in the new MAC in most ciliates. In Tetrahymena, this process downsizes the 200 Mb MIC genome to the 103 Mb MAC genome by removing ~12,000 internal elimination sequences (IESs), many of which include transposons, followed by re-ligation of the remaining macronuclear-destined sequences (MDSs) forming the MAC chromosomes (Noto and Mochizuki, 2018).

DNA elimination in Tetrahymena and Paramecium is regulated by three types of lncRNAs, which occur in different nuclei at distinct times (Aronica et al., 2008; Schoeberl and Mochizuki, 2011). During meiotic prophase in Tetrahymena, lncRNAs are transcribed bidirectionally in the MIC in a genome-wide manner by RNA polymerase II and dedicated conjugation-specific Mediator-associated proteins (Chalker and Yao, 2001; Garg et al., 2019; Mochizuki and Gorovsky, 2004a; Schoeberl et al., 2012; Tian et al., 2019). The sexual reproduction-specific Spt4 and Spt5 paralogs are specifically involved in the MIC transcription in Paramecium (Gruchota et al., 2017; Owsian et al., 2022), while Tetrahymena genome does not encode such specialized Spt4/Spt5 paralogs. These micronuclear long non-coding RNAs (MIC-lncRNAs) are processed into small RNAs (~29 nt in Tetrahymena and 25-nt in Paramecium), called scnRNAs, by Dicer homologs (Lepère et al., 2009; Malone et al., 2005; Mochizuki and Gorovsky, 2005) and loaded into Piwi-clade Argonaute proteins (Bouhouche et al., 2011; Mochizuki et al., 2002; Noto et al., 2010).

The Piwi-scnRNA complex then moves into the parental MAC, where parental macronuclear long non-coding RNAs (pMAC-lncRNAs) are transcribed bidirectionally during the mid-conjugation stages (Woo et al., 2016). The RNA helicase Ema1 in Tetrahymena promotes the interaction between pMAC-lncRNAs and scnRNAs (Aronica et al., 2008). Reminiscently of target-directed micro RNA degradation (TDMD) (Han and Mendell, 2023), this interaction induces TDSD, leading to the selective retention of IES-derived scnRNAs (Aronica et al., 2008; Mochizuki and Gorovsky, 2004b; Noto and Mochizuki, 2018; Noto et al., 2015; Schoeberl et al., 2012). A similar TDSD has also been suggested in Paramecium, and the importance of pMAC-lncRNAs for DNA elimination was demonstrated by disrupting certain pMAC-lncRNAs by RNAi in this ciliate (Lepère et al., 2008). Although mRNAs are transcribed in the parental MAC, it remains unclear whether they also can induce TDSD and how mRNAs and pMAC-lncRNAs can be transcribed from overlapping locations. Also, although trimethylation of histone 3 lysine 27 (H3K27me3) occurs in the parental MAC in a scnRNA- and Polycomb repressive complex 2 (PRC2)-dependent manner in both Tetrahymena and Paramecium (Lhuillier-Akakpo et al., 2014; Liu et al., 2007), the role of H3K27me3 in the parental MAC, if any, is unclear.

When the new MAC develops, the retained Piwi-scnRNA complexes translocate to the new MAC, where yet another type of lncRNA, new macronuclear non-coding RNAs (nMAC-lncRNAs), is transcribed. The interaction between nMAC-lncRNA and scnRNA, which is also dependent on Ema1 (Aronica et al., 2008), is believed to recruit PRC2, which catalyzes both H3K9me2/3 and H3K27me3 for IES-specific heterochromatin assembly (Frapporti et al., 2019; Liu et al., 2007; Miró-Pina et al., 2022; Wang et al., 2022; Xu et al., 2021), and facilitate the secondary production of scnRNAs from nMAC-lncRNAs, which further promotes heterochromatin assembly (Allen et al., 2017; Noto et al., 2015). Then, IESs are excised by domesticated PiggyBac transposases (Baudry et al., 2009; Bischerour et al., 2018; Cheng et al., 2010; Vogt and Mochizuki, 2013). While nMAC-lncRNA production occurs prior to the excision of IESs in Tetrahymena (Mutazono et al., 2019), it also occurs from excised IESs that are concatenated and circularized in Paramecium (Allen et al., 2017).

Among the above three lncRNAs, pMAC-lncRNA does not produce small RNAs and is believed to be specialized for receptor function. Therefore, pMAC-lncRNA transcription and the following TDSD in the parental MAC provide a unique paradigm to investigate how the genome is transcribed to communicate with small RNAs. In this study, we show that Ema2, the conjugation-specific E3 ligase for a small ubiquitin-like modifier (SUMO), is required for pMAC-lncRNA transcription in Tetrahymena and thus provides a tool to dissect the role of and the molecular mechanism for the transcription of this lncRNA.

Results

Ema2 is exclusively expressed during conjugation and localized in the MAC

As part of our systematic investigation into genes highly upregulated during conjugation (Loidl, 2021), we explored the function of EMA2 (TTHERM_00113330). EMA2 mRNA is exclusively expressed during conjugation (Figure 1A). The encoded Ema2 protein tagged with HA at the endogenous EMA2 locus was not detectable in the vegetative cells (Figure 1B, Vg) and first appeared in the MAC during conjugation (Figure 1B, 3 hr post-induction of mating [hpm] and 6 hpm). At the onset of new MAC development, Ema2 disappeared from the parental MAC and appeared in the new MAC (Figure 1B, 8 hpm), which later faded away (Figures 1B, 12, and 14 hpm). The conjugation-specific expression and the localization switch from the parental to the new MAC are reminiscent of the factors involved in DNA elimination such as the Piwi protein Twi1, which is loaded by scnRNAs, and PRC2 (Liu et al., 2007; Mochizuki et al., 2002; Noto et al., 2010).

Ema2 is expressed during conjugation and localized in the macronucleus (MAC).

(A) EMA2 mRNA expression levels (in an arbitrary unit) in growing Tetrahymena cells in low (l), middle (m), and high (h) cell concentrations, starved cells from 0 to 24 hr, and cells in the conjugation and post conjugation stages from 0 to 18 hr post-mixing (hpm) are shown. The mRNA expression data were obtained from Miao et al., 2009. (B) Ema2 localization. Two Ema2-HA strains were mated and fixed at the indicated time points (Vg = vegetative cell). An anti-HA antibody was used to localize Ema2-HA (green), and DNA was stained with DAPI (magenta). The micronucleus (MIC), the parental MAC, and the newly formed MAC are marked with arrowheads with ‘i,’ ‘a,’ and ‘na,’ respectively. All pictures share the scale bar.

Ema2 is required for completing DNA elimination

DNA elimination of exconjugants (sexual progeny) at 36 hpm was analyzed by DNA fluorescent in situ hybridization (FISH) using probes complementary to the transposable element Tlr1 (Wuitschick et al., 2002). DNA elimination is completed by ~14–18 hpm in wild-type cells (Austerberry et al., 1984; Mutazono et al., 2019), and the Tlr1 element was detected only in the MICs in the exconjugants from wild-type cells (Figure 2A, WT). In contrast, the Tlr1 element was detected in both the MICs and the MACs in the exconjugants from the EMA2 somatic KO strains, in which all EMA2 copies in the MAC were disrupted (Shehzada and Mochizuki, 2022; Figure 2A, KO). The intensity of the FISH signal in the new MACs was lower in the exconjugants from the EMA2 KO cells than in those from the TWI1 KO cells (Figure 2B), in the latter of which DNA elimination is known to be completely blocked (Noto et al., 2015). Therefore, DNA elimination was partially blocked in the exconjugants of EMA2 KO cells. Consistent with the requirement of DNA elimination in the viability of sexual progeny (Cheng et al., 2010; Vogt and Mochizuki, 2013), EMA2 KO cells did not produce viable progeny (Figure 2C). Altogether, we conclude that maternally expressed EMA2 is required for completing DNA elimination.

Ema2 is required for completing DNA elimination.

(A) Two wild-type (WT) or two EMA2 somatic KO (KO) cell lines were mixed, and their exconjugants at 36 hpm were analyzed by DNA-fluorescent in situ hybridization (FISH) with fluorescent probes complementary to the Tlr1 element (green). DNA was counterstained with DAPI (magenta). The micronucleus (MIC) and the new macronucleus (MAC) are marked with arrowheads with ‘i’ and ‘na’, respectively. All pictures share the scale bar. (B) Exconjugants from wild-type (WT) cells, EMA2 somatic KO cells, and TWI1 KO cells were stained as in (A), the IES retention index was calculated (see Materials and Methods for details) from 20 cells each, and shown as box plots. The whiskers represent 10-90 percentile. Three asterisks (***) indicate a p-value of less than 0.001 in the Welch two-sample t-test. (C) Two wild-type (WT) cells and two independent crosses of EMA2 somatic KO cells (Cross 1 and 2) were mated, and the conjugating pairs were isolated for the viability test. The percentages of pairs that gave rise to viable sexual progeny are shown. ‘n’ represents the number of total pairs tested.

Ema2 is required for target-directed small RNA degradation (TDSD)

We next asked whether EMA2 is involved in TDSD. The production of scnRNAs occurs from IESs and their surrounding MDS regions in the MIC at the early conjugation stages (~2–3 hpm), and scnRNAs complementary to the MAC genome (=MDSs) are subjected to TDSD in the parental MAC in the mid-conjugation stages (~3–7 hpm), resulting in the selective retention of IES-derived scnRNAs that later target IESs for DNA elimination in the new MAC (Aronica et al., 2008; Mochizuki and Gorovsky, 2004b; Noto et al., 2015; Schoeberl et al., 2012). We first compared scnRNAs at different conjugation stages by northern blot analysis using the 50-nt Mi-9 probe that is complementary to a repetitive sequence found in both IESs and MDSs (Aronica et al., 2008). Because scnRNAs detected by this probe are complementary to the MAC genome, in wild-type cells, the Mi-9-complementary scnRNAs were detected at 3 hpm, reduced at 4.5 hpm, and became undetectable at 6 hpm due to TDSD (Figure 3A, WT). In contrast, the Mi-9-complementary scnRNAs remained detectable at 6 hpm in the EMA2 KO cells (Figure 3A, KO), suggesting that Ema2 is required for TDSD.

Ema2 is required for target-directed small RNAs (scnRNA) degradation (TDSD).

(A) Total RNA was isolated from conjugating wild-type (WT) and EMA2 somatic KO (KO) cells at 3, 4.5, and 6 hr post-mixing (hpm), separated in denaturing gel and stained with the nucleic acid dye Gel-Red (right). Then, RNA was transferred to a membrane and hybridized with the radioactive Mi-9 probe, which is complementary to a repetitive sequence in MDSs (right). (B) Small RNAs from conjugating wild-type (WT), EMA2 somatic KO, and EMA1 somatic KO strains were isolated at 3 and 8 hpm and analyzed by high-throughput sequencing. Normalized numbers (RPKM [read per kilobase of unique sequence per million]) of sequenced small RNAs (26–32 nt) that uniquely matched to the macronuclear-destined sequences (MDS) (left) or internal elimination sequence (IES) (right) genomic tiles (see Materials and methods) are shown as box plots. The median value is represented by the horizontal bar in the box. The minimum and maximum values are indicated by the bars on top and bottom of the box, respectively, with 1.5 x the interquartile range (IQR). Three asterisks (***) and ‘ns’ respectively indicate a p-value of less than 0.001 and more than 0.05 in the Wilcoxon rank sum test.

Figure 3—source data 1

The raw data of northern blot (top) and Gel-red stained gel (bottom) without (Figure_3 A_Original) and with (Figure_3 A_Original-marked) marks of the positions of regions used for Figure 3A.

https://cdn.elifesciences.org/articles/95337/elife-95337-fig3-data1-v2.zip

We also analyzed TDSD by small RNA sequencing at 3 and 8 hpm. Sequenced 26- to 32-nt small RNAs, which correspond to scnRNAs (Schoeberl et al., 2012), were mapped to the genomic tiles of MDSs and IESs (see Materials and methods). Although scnRNAs that mapped many of the MDS tiles were greatly reduced from 3 to 8 hpm due to TDSD in the wild-type cells (Figure 3B, left, WT), those in the EMA2 KO cells were only slightly reduced by 8 hpm (Figure 3B, left, EMA2 KO). Because TDSD takes place concurrently with the scnRNA production (Schoeberl et al., 2012), the increased abundance of MDS-complementary scnRNAs at 3 hpm in the EMA2 KO cells compared to the wild-type cells can also be attributed to the necessity of Ema2 in TDSD. This TDSD defect in the EMA2 KO cells seems milder than that in the EMA1 KO cells, in which the amount of MDS-complementary scnRNAs remained constant until 8 hpm (Figure 3B, left, EMA1 KO), indicating that Ema1 and Ema2 act differently in TDSD. In contrast, the amounts of scnRNAs complementary to IES tiles at 3 hpm and 8 hpm were comparable in all strains (Figure 3B, right), which is consistent with previous observations that only scnRNAs complementary to MDSs are targeted for TDSD. These results suggest that Ema2 is required for TDSD at the genome-wide level. Because it is known that loss of TDSD results in a partial block of DNA elimination (Aronica et al., 2008), the mild DNA elimination defect in EMA2 KO cells (Figure 2B) can be explained by the requirement of Ema2 in TDSD.

Ema2 is required for the accumulation of SUMOylated proteins during conjugation

Ema2 possesses an SP-RING domain that has been found in many SUMO E3 ligases (Hochstrasser, 2001). The SP-RING domain of Ema2 is atypical (Figure 4A) in that the first cysteine of the zinc ion-binding residues in typical counterparts is replaced by histidine and some of the stabilizer residues (Duan et al., 2009; Yunus and Lima, 2009) are likely missing. SUMOylation is catalyzed by the sequential actions of E1, E2, and in most cases, E3 enzymes and thus Ema2 should interact with the E2 enzyme if it acts in SUMOylation. We indeed found that Ema2 can directly interact with the Tetrahymena SUMO E2 enzyme Ubc9 in vitro (Figure 4B), suggesting that Ema2 is a bona fide SUMO E3 ligase.

Figure 4 with 1 supplement see all
Ema2 acts as a SUMO E3 ligase.

(A) The SP-RING domain of Ema2 is compared with that of MMS21 and SIZ1 in S. cerevisiae, Su(var)2–10 in D. melanogaster and PIAS1 in H. sapiens. The conserved cysteine and histidine residues that are involved in zinc ion binding are highlighted in yellow. The residues that stabilize the domain structure of some SP-RING domain proteins are marked with pink. (B) GST alone (GST), GST-tagged Ema2 (GST-Ema2), and His-tagged Ubc9 (His-Ubc9) were recombinantly expressed in E. coli and purified. GST and GST-Ema2 were immobilized on glutathione beads and incubated with His-Ubc9. Proteins retained on the beads were eluted, and the input and eluted proteins (PD) were analyzed by western blotting using anti-GST (left) and anti-His (right) antibodies. (C) An EMA2 somatic KO strain expressing HA-tagged Smt3 was crossed with a wild-type strain (WT cross, WT) or another EMA2 somatic KO strain (EMA2 KO cross, KO), and their total proteins at the indicated time points were analyzed by western blotting using anti-HA (top), anti-Twi1 (middle) and anti-alpha tubulin (bottom) antibodies. The signal intensities of the anti-HA blots in the individual entire lanes were quantified in three independent experiments. The values in the WT cross were normalized to 1, and their means and standard deviations are presented as a bar graph, with p-values determined by the Welch two-sample t-test.

Figure 4—source data 1

The raw data of western blot without (Figure_4B_Original) and with (Figure_4B_Original-marked) marks of the positions of regions used for Figure 4B.

https://cdn.elifesciences.org/articles/95337/elife-95337-fig4-data1-v2.zip
Figure 4—source data 2

The raw data of western blot without (Figure_4 C_Original) and with (Figure_4 C_Original-marked) marks of the positions of regions used for Figure 4C.

https://cdn.elifesciences.org/articles/95337/elife-95337-fig4-data2-v2.zip

To examine the role of Ema2 in SUMOylation, we expressed HA-tagged Smt3 (HA-Smt3) in an EMA2 KO strain. SUMO is solely encoded by SMT3 in Tetrahymena (Nasir et al., 2015), and HA-Smt3 could replace the essential function of endogenous Smt3 (Figure 4—figure supplement 1). We then crossed this strain with either a wild-type strain (called the WT cross) or another EMA2 KO strain (called the EMA2 KO cross). Because proteins and mRNAs are exchanged between two mating pairs through the conjugation junction, EMA2 mRNA/Ema2 protein expressed in the wild-type partner of the WT cross is expected to move into the EMA2 KO partner and restore the EMA2 KO phenotypes.

Total proteins were harvested at 4.5 and 6 hpm, and SUMOylated proteins were detected by western blotting using an anti-HA antibody (Figure 4C). At both time points, high molecular weight proteins (mainly >200 kDa) were detected in the WT cross (Figure 4C, WT), and they were reduced to ~50% in the EMA2 KO cross (Figure 4C, KO). These results indicate that Ema2 is the major SUMO E3 ligase during the mid-conjugation stages. The remaining Ema2-independent SUMOylation is likely mediated by other SUMO E3 ligases (including the SP-RING containing proteins TTHERM_00227730, TTHERM_00442270 and TTHERM_00348490), and/or E3-independent SUMOylation (Sampson et al., 2001). The requirement of protein SUMOylation in DNA elimination was previously demonstrated in Paramecium by RNAi knockdown of UBA2, the gene encoding the SUMO E1 enzyme, and SUMO (Matsuda and Forney, 2006). Therefore, the involvement of a SUMO pathway in DNA elimination is likely conserved among ciliates.

Ema2 is required for SUMOylation of Spt6

Next, to identify the SUMOylation target(s) of Ema2, we introduced a construct expressing His-tagged Smt3, which can also replace the essential function of Smt3 (Figure 4—figure supplement 1 ), into an EMA2 KO strain and crossed it with a wild-type strain (WT cross) or another EMA2 KO strain (KO cross). Then, SUMOylated proteins at 6 hpm were concentrated using nickel-NTA beads and identified by mass spectrometry. We additionally examined the mating of wild-type strains without His-Smt3 expression and excluded any proteins identified with a log2 label-free quantification (LFQ) score above 25 or those possessing more than six consecutive histidine residues in this analysis, considering them as proteins binding to the nickel-NTA beads without His-Smt3 conjugation. Although most of the proteins were detected similarly between the WT cross and EMA2 KO cross, Spt6, the most abundantly detected protein in the WT cross, was detected at a much lower level in the EMA2 KO cross (Figure 5A). This result suggests that Spt6 is the major Ema2 target for SUMOylation.

Figure 5 with 1 supplement see all
Ema2 promotes SUMOylation of Spt6.

(A) A construct expressing His-tagged Smt3 (His-Smt3) was introduced into a wild-type and an EMA2 somatic KO strain and crossed with another wild-type (WT cross) or EMA2 somatic KO strain (EMA2 KO cross). Proteins were harvested at 6 hpm, and His-Smt3-conjugated proteins were purified with Ni-NTA beads from conjugating cells and analyzed by mass spectrometry. Values of log base 2 of label-free quantification (LFQ) intensities of each identified protein between the WT cross (WT) and EMA2 KO cross (KO) were compared. (B) A construct expressing HA-tagged Spt6 (Spt6-HA) was introduced into an EMA2 somatic KO strain and crossed with a wild-type (WT-cross, WT) or another EMA2 somatic KO (KO-cross, KO) strain. Total proteins were harvested at 4.5 and 6 hpm, and Spt6-HA was detected by western blotting using an anti-HA antibody. Twi1 and alpha-tubulin (Tub) were also analyzed to monitor mating efficiency and loading, respectively. The positions of modified and unmodified Spt6-HA proteins are marked with a bracket and an arrowhead, respectively. (C) Total proteins were harvested from WT and KO crosses at 6 hpm (input), and Spt6-HA was immunoprecipitated using an anti-HA antibody (IP). The purified proteins were analyzed by western blotting using an anti-HA (left) or an anti-Smt3 (right) antibody. SUMOylated Spt6 proteins are marked with brackets. Unidentified protein cross-reacting with the anti-HA antibody is marked with an asterisk.

Figure 5—source data 1

The raw data of western blot without (Figure_5B_Original) and with (Figure_5B_Original-marked) marks of the positions of regions used for Figure 5B.

https://cdn.elifesciences.org/articles/95337/elife-95337-fig5-data1-v2.zip
Figure 5—source data 2

The raw data of western blot without (Figure_5 C_Original) and with (Figure_5 C_Original-marked) marks of the positions of regions used for Figure 5C.

https://cdn.elifesciences.org/articles/95337/elife-95337-fig5-data2-v2.zip

To confirm the Ema2-dependent SUMOylation of Spt6, we introduced a construct expressing HA-tagged Spt6 (Spt6-HA) from the endogenous SPT6 locus into an EMA2 KO strain and mated it with a wild-type strain (WT cross) or another EMA2 KO strain (EMA2 KO cross). Then, total proteins harvested at 4.5 and 6 hpm were analyzed by western blotting using an anti-HA antibody (Figure 5B). In the WT cross, a slower migrating population of Spt6-HA was detected in addition to a band corresponding to unmodified Spt6-HA at both time points (Figure 5B, WT). In contrast, slower migrating Spt6-HA was barely detectable in the EMA2 KO cross (Figure 5B, KO). Then, to examine the timing of the appearance of the slower migrating Spt6 species, we introduced the same Spt6-HA-expressing construct into a wild-type strain and Spt6-HA was analyzed by western blotting (Figure 5—figure supplement 1). Consistent with the Ema2-dependent appearance of the slower migrating Spt6-HA, they were not detected in growing and starved vegetative wild-type cells (Figure 5—figure supplement 1, Veg and 0 hpm, respectively) when Ema2 was not expressed (Figure 1). The slower migrating Spt6-HA was also detected at 8 hpm when the new MAC was already formed (Figure 5—figure supplement 1, 8 hpm) suggesting that Spt6 is possibly SUMOylated also in the new MAC.

The nature of slower migrating species of Spt6-HA was further examined by immunoprecipitating Spt6-HA using an anti-HA antibody. Among the total purified Spt6-HA (Figure 5C, left, IP-WT), the slower migrating species were detected by an anti-Smt3 antibody in the WT cross (Figure 5C, right, IP-WT), and such SUMOylated Spt6-HA species were greatly reduced in the EMA2 KO cross (Figure 5C, KO). These results indicate that the slower migrating species of Spt6 are SUMOylated Spt6 and Ema2 is required for the majority of SUMOylation of Spt6 during the mid-conjugation stages. The remaining SUMOylation observed on Spt6 in the absence of Ema2 is likely facilitated by other SUMO E3 ligases and/or E3-independent SUMOylation, as discussed earlier for the other instances of Ema2-independent SUMOylations.

Ema2 is required for the accumulation of lncRNA in the parental MAC

Spt6 is a conserved regulator of several steps of transcription in various eukaryotes. Because TDSD was proposed to be triggered by the base-pairing interaction between scnRNAs and nascent pMAC-lncRNAs in the parental MAC (Aronica et al., 2008; Noto and Mochizuki, 2018), we hypothesized that Ema2-dependent Spt6 SUMOylation promotes pMAC-lncRNA transcription.

To examine pMAC-lncRNAs, we amplified transcripts spanning IES-MDS borders by RT‒PCR in which MAC-lncRNAs can be distinguished from MIC-lncRNAs by their lengths (Figure 6A). For the three loci examined, pMAC-lncRNAs were detected in wild-type cells but not in EMA2 KO cells at 6 hpm, although the control RPL21 mRNA was detected in both conditions (Figure 6A). The lack of detection of pMAC-lncRNAs in EMA2 KO cells was not due to loss of the primer binding sites by alternative DNA elimination in their prior sexual reproductions, as genomic PCR with the same primer sets detected the corresponding MAC loci in the EMA2 KO cells (Figure 6—figure supplement 1). We, therefore, conclude that Ema2 is required for the accumulation of pMAC-lncRNAs at least for the three tested loci.

Figure 6 with 1 supplement see all
Ema2 is required for the accumulation of long non-coding RNA (lncRNA) transcripts from the parental macronucleus (MAC).

(A) (Left) Schematic representation of the RT‒PCR assay. The black bars and the open box represent the macronuclear-destined sequence (MDS) and internal elimination sequence (IES), respectively. The arrows represent the primers used for RT‒PCR. The lengths of the PCR amplicons are shown with double-sided arrows. (Right) Wild-type (WT) or EMA2 somatic KO (KO) cells were mated, and their total RNAs at 6 hpm were used for RT‒PCR. The positions corresponding to the PCR products of lncRNAs from the MAC M, L8, and R2 loci are marked. RPL21 mRNA was also analyzed as a positive control. (B) Conjugating wild-type (WT), EMA2 somatic KO, and EMA1 somatic KO cells at the indicated time points were analyzed by immunofluorescence staining using the anti-long dsRNA antibody J2 (dsRNA, green). DNA was counterstained with DAPI (magenta). The MIC, the parental MAC, and the new MAC are indicated with arrowheads with ‘i,’, ‘a,’and ‘“na,’ respectively. All pictures share the scale bar. (C) Heterochromatin formation in the parental MAC. Conjugating wild-type (WT), EMA2 somatic KO, EMA1 somatic KO, TWI1 complete (somatic + germline) KO, and EZL1 somatic KO cells at 6 hpm were analyzed by immunofluorescence staining using an anti-H3K27me3 antibody (green). DNA was counterstained with DAPI (magenta). The micronucleus (MIC) and the parental MAC are marked with arrowheads with ‘i’ and ‘a,’ respectively. All pictures share the scale bar.

Figure 6—source data 1

The raw data of ethidium bromide-stained PCR products separated in agarose gels without (Figure_6 A_Original) and with (Figure_6 A_Original-marked) marks of the positions of regions used for Figure 6A.

https://cdn.elifesciences.org/articles/95337/elife-95337-fig6-data1-v2.zip

The accumulation of lncRNAs was also examined cytologically. As the consequence of the bidirectional lncRNA transcription in the three different nuclei, double-stranded RNAs are accumulated in these nuclei (Aronica et al., 2008; Malone et al., 2005; Mochizuki and Gorovsky, 2005; Woo et al., 2016). As previously reported, immunostaining of wild-type cells using the long double-stranded (ds)RNA-specific J2 antibody (Schönborn et al., 1991; Figure 6B, WT) detected long dsRNAs first in the MIC at its premeiotic stage (3 hpm), then in the parental MAC at the mid stages of conjugation (6 hpm), and finally in the new MAC at late conjugation (9 hpm). In the EMA2 KO cells, long dsRNAs were detected in the MIC at 3 hpm and in the new MAC at 9 hpm but were undetectable in the parental MAC at 6 hpm (Figure 6B, EMA2 KO), indicating that Ema2 is specifically required for the accumulation of pMAC-lncRNAs.

In contrast, EMA1 was dispensable for the accumulation of dsRNAs in the parental MAC (Figure 6B, EMA1 KO), which is consistent with the previous notions that Ema1 is required for pMAC-lncRNAs to interact with scnRNAs but not their transcription (Aronica et al., 2008). Although it is unclear whether lncRNAs are single or double-stranded when Ema1 promotes the lncRNA-scnRNAs interaction, the less severe TDSD defect observed in the EMA2 KO cells compared to the EMA1 KO cells (Figure 3B) indicates that certain Ema1-dependent TDSD may be initiated by single-stranded lncRNAs or mRNAs that are transcribed independently of Ema2.

In parallel to TDSD, scnRNA-dependent accumulation of H3K27me3, which is catalyzed by the histone methyltransferase Ezl1 within PRC2, occurs in the parental MAC (Liu et al., 2007). We found that H3K27me3 was undetectable in the parental MAC in the EMA2 KO cells at 6 hpm or in the EMA1, TWI1, and EZL1 KO cells (Figure 6C). Therefore, Ema2 is required for the scnRNA-directed deposition of H3K27me3 in the parental MAC, which can be explained by the loss of the chromatin recruitment of PRC2 by the scnRNA-lncRNA interaction. Altogether, these results indicate that Ema2 is required for the genome-wide accumulation of pMAC-lncRNAs.

Ema2 facilitates the chromatin association of Spt6 and RNA polymerase II

Next, we analyzed the localization of Spt6 by immunofluorescence staining. In both WT and EMA2 KO cross described above, Spt6-HA was similarly detected in the MAC at 4.5 hpm (Figure 7A, left) and in the new MAC at 9 hpm (Figure 7—figure supplement 1). We also found that the MAC localization of Rpb3, the third largest subunit of RNA polymerase II (RNAPII) was not affected in the absence of Ema2 (Figure 7A, right). Therefore, at least at the cytological level, the absence of Ema2 does not affect the localization of Spt6 and RNAPII.

Figure 7 with 3 supplements see all
Ema2 facilitates the chromatin association of Spt6 and RNA polymerase II.

(A) A construct expressing HA-tagged Spt6 (Spt6-HA) was introduced into an EMA2 somatic KO strain and crossed with a wild-type (WT-cross, WT) or another EMA2 somatic KO (KO-cross, KO) strain. The localizations of Spt6-HA and Rpb3, the third largest subunit of RNA polymerase II (RNAPII), were analyzed by immunofluorescence staining at 4.5 hpm using anti-HA (left) or anti-Rpb3 (right) antibodies, respectively. DNA was counterstained with DAPI (magenta). The parental macronucleus (MACs) are marked with arrowheads with ‘a.’ All other structures stained by DAPI are micronucleus (MICs). All pictures share the scale bar. (B) (Left) Schematic representation of the cell fractionation assay. Conjugating cells were incubated with a lysis buffer that releases cytoplasmic (green) and nucleoplasmic (magenta) proteins to the soluble fraction (S1). Then, the insoluble fraction was resuspended in fresh lysis buffer and sonicated. The solubilized fraction after sonication (S2) contains fragmented chromatin. (Right) S1 and S2 fractions as well as total cellular proteins (W) from WT-cross (WT) and EMA2 KO-cross (KO) explained in (A) at 4.5 hpm were analyzed by western blotting using anti-HA (Spt6-HA), anti-Rpb3, and anti-histone H3 antibodies. The position of SUMOylated Spt6-HA is marked with a bracket. (C) (Left) Schematic representation of the cell fractionation assay with RNase treatment. Conjugating cells were incubated with a lysis buffer that releases cytoplasmic (green) and nucleoplasmic (magenta) proteins to the soluble fraction (S1). Then, the insoluble fraction was incubated in fresh lysis buffer with (+) or without (-) RNase A, and the solubilized (S2) and insoluble (I) fractions were obtained. (Right) S1, S2, and I fractions as well as total cellular proteins (W) from SPT6 germline (MAC + MIC) KO strains rescued with the HA-SPT6-WT construct (see Figure 8) were analyzed by western blotting using anti-HA (HA-Spt6), anti-Twi1p, anti-Rpb3, and anti-histone H3 antibodies. The position of SUMOylated HA-Spt6 is marked with a bracket.

Figure 7—source data 1

The raw data of western blot without (Figure_7B-Original) and with (Figure_7B-Original-marked) marks of the positions of regions used for Figure_7B.

https://cdn.elifesciences.org/articles/95337/elife-95337-fig7-data1-v2.zip
Figure 7—source data 2

The raw data of western blot without (Figure_7C-Original) and with (Figure_7C-Original-marked) marks of the positions of regions used for Figure_7 C.

https://cdn.elifesciences.org/articles/95337/elife-95337-fig7-data2-v2.zip

We then examined their subnuclear localization by cell fractionation (Figure 7B, left), in which cells at 4.5 hpm were first incubated with lysis buffer to extract soluble cyto- and nucleoplasmic proteins, and then, the insoluble fraction was sonicated to extract chromatin-bound proteins (Ali et al., 2018). In the WT cross (Figure 7B, WT), Spt6-HA was detected in both the soluble (S1) and chromatin-bound (S2) fractions, but slower-migrating SUMOylated Spt6-HA was detected only in the latter fraction, indicating that SUMOylated Spt6 is associated with chromatin. A similar distribution was detected for Rpb3. This chromatin association of Spt6 and RNAPII does not require RNA, as they were retained in the insoluble fraction after RNase A treatment without sonication (Figure 7C), although Twi1, which interacts with chromatin through nascent pMAC-lncRNA (Aronica et al., 2008), was mostly detected in the soluble fraction after the treatment (Figure 7C). In the EMA2 KO cross, Spt6-HA and Rpb3 in the chromatin-bound fraction were greatly reduced (Figure 7B, KO, S2). These findings were further validated in two additional replicated experiments (Figure 7—figure supplements 2 and 3). We, therefore, conclude that Ema2 promotes the association of Spt6 and RNAPII with chromatin in the parental MAC at the mid-conjugation stage.

Spt6 is not necessary to be SUMOylated in Ema2-directed lncRNA transcription

Next, to directly examine the importance of SUMOylation of Spt6, we intended to produce a SUMOylation-defective Spt6 mutant. Tetrahymena Spt6 consists of an N-terminal domain of low complexity (DOL) and a downstream region that is conserved among eukaryotes (Figure 8A, WT). We produced constructs expressing HA-tagged Spt6 with lysine to arginine substitutions either for all lysine residues in the DOL (HA-SPT6-DOL-KR) or for lysine residues that were on the surface of an AlphaFold predicted Tetrahymena Spt6 structure in one of the three non-DOL parts (N, M, and C in Figure 8A) in addition to those that were also predicted to be SUMOylation targets by the JASSA algorithm (Beauclair et al., 2015) in all three parts (HA-SPT6-N-KR, HA-SPT6-M-KR, and HA-SPT6-C-KR, Figure 8A).

Figure 8 with 3 supplements see all
Spt6 SUMOylation is dispensable for parental macronucleus (MAC) long non-coding RNA (lncRNA) transcription.

(A) Schematic representation of SPT6 mutant constructs with lysine (K)-to-arginine (R) substitutions. Spt6 has a domain of low complexity (DOL) region followed by conserved TEX-like and SH2 domains. The DOL-KR mutant has K to R substitutions for all lysine residues in the DOL region. N-KR, M-KR, and C-KR mutants have K to R substitutions for lysine residues that were on the surface of a predicted Spt6 structure in one of the three non-DOL regions (N, M, and C-regions) in addition to those surface lysine residues that were also predicted to be SUMOylatable by an algorithm in all three regions. (B) Total proteins were harvested at 4.5 hpm from conjugating SPT6 germline (MAC + MIC) KO cells rescued with wild-type (HA-SPT6-WT), HA-SPT6-DOL-KR, or HA-SPT6-C-KR constructs. Because mating was low in the HA-SPT6-C-KR rescued cells, three times (3 x) more total protein sample was loaded for these cells. HA-Spt6 was detected by western blotting using an anti-HA antibody. The positions of modified and unmodified HA-Spt6 proteins are marked with ‘M’ and ‘UM,’ respectively. (C) HA-SPT6-WT (WT) and HA-SPT6-C-KR (C–KR) rescued cells at 4.5 hpm were analyzed by cell fractionation as described in Figure 7B. The position of SUMOylated HA-Spt6 is marked with a bracket. (D) Accumulation of long dsRNAs in HA-SPT6-WT (WT) and HA-SPT6-C-KR (C–KR) rescued cells at 4.5 hpm was analyzed by immunofluorescence staining using the J2 antibody as described in Figure 6B. The parental MACs are marked with arrowheads with ‘a.’ All other structures stained by DAPI are micronucleus (MICs). All pictures share the scale bar. (E) HA-SPT6-WT (WT) and HA-SPT6-C-KR (C–KR) rescued cells at 36 hpm were analyzed by DNA- FISH with fluorescent probes complementary to the Tlr1 element (red). DNA was counterstained with DAPI (magenta). The MIC and the new MAC are marked with arrowheads with ‘i’ and ‘a,’ respectively. All pictures share the scale bar.

Figure 8—source data 1

The raw data of western blot without (Figure_8B-Original) and with (Figure_8B-Original-marked) marks of the positions of regions used for Figure 8B.

https://cdn.elifesciences.org/articles/95337/elife-95337-fig8-data1-v2.zip
Figure 8—source data 2

The raw data of western blot without (Figure_8C-Original) and with (Figure_8C-Original-marked) marks of the positions of regions used for Figure 8C.

https://cdn.elifesciences.org/articles/95337/elife-95337-fig8-data2-v2.zip

We introduced these mutant constructs as well as the construct expressing wild-type Spt6 (HA-SPT6-WT) into SPT6 KO cells and found that all of them were able to restore the lethality of SPT6 KO cells (Figure 8—figure supplement 1). However, the cells rescued by HA-SPT6-N-KR and HA-SPT6-M-KR showed severe defects in meiotic progression and mating initiation, respectively, making their SUMOylation status during conjugation uninvestigable. In contrast, the cells rescued by HA-SPT6-DOL-KR and HA-SPT6-C-KR showed a seemingly normal progression of conjugation, although the HA-SPT6-C-KR cells showed lower mating efficiency. We found that while HA-Spt6-DOL-KR was SUMOylated like HA-Spt6-WT, SUMOylation was not detected on HA-Spt6-C-KR (Figure 8B). Therefore, Spt6-C-KR represents a SUMOylation-defective Spt6 mutant, exhibiting at least a reduced level of SUMOylation compared to Spt6 in the absence of Ema2 (compare Figure 8B and Figure 5B).

Cell fractionation experiments showed that HA-Spt6 and RNAPII were detected in the chromatin-bound fraction in HA-SPT6-C-KR cells as in the HA-SPT6-WT cells consistently in three independent experiments (Figure 8C, Figure 8—figure supplement 2), and long dsRNAs were detected in the parental MAC (Figure 8D) as well as in the new MAC (Figure 8—figure supplement 3) in the HA-SPT6-C-KR cells. These results suggest that, contrary to our expectation, Spt6 SUMOylation per se is not required for the lncRNA transcription in the parental MAC. Nonetheless, exconjugants from the HA-SPT6-C-KR cells showed a mild DNA elimination defect (Figure 8E), indicating that Ema2-directed Spt6-SUMOylation plays a role in DNA elimination other than promoting pMAC-lncRNA transcription.

Discussion

In this study, we showed that the conjugation-specific SUMO E3 ligase Ema2 is required for the accumulation of lncRNAs (Figure 6), TDSD (Figure 3A and B), and heterochromatin formation (Figure 3C) in the parental MAC and eventually for completing DNA elimination (Figure 1) in Tetrahymena. We found that Ema2 is responsible for SUMOylation of the transcriptional regulator Spt6 (Figure 5) and promotes the interaction of Spt6 and RNAPII with chromatin (Figure 7). We, therefore, conclude that Ema2 facilitates genome-wide lncRNA transcription in the parental MAC, which is a prerequisite for scnRNA-chromatin communication and thus for the downstream TDSD that regulates DNA elimination.

Our observation that Ema2 enhances the chromatin interaction of Spt6 and RNAPII (Figure 7) seems to contradict the fact that Spt6 and RNAPII are essential for vegetative cell viability (Figure 8—figure supplement 1; Mochizuki and Gorovsky, 2004a), but Ema2 is expressed exclusively during conjugation (Figure 2). Moreover, as EMA2 KO cells did not significantly impede the progression of conjugation processes, any essential mRNA transcriptions for these processes must take place in the parental MAC during conjugation even in the absence of Ema2. Therefore, the observed loss of the majority of Spt6 and RNAPII from chromatin in the absence of Ema2 (Figure 7B) must be a temporal event during the mid-conjugation stage. This suggests that Spt6 and RNAPII might be specifically engaged in pMAC-lncRNA transcription at this particular time window in wild-type cells. It is likely that some radical change in the chromatin environment in the parental MAC is required for the global transcription of pMAC-lncRNAs, and Ema2-dependent SUMOylation might maintain Spt6 and RNAPII on chromatin for transcription in such an environment. Alternatively, because the expression of many of the transcriptional machineries, including Spt6 and RNAPII subunits, is highly upregulated during conjugation (Miao et al., 2009; Mochizuki and Gorovsky, 2004a; Xiong et al., 2012), the transcription of pMAC-lncRNAs might require a greater amount of transcriptional machinery than other types of transcription, and Ema2-dependent SUMOylation might be required for committing these excess machineries to chromatin for transcription.

Our investigation of the SUMOylation-defective Spt6 mutant suggested that although Spt6 is SUMOylated in an Ema2-dependent manner (Figure 5), SUMOylation of Spt6 by itself is not required for pMAC-lncRNA transcription (Figure 8). This observation suggests that Ema2 may facilitate pMAC-lncRNA transcription through SUMOylation of some protein(s) other than Spt6. Although, we failed to detect any other protein that was SUMOylated in an Ema2-dependent manner (Figure 5A), some Ema2-dependent SUMOylation events might not be detected in our current approach if Ema2-dependent and Ema2-independent SUMOylation occur in the same proteins and/or if Ema2-dependent SUMOylation results in protein degradation, such as through SUMO-targeted ubiquitination (Praefcke et al., 2012; Staudinger, 2017). Alternatively, SUMOylation of Spt6 may promote pMAC-lncRNA transcription by competing with some other modification that occurs at the same lysine residue(s) and our SUMOylation-defective Spt6 mutant might actually mimic the SUMOylated state of Spt6 by preventing such modification. To further investigate the role of Ema2, future studies will need to identify individual Ema2-dependent SUMOylated lysine residues under conditions of in vivo proteasome inhibition and to compare the status of other modifications of Spt6 in the presence and absence of Ema2. These are also important to understand the role of Spt6 SUMOylation in DNA elimination (Figure 8E).

It was reported that SUMOylation of the heterochromatin components Swi6, Chp2, and Clr4 is important for RNAi-directed heterochromatin silencing in fission yeast (Shin et al., 2005). Additionally, SUMOylation of the histone deacetylase HDAC1 promotes small RNA-directed transcriptional silencing in C. elegans (Kim et al., 2021). Furthermore, in the piRNA-directed transposon silencing of Drosophila, the SUMO E3 ligase Su(var)2–10 plays an essential role in the recruitment of the histone methyltransferase complex SetDB1, which deposits H3K9me3 (Ninova et al., 2020a; Ninova et al., 2020b), and Su(var)2–10-independent SUMOylation of Panoramix (Panx) also promotes transcriptional silencing by recruiting the general heterochromatin effector Sov (Andreev et al., 2022). All these SUMOylation events seem to require pre-existing lncRNA transcription. In contrast, this study showed that Ema2-dependent SUMOylation during the process of DNA elimination in Tetrahymena occurs upstream of lncRNA transcription (Figure 6A and B). In addition, the fact that the loss of the Piwi protein Twi1 blocks the accumulation of H3K27me3 in the parental MAC (Liu et al., 2007; Xu et al., 2021) without affecting lncRNA accumulation (Woo et al., 2016) indicates that heterochromatin formation is not a prerequisite for lncRNA transcription in Tetrahymena, in contrast to heterochromatin-dependent lncRNA transcription of piRNA clusters in Drosophila (Andersen et al., 2017). Altogether, Tetrahymena likely uses a unique evolutionary solution for transcribing lncRNAs to promote TDSD and small RNA-directed heterochromatin formation. We, therefore, believe that further investigations of the role of Ema2-dependent SUMOylation in lncRNA transcription will clarify an undescribed layer of regulatory mechanisms in small RNA-directed chromatin regulation.

Materials and methods

Strains and cell culture condition

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The Tetrahymena thermophila wild-type strains B2086, CU427, and CU428 and the MIC defective ‘star’ strains B*VI and B*VII were obtained from the Tetrahymena stock center. The production of the EMA2 KO strains was described previously (Shehzada and Mochizuki, 2022). The other transgenic Tetrahymena strains are described below. The Tetrahymena strains reported in this manuscripts are available from the corresponding author upon reasonable request. Cells were grown in SPP medium (Gorovsky et al., 1975) containing 2% proteose peptone at 30 °C. For conjugation, growing cells (~5–7 × 105 /mL) of two different mating types were washed, pre-starved (~12–24 hr), and mixed in 10 mM Tris (pH 7.5) at 30 °C.

Oligo DNA, synthetic DNA, and antibodies

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The primers and synthetic DNAs used in this study are listed in Supplementary file 1. The following antibodies were obtained from manufacturers: anti-H3K27me3 (Millipore, 07–449, RRID:AB_310624); anti-HA HA11 (Covance, clone 6B12, RRID:AB_291231); anti-H3K9me3 (Active Motif, 39162); anti-GST (BD Biosciences, 554805, RRID:AB_395536); anti-His (Proteintech, 66005–1-Ig, RRID:AB_11232599); anti-long dsRNA J2 (Jena Bioscience RNT-SCI-10010200, RRID:AB_2651015); anti-α-tubulin 12G10 (Developmental Studies Hybridoma Bank of the University of Iowa). The anti-Rpb3 antibody was described previously (Kataoka and Mochizuki, 2017). The anti-Smt3 antibody was a gift from Dr. James Forney (Purdue University) and described previously (Nasir et al., 2015).

Establishment of EMA2-HA strains

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The C-terminal part of the EMA2 coding sequence was amplified from the genomic DNA of B2086 using EMA2-C-FW and EMA2-HA-C-RV. Similarly, the 3' flanking sequence of EMA2 was amplified using EMA2-F-FW and EMA2-F-RV. The two PCR products were combined by overlapping PCR using EMA2-C-FW and EMA2-F-RV and cloned into XbaI and XhoI sites of pBlueScriptSK (+) to obtain pEMA2. Next, the HA-neo3 construct was excised out from pHA-neo3 (Kataoka et al., 2010) using SalI and BamHI and cloned into the same restriction sites of pEMA2 to obtain pEMA2-HA-neo3. Finally, pEMA2-HA-neo3 were digested with XbaI and XhoI and introduced into the EMA2 MAC locus of B2086 and CU428 by homologous recombination using biolistic transformation (Bruns and Cassidy-Hanley, 2000) followed by the selection with 100 μg/mL paromomycin and 1 μg/mL CdCl2. Then, the endogenous EMA2 MAC copies were replaced by the EMA2-HA-neo3 locus through phenotypic assortment (Hamilton and Orias, 2000).

Immunofluorescent staining and western blotting

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Immunofluorescent staining of long dsRNA was performed as described (Woo et al., 2016). The other immunofluorescent staining experiments were performed as described (Loidl and Scherthan, 2004). For immunofluorescent staining, all the primary and respective secondary antibodies (Alexa-488-coupled anti-rabbit or mouse IgG, Invitrogen) were diluted 1:1000. For western blotting, all the primary and respective secondary antibodies (HRP-coupled anti-rabbit or mouse IgG, Jackson ImmunoResearch Lab) were diluted 1:10,000.

DNA elimination assay by FISH

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DNA elimination assay by FISH was performed as described (Loidl and Scherthan, 2004). The plasmid DNAs pMBR 4C1, pMBR 2, and Tlr IntB, which contain different parts of the Tlr1element were used to produce Cy5-labeled FISH probes by nick translation (Wuitschick et al., 2002). Images of stained cells were analyzed by Fiji software. Whole areas of the new MAC (A) and MIC (I) and a part of the cytoplasm (C) were manually encircled in the images; and the intensities of DAPI and Cy5 (FISH) signals in each area were measured. Then, the IES retention index was calculated as follows: 0.35 × {[(mean Cy3 intensity A - mean Cy3 intensity C)]/[(mean Cy3 intensity I - mean Cy3 intensity C)]}/{[(mean DAPI intensity A - mean DAPI intensity C) ]/[(mean DAPI intensity I - mean DAPI intensity C)]}. Based on our previous observation, DNA elimination is completely blocked in the absence of TWI1 KO, therefore, the correction factor of 0.35 was obtained experimentally to make the average IES retention index of TWI1 KO cells to 1.

Viability test for progeny

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To test the progeny viability, mating pairs were isolated into drops of 1 x SPP at 6 hpm and incubated for ~3 days. Then, the completion of the conjugation of grown-up cells from wild-type and EMA2 KO pairs were examined for their 6-methylpurine (6-mp) resistance and paromomycin sensitivity, respectively as described previously (Mochizuki et al., 2002).

Small RNA analyses

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For northern hybridization analysis of scnRNAs, 10 µg of total RNAs isolated from conjugating wild-type and EMA2 KO cells using TRIzol reagent were separated in a 15% acrylamide-urea gel and analyzed by northern blot using the radio-labeled Mi-9 probe as previously described (Aronica et al., 2008). The hybridized probe was detected by Typhoon IP Phosphorimager (GE healthcare). High-throughput sequencing and analyses of small RNAs were performed as described (Mutazono et al., 2019). The 2020 version of the MAC genome assembly (Sheng et al., 2020) was fragmented into 10 kb pieces. Each 10 kb fragment that contains longer than 3 kb mappable sequence was used as an MDS tile (total 10,235 tiles). Each Type-A IES (Noto et al., 2015) was used as an IES tile (total of 4691 tiles). Normalized numbers (RPKM [reads per kilobase of unique sequences per million]) of 26- to 32-nt small RNAs that uniquely matched one of the MDS and IES tiles were counted.

GST pull down assay

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E. coli codon-optimized synthetic EMA2 and UBC9 genes (EMA2-Ec and UBC9-Ec) were cloned into the BamHI and XhoI sites of pGEX-4T-1 and NdeI and BamHI sites of pET28a (+), respectively to obtain pGEX-4T-1-EMA2-Ec and pET28-UBC9-Ec. pET28-UBC9-Ec was introduced into BL21 E. coli strain, His-Ubc9 expression was induced ~16 hr at 25 °C with 0.05 mM isopropylthio-β-galactoside (IPTG), the cells were lysed in HEPES buffer (50 mM HEPES pH 7.5, 800 mM KCL, 10% glycerol, 0.2 mM PMSF, 1 x cOmplete ETDA-free [Roche]) including 10 mM imidazole, incubated with Ni-NTA beads for 2 hr at 4 °C, washed four times with HEPES buffer including 20 mM imidazole, eluted with HEPES buffer including 250 mM imidazole, dialyzed with 50 mM Tris pH 7.5, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT, 50% glycerol and finally passed through Microcon 10 kDa filter (Merck). For GST pull-down assay, BL21 E. coli cells were transformed with pGEX-4T-1 or pGEX-4T-1-EMA2-Ec, and expression of GST-alone or GST-Ema2 was induced ~16 hr at 25 °C, lysed with 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, 0.2 mM PMSF, 1x cOmplete EDTA-free and incubated with Glutathione Sepharose 4B for 90 min at 4 °C. Then, the beads were incubated with 10 µg of His-Ubc9 in GST-pull down buffer (20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1% NP-40, 5 mM MgCl2, 1 mM DTT, 0.2 mM PMSF, 1 x cOmplete EDTA-free) for 90 min at 4 °C, washed four times with GST-pull down buffer and eluted with 2 x SDS sample buffer. The eluted proteins were analyzed by western blotting.

Functionality test of HA-SMT3 and His-SMT3 using SMT3 germline KO strains

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The SMT3 homozygous heterokaryon KO strain GC6 (Nasir et al., 2015) was obtained from Dr. James Forney (Purdue University). This strain was crossed with B2086, and sexual progeny were selected for their paromomycin resistance. After sexual maturation, clones showing paromomycin sensitivity (i.e. the SMT3 KO locus was removed from the MAC by phenotypic assortment) were established as SMT3 heterozygous heterokaryon KO strains. Then they were crossed with B*VII to obtain their round I exconjugants. Finally, their genotypes were examined by PCR to select two SMT3 homozygous heterokaryon KO strains, B1-1 and B5-17. They were then mated and pBCMB1-His-SMT3 (or pBCMB1-His for negative control) or pBCS3B1-HA-SMT3 (or pBCS3B1-HA for negative control) were introduced into the new MAC at 8 hpm by particle gun. The cells were incubated in 10 mM Tris pH 7.5 at 30 °C until 24 hpm and fed by adding the equal volume of 2x SPP. After 3 hr incubation at 30 °C, the neo expression (and His-Smt3 expression for pBCMB1-His-SMT3) was induced by adding 1 µg/mL CdCl2 and cells were incubated an additional 1 hr. Then 100 µg/mL paromomycin was added and aliquoted to 96-well plates (150 µL/well, seven plates). The plates were incubated at 30 °C for 4–5 days and paromomysin-resistant cells were further examined by genomic PCR to confirm genetic rescue by the introduced constructs (but not by the endogenous SMT3 copy from the parental MAC).

Establishment of HA-Smt3 and His-Smt3 expressing strains

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STM3 cDNA was amplified by RT-PCR from the total RNA of vegetatively growing CU428 using SMT3_HA_FW and SMT3_HisExt_RV and cloned into the BamHI and SpeI sites of pBNMB1-HA using NEBuilder HiFi DNA assembly kit to obtain pBNMB1-HA-SMT3. Next, neo5 was excised with SalI and XmaI and replaced with pur6, which was amplified from pBP6MB1 with BTU1-LCFW and MTT1_5UTR-SeqRV, using NEBuilder HiFi DNA assembly kit to obtain pBP6MB1-HA-SMT3. For His-SMT3 expression, the HA-tag encoding sequence of pBP6MB1-HA-SMT3 was replaced by a 6x His tag-encoding sequence. Then, pBP6MB1-HA-SMT3 and pBP6MB1-His-SMT3 were digested by XhoI and introduced into the MAC BTU1 locus of the EMA2 KO cells. Transformation, selection, and phenotypic assortment were performed as described for the SPT6-HA strains.

Affinity purification of SUMOylated proteins

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For purification of His-Smt3-conjugated proteins, EMA2 KO cells containing the BP6MB1-His-SMT3 construct were mated with either a wild-type or another EMA2 KO strain and His-SMT3 was induced by adding 0.05 μg/mL CdCl2. The purification was performed as descried (Hendriks and Vertegaal, 2016; Liebelt et al., 2019). Briefly, the cells were lysed in Guanidine Lysis Buffer (6 M Guanidine-HCl, 93.2 mM Na2HPO4, 6.8 mM NaH2PO4, 10 mM Tris-HCl pH 8.0) by sonication (2 × 25 times, 20% power, 60% pulse) using a probe sonicator (Omni-Ruptor 250 Ultrasonic Homogenizer). After centrifugation, the cleared lysate was supplemented with 50 mM imidazole and 5 mM β-mercaptoethanol and incubated with Ni-NTA agarose beads. The beads were washed, and the bound proteins were eluted with Elution buffer (6 M Urea, 58 mM Na2HPO4, 42 mM NaH2PO4, 10 mM Tris-HCl pH 8.0, 500 mM imidazole). The eluted proteins were precipitated in 10% TCA, resuspended in 1 x SDS buffer, and analyzed by western blot and mass-spectrometry. Wild-type cells that do not express His-Smt3 was also analyzed in parallel to identify proteins that intrinsically bind to the Ni-NTA agarose beads. For purification of HA-Smt3-conjugated proteins, total proteins were precipitated by incubating cells in 6% trichloroacetic (TCA) on ice for 10 min and then spinned down at 13,000 rpm for 5 min at 4 °C. The precipitate was dissolved in 1 x SDS sample buffer, neutralized with 2 M Tris, and then incubated for 5 min at 95 °C. The dissolved proteins (600 µl) were then suspended in 7.4 mL of 20 mM Tris pH 7.5, 100 mM NaCl, 2 mM MgCl2, 2 mM CaCl2 and incubated with Ezview Red anti-HA agarose beads (Sigma) for overnight at 4 °C. Then the beads were washed five times with 20 mM Tris pH 7.5, 100 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 0.1% tween 20 and the bound proteins were eluted by incubating with 1 x SDS buffer for 5 min at 95 °C. The eluted proteins were analyzed by western blotting.

Establishment of Spt6-HA expressing strains

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To obtain the SPT6-HA construct, a part of the coding sequence of SPT6 was amplified from the genomic DNA of B2086 using SPT6-FW1-SacII and SPT6-RV2-overlap. Similarly, the 3' flanking sequence was amplified using SPT6-3F-FW2-overlap and SPT6-3F-RV1-KpnI. The two PCR products were combined by overlapping PCR using SPT6-FW1-SacII and SPT6-3F-RV1-KpnI and cloned into the SacII and KpnI sites of pBlueScriptSK (+) to obtain pSPT6. Next, the NheI and XhoI fragment of pHA-neo3 was cloned into the same restriction sites of pSPT6 to obtain pSPT6-HA-neo3. Then, neo3 was excised from pSPT6-HA-neo3 with XmaI and SalI and replaced by pur6 from pBP6MB1-TR-TUBE to obtain pSPT6-HA-pur6. Finally, the pSPT6-HA-pur6 plasmid were digested by SacII and KpnI and introduced into the MAC SPT6 locus of an EMA2 KO and a wild-type strain by biolistic transformation. The transformed cells were selected in 300 μg/mL puromycin (Invitrogen) and 1 μg/mL CdCl2 and cells were phenotypically assorted as previously described (Hamilton and Orias, 2000), until cells grew in 1.2 mg/mL of puromycin.

RT-PCR analyses of lncRNA transcripts

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Total RNA from mating wild-type and EMA2 KO cells were isolated at 6 hpm using TRIzol reagent (Invitrogen). The RNA was treated with Turbo DNase (Ambion) and cDNA was synthesized from 1 µg of RNA using SuperScript II (Invitrogen) with random 6-mer as primers. The lncRNA transcripts from the MAC loci containing the M-, L8-, and R2 IES boundaries (see also Figure 6A) were amplified by nested PCR (95 °C for 20 s/50 °C for 30 s/68 °C for 1 min; 30 cycles each) using the primers M5'–3+M3'–3 (M-1st), M5'–4+M5'–4 (M-2nd), L8_5'–1+L8_3'–1 (L8-1st), L8_5'–2+L8_3'–2 (L8-2nd), R2_5'–1+R2_3'–1 (R2-1st), and R2_5'–2+R2_3'–2 (R2-2nd) and analyzed in 1% agarose gel. As a positive control, the constitutively expressed RPL21 mRNA was detected with RPL21-FW and RPL21-RV.

Cell fractionation

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Cell fractionation was performed as described (Ali et al., 2018). 1.4 x 106 cells were incubated with 1 mL of Lysis buffer (50 mM Tris pH 8.0, 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 0.5% Triton X-100, 1 x Complete Protease Inhibitor) on ice for 30 min and spun down at 15,000 g for 10 min at 4 °C. The proteins in 500 µL of supernatant were precipitated by adding 10% (final) TCA, washed three times with ice-cold acetone, and dissolved in 70 µL 1x SDS sample buffer, while the precipitate was dissolved in 2 mL of fresh Lysis buffer, sonicated (five times of 20% power-50% pulse-5 pulses with >20 s interval) using a probe sonicator (Omni-Ruptor 250 Ultrasonic Homogenizer), and then centrifuged at 20,000 g for 10 min at 4 °C. Proteins in 1 mL of the supernatant (solubilized chromatin) were precipitated by adding 10% (final) TCA, washed three times with ice-cold acetone and dissolved in 70 µL 1x SDS sample buffer. For cell Fractionation with RNase treatment, 1.4 × 106 cells were incubated with 1 mL of Lysis buffer containing 20 µg/mL RNase A on ice for 30 min and spun own at 15,000 g for 10 min at 4 °C. The proteins in 1 mL of the supernatant were precipitated by adding 10% (final) TCA, washed three times with ice-cold acetone, and dissolved in 70 µL 1x SDS sample buffer, while the precipitate was directly dissolved in 140 µL of 1 x SDS sample buffer.

Establishment of SPT6 germline KO strains

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To create the knockout construct for SPT6, the 5’ and 3’ flanking regions of the SPT6 gene were amplified by PCR using the primer sets SPT6-KO-5F-FW/SPT6-KO-5F-RV and SPT6-KO-3F-FW/SPT6-KO-3F-RV, respectively and concatenated by overlap PCR using SPT6-KO-5F-FW and SPT6-KO-3F-RV. The PCR product was cloned into the XhoI and BamHI digested pBlueScriptSK(+). Then the obtained plasmid was digested with BamHI and the pm-resistant cassette neo4, which was isolated from the pNeo4 plasmid (Aronica et al., 2008) by SmaI digestion (Bruns and Cassidy-Hanley, 2000), was inserted. All assembly reactions were done using NEBuilder HiFi DNA assembly mix (NEB). The resulting plasmid, pSPT6-KO-neo4, was digested with SacII and XhoI to release the targeting construct from the vector backbone and introduced into the MIC of mating UMPS214 and UMPS811 cells (Vogt and Mochizuki, 2013) by biolistic transformation. Heterozygous progenies were selected with 100 µg/mL pm and 500 µg/mL 5-Fluoroorotic Acid (5-FOA, Fermentas) in the presence of 1 μg/ml CdCl2. The resulting heterozygous strains were cultured for 10 passages without pm and 5-FOA, and pm-sensitive (pm-s) heterozygous strains were isolated. The pm-s heterozygous cells were then mated with B*VI or B*VII to obtain their round I exconjugants. Finally, their genotypes were examined by PCR to select SPT6 homozygous heterokaryon KO strains.

Establishment of wild-type and mutant SPT6 strains

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SPT6 cDNA was amplified from total RNA of mating B2086 and CU428 at 6 hpm with SPT6-FW-Turbo and SPT6-RV-Turbo, cloned into SpeI and BamHI sites of pBCMB1-HA-Turbo to obtain pBCMB1-HA-Turbo-SPT6. Then, the MTT1 promoter and the HA-Turbo-tag were removed using AgeI and XmaI and replaced by the SPT6 promoter and the N-terminal HA tag, using the overlapping product of two genomic PCR fragments amplified with SPT6-5F-FW, SPT6-5F-RV and with SPT6-N-FW-HA and SPT6-N-RV to obtain pBCSB-HA-SPT6. To produce pBCSB-HA-SPT6-DOL-KR, the SPT6 cDNA lacking the first 248 amino acids were amplified using SPT6_M249_FW and SPT6_M249_RV, replaced with the NdeI-BamHI fragment of pBCSB-HA-SPT6 and then the synthetic DNA SPT6-DOL-KtoR was inserted into the BamHI site. To make pBCSB-HA-SPT6-N-KR, pBCSB-HA-SPT6-M-KR and pBCSB-HA-SPT6-C-KR, the conserved region of SPT6 was excised from pBCSB-HA-SPT6 using NdeI and AgeI and replaced respectively with Synthetic DNA Set 1 (SPT6-gBlock-118KR-1, SPT6-gBlock-42KR-2, and SPT6-gBlock-42KR-3), Synthetic DNA Set 2 (SPT6-gBlock-42KR-1, SPT6-gBlock-118KR-2, and SPT6-gBlock-42KR-3), or Synthetic DNA Set 3 (SPT6-gBlock-42KR-1, SPT6-gBlock-42KR-2, and SPT6-gBlock-118KR-3). All assembly reactions were done using NEBuilder HiFi DNA assembly mix (NEB). The constructs were digested with XhoI and introduced into the BTU1 locus of the new MAC of conjugating cells of two SPT6 KO homozygous heterokaryon strains at 10 hpm by electroporation. Progeny rescued by the introduced constructs were selected in 100 μg/mL paromomycin and 1 µg/mL CdCl2, and cells were phenotypically assorted until cells grew in ~100–120 µg/mL cycloheximide.

Data availability

The small RNA sequencing data have been deposited in the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo) with the accession no. GSE243435. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the MassIVE partner repository (https://massive.ucsd.edu/) with the database identifier MSV000092977.

The following data sets were generated
    1. Shehzada S
    2. Noto T
    3. Mochizuki K
    (2023) NCBI Gene Expression Omnibus
    ID GSE243435. A SUMO E3 ligase promotes long non-coding RNA transcription to regulate small RNA-directed DNA elimination.
    1. Mochizuki K
    (2023) MassIVE partner repository
    ID MSV000092977. Comparison of SUMOylated proteins between wild-type and EMA2 KO Tetrahymena cells.

References

Decision letter

  1. Adèle L Marston
    Senior and Reviewing Editor; University of Edinburgh, United Kingdom

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

[Editors' note: this paper was reviewed by Review Commons.]

https://doi.org/10.7554/eLife.95337.sa1

Author response

General Statements [optional]

We would like to thank all reviewers for their valuable and constructive comments, which helped us a lot to improve the manuscript.

Point-by-point description of the revisions

Reviewer #1 :

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

The authors convincingly show that Ema2, a conjugation-specific SUMO E3 ligase, localizes in the parental MAC during early conjugation stages, then moves to the new MAC. Using somatic EMA2 KO strains, they show that Ema2 is necessary for IES elimination and the recovery of viable progeny. They demonstrate that MAC scnRNAs do not disappear in an EMA2 KO and conclude that Ema2 is required for TDSD. They also show that ds lncRNA amounts in the parental MAC drop to background levels in an EMA2 KO, while they remain similar to WT in meiotic MICs or the new MACs.

They also present evidence supporting that the transcription factor Spt6 is one of the targets of Ema2-mediated sumoylation. Spt6 is found in the parental MAC of conjugating cells, regardless of Ema2. However, Ema2 is crucial for the stable chromatin association of both Spt6 and Rpb3 (a subunit of RNA polymerase II). Unexpectedly, a non-sumoylatable Spt6 mutant is able to complement a SPT6 KO, since it maintains the synthesis of lncRNA in the parental MAC. Nonetheless, this mutant strongly impairs new MAC development and IES elimination. As a whole, the role of Spt6 sumoylation in programmed DNA elimination is not clearly established, and it probably affects another step than pMAC-lncRNA synthesis.

Strong points

The demonstration that pMAC-lncRNA accumulation depends upon Ema2 is convincing. This finding provides novel insights into the mechanism involved in TDSD in Tetrahymena. An important point that would be worth discussing is how ds pMAC-lncRNAs may pair with scnRNAs.

An RNA helicase (Ema1?) may play an important role in this process.

The requirement of Ema1 in the interaction between pMAC-lncRNAs and scnRNAs was reported previously by us (Aronica et al. 2008), which has been cited in this manuscript. Related to this point, we have added the following discussion in the revised manuscript (Page 10, Line 30): “Although it is unclear whether lncRNAs are single or double stranded when Ema1 promotes the lncRNA-scnRNAs interaction, the less severe TDSD defect observed in the EMA2 KO cells compared to the EMA1 KO cells (Figure 3B) indicates that certain Ema1-dependent TDSD may be initiated by single-stranded lncRNAs or mRNAs that are transcribed independently of Ema2”.

The manuscript is very well written. I noticed only a few typos (see minor comments below).

The pointed typos have been corrected in the revised manuscript.

The experiments are overall well done and well described. For non-Tetrahymena readers, it would be useful to clarify in the Results section (or in figure captions) whether the different KOs are in the MAC and/or also in the MIC

We have indicated whether each KO line is somatic or germline (MAC+MIC) in the figure legends whenever these lines are referenced.

The search for Ema2 targets using mass spectrometry was performed in a wild-type SMT3 background. This implies that endogenous wild-type Smt3 may have competed with His-Smt3 for protein sumoylation. To what extent may this have been a problem for the enrichment of sumoylated proteins on nickel columns? This point is critical, since the authors discuss that other proteins involved in pMAC-lncRNA transcription may be modified by Ema2 (p. 12). They should repeat the experiment in an SMT3 KO, or use anti-Smt3 antibodies to enrich for sumoylated proteins. If this is not possible, they should at least provide additional explanations.

We agree that a competition between His-tagged and non-tagged Smt3 lowered the sensitivity for the identification of SUMOylated proteins and we might miss some Ema2-dependent SUMOylated protein in the current study. However, we believe such protein, if any, is SUMOylated at very low level and not highly likely to be involved in the genome-wide orchestration of lncRNA transcription. We rather think that a critical Ema2-dependent SUMOylation event might be missed because some other residues of the same protein are SUMOylated by Ema2-independent manner and it was detected as a protein that was SUMOylated in both wild-type and EMA2 KO condition. Therefore, as was explained in Discussion, it is important to identify individual residues that are SUMOylated in Ema2-dependent manner. We are on our way to set up an experimental system that allows us to detect individual SUMOylated residues in Tetrahymena and we hope to analyze the functions of Ema2-dependent SUMOylated residues in future studies.

In Figure 7A, the authors only show the localization of Spt6 in early exconjugants. Since Spt6 is essential for vegetative growth, one can expect that it also localizes in the vegetative MAC. Is it also found in the new developing MACs? The authors should complete the figure with additional panels showing vegetative cells and exconjugants at later stages (with their new MAC).

The Spt6 is indeed localized in the MAC during vegetative growth and in the new MAC at late conjugation stage in the wild-type condition. We did not detect any anomaly of Spt6 localization in the EMA2 KO cells at least at the cytological level. The immunostaining results at the late conjugation stage are shown in Figure 7—figure supplement 1 in the revised manuscript and mentioned in the revised text (Page 11, Line 13). The immunostaining results of vegetatively growing cells are only attached below because Spt6 localization at vegetative stage when EMA2 is not expressed is not highly relevant to this study.

Along the same line, the authors show that the non sumoylatable Spt6 mutant does not inhibit pMAC-lncRNA synthesis. No scnRNA analysis is shown under these conditions: does TDSD still take place? It would also be interesting to check whether lncRNAs are still produced in the new MACs.

The nonSUMOylatable Spt6 mutant (we now call SUMOylation defective Spt6 mutant according to one of the Reviewer 3’s suggestions) show lower mating, making us difficult to investigate its effect on TDSD. Because we did not detect Spt6 SUMOylation prior to mating, we believe the low mating phenotype of this mutant is not directly due to the loss of SUMOylation but instead some of the 77 K to R mutations affect the functions of Spt6 in efficient initiation of mating. Therefore, to precisely measure the effect of Ema2-dependent Spt6 SUMOylation, we need to identity exact Ema2-dependent SUMOylated residues of Spt6 to produce another nonSUMOylatable Spt6 mutant with fewer number of mutations that does not affect the mating process. Engaging in such work demands a substantial time investment, and we believe that the reviewers will concur that these experiments are components of our future projects.

Long dsRNA accumulation in the new MACs detected by the J2 antibody was comparable between wild-type and the SUMOylation-defective Spt6 mutant, suggesting that Spt6 SUMOylation is not necessary to produce lncRNAs in the new MAC. The data have been shown in Figure EV9 and mentioned in the main text (Page 12, Line 24) in the revised manuscript.

The experiment shown in Figure 4C indicates that high-molecular weight (possibly sumoylated) proteins decrease to 50% in the EMA2 KO: this suggests that another sumoylation activity exists in the cell. A search for other putative SUMO E3 ligases is missing in this study.

A few other putative SUMO E3 ligases indeed encoded in the Tetrahymena genome. Moreover, it is known that some substrates are SUMOylated without any SUMO E3 ligase in other eukaryotes. These points have been described in the revised text as follows (Page 8, Line 22): “The remaining Ema2-independent SUMOylation is likely mediated by other SUMO E3 ligases (including the SP-RING containing proteins TTHERM_00227730, TTHERM_00442270 and TTHERM_00348490) and/or E3-independent SUMOylation (Sampson et al. 2001).”

We agree that exploring the roles of other SUMO E3 ligases in Tetrahymena would be important and interesting, and we believe it will be one of our future projects.

Can one exclude that Spt6 is sumoylated at other stages (vegetative or during new MAC development) in an Ema2-independent manner?

We have now included western blot observation of Spt6 at different life stages of wild-type cells as Figure EV2. We did not detect any slower-migrating Spt6 species in vegetative cells. This has been mentioned in the revised text as follows (Page 9, Line 17):

“Then, to examine the timing of the appearance of the slower migrating Spt6 species, we introduced the same Spt6-HA-expressing construct into a wild-type strain and Spt6-HA was analyzed by western blotting (Figure EV2). Consistent with the Ema2-dependent appearance of the slower migrating Spt6-HA, they were not detected in growing and starved vegetative wild-type cells (Figure EV2, Veg and 0 hpm, respectively) when Ema2 was not expressed (Figure 1). The slower migrating Spt6-HA was also detected at 8 hpm when the new MAC was already formed (Figure EV2, 8 hpm) suggesting that Spt6 is possibly SUMOylated also in the new MAC.”

In which nucleus does coding transcription take place between 4.5 and 6 hpm? Can we exclude that the weaker association of Rpb3 with chromatin in the EMA2 KO cross also impairs coding transcription?

Coding transcription takes place in the parental MAC at 4.5 and 6 hpm in wild-type cells. Also, because EMA2 KO cells did not show obvious defect in the progression of the conjugation processes, any essential mRNA transcriptions for these processes must occur even in the absence of Ema2. These points prompted us to add the following discussion in the Discussion section (Page 13, Line 14):

“Moreover, as EMA2 KO cells did not significantly impede the progression of conjugation processes, any essential mRNA transcriptions for these processes must take place in the parental MAC during conjugation even in the absence of Ema2. Therefore, the observed loss of the majority of Spt6 and RNAPII from chromatin in the absence of Ema2 (Figure 7B) must be a temporal event during the mid-conjugation stage. This suggest that RNAPII might be specifically engaged in pMAC-lncRNA transcription at this particular time window in wild-type cells.”

The authors do not explain how they found Ema2. More information could be useful.

Ema2 was identified as a protein involved in DNA elimination during our systematic genetic investigation of genes exclusively expressed during conjugation. This has been mentioned in the revised manuscript (Page 6, Lines 4-5).

In Figures 2B and 3B: the statistical significance of the differences observed for the IES retention index and small RNA amounts should be evaluated using appropriate tests.

The result shown in Figure 2B (IES retention analysis) has been tested by Welch two-sample ttest and outcomes have been shown in the revised Figure 2B.

The result shown in Figure 3B (small RNA seq) has been tested by Wilcoxon rank sum test and outcomes have been shown in the revised Figure 3B.

Figure 3 caption: define acronym "IQR".

The definition of IQR (the interquartile range) has now been mentioned in the figure legend in the revised manuscript.

Figure 5 caption (line 4): there may be a word missing ("from conjugating cells?")

We have corrected the sentence by adding “cells” after “from conjugating” in Page30-Line 34.

Figure 8C: what does the asterisk stand for?

We realized that the asterisk is not necessary in the figure and thus it have been removed in the revised figure.

p. 10 (bottom): an "o" is missing in "Aronica et al. 2008".

We have corrected the error.

p. 13 (2nd line): remove final "s" in "mimic".

We have corrected the error.

p. 14: change "were" to "was" in "the production of the EMA2 KO strains was described previously"

We have corrected the error.

p. 14: remove capital letters in "Gorovsky"

We have corrected the error.

p. 15 (Viability test for progeny): what does "6-mp" stand for?

It is 6-methylpurine. We have added this information to the revised manuscript.

p. 17 (end of first paragraph): change "contracts" to "constructs".

We have corrected the error.

p. 17 (2nd line of last paragraph): change "was" to "were " in "EMA2 cells containing the BP6MB1His-SMT3 construct were mated…"

We have corrected the error.

p. 19 (3rd line of 2nd paragraph"): "spined own" should be replaced by "spinned down".

We have corrected the error.

Reviewer #1 (Significance (Required)):

In this manuscript Shehzada et al. report important novel findings on the molecular mechanisms involved in RNA-mediated control of programmed DNA elimination in the ciliate Tetrahymena thermophila. In this organism, non-coding transcription takes place in distinct nuclei and produces double-stranded (ds) long non-coding RNAs (lncRNAs) at different stages during conjugation. First, bidirectional transcription in the MIC during meiosis produces ds lncRNAs that are processed to short scnRNAs. Second, lncRNAs from the parental MAC (pMAC-lncRNAs) are thought to drive the degradation of scnRNAs homologous to parental MAC DNA, in a process called TDSD (target-directed scnRNA degradation). Third, the remaining MIC-specific scnRNAs are imported to the new MACs, where their pair with lncRNAs and drive heterochromatin formation and DNA elimination.

The present study focuses on TDSD, a process that has been poorly described at the molecular level. The strongest part of the work is the demonstration that the SUMO E3 ligase Ema2 is necessary for the production of pMAC-lncRNAs, which in turn impairs the selective degradation of MAC scnRNAs. A less convincing part is the identification of Ema2 targets. The authors identify Spt6 as one of the Ema2-dependent sumoylated proteins. However, they show that Spt6 sumoylation is not necessary for pMAC-lncRNA transcription.

In principle, the results presented in this manuscript should be of broad interest for the scientific communities working on non-coding RNA biology and the epigenetic control of programmed genome rearrangements.

Reviewer #2

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary

During conjugation (the sexual reproduction stage in the Tetrahymena ciliates), programmed DNA elimination guided by small RNAs termed scnRNAs results in the specific elimination of many repetitive sequences. This specificity relies on the target-directed scan RNA degradation (TDSD) pathway where scnRNAs matching the active parental macronucleus are eliminated.

The manuscript by Shehzada et al. identifies a novel player in Tetrahymena TDSD: SUMO E3 ligase Ema2. The authors show by northen and small RNA-seq that Ema2 is required for TDSD. Furthermore, the paper describes how Ema2 post-translationally modifies the transcription elongation factor Spt6 by SUMOylation and that Ema2 is required to produce long doublestranded scnRNA precursor transcripts from the parental macronucleus, possibly via its modification of Spt6.

Major comments

From Figure 4C, the authors conclude that "Ema2 is the major SUMO E3 ligase during the midconjugation stages.", yet in Figure 5 show that only Spt6-SUMOylation is affected in Ema2 mutants. These conclusions seem inconsistent and should be reconciled as it is a central point in the paper. E.g. is Spt6 protein abundance based on the MS data supporting that this protein constitutes a major fraction of the (high mol weight) SUMOylated proteins? Of note, the discussion contains a very balanced discussion of this but the current description in the results should be improved.

Some of the proteins detected from both the wild-type and EMA2 KO conditions were possibly poly-histidine-containing proteins that bound intrinsically to the nickel-NTA beads or proteins unpacifically bound to some of the bead material. Taking these possibilities into account, a control experiment with wild-type cells not expressing His-Smt3 in the same condition is now included in the study and any proteins that were also identified in this experiment with log2 LFQ score above 25 were excluded in the new Figure 5A. We also removed any identified proteins containing more than 6 consecutive histidine residues from the plot. After these filtering processes, it is now clear that Spt6 is the major SUMOylated protein detected in the wild-type (with His-Smt3) condition and the LFQ intensities of other proteins (except Smt3) were ~16 or more hold less than that of Spt6. Together with the fact that the molecular weight range of most of the SUMOylated proteins fits very well to that of SUMOylated Spt6, we are now more confident to conclude that Ema2 is the major SUMO E3 ligase during the mid-conjugation stages and Spt6 is the major target of Ema2. We have modified the corresponding figure and texts to explain this filtering and the outcomes (Page 9, Lines 2-9).

The western blots carried out for the chromatin fraction and presented in Figures 7B, 7C, and 8B have variable levels of histone H3 which serves as a fractionation control, thus indicating some experimental variability. To support the quantitative conclusions, the authors should indicate how many times were these fractionation experiments repeated and should also provide experimental replicate data in the supplements. These data are important to firmly support the quantitative conclusions the authors currently draw from the experiments.

Each of these fractionation experiments was done three times and gave comparative results. The replicate data have been shown in Figures EV5, EV6 and EV8.

Minor comments

Page 3: "Because small RNA-producing loci are also small RNA targets … " It should be specified that this is the case specifically for the studied system as it is not generally the case for small RNA loci. Overall, this third intro paragraph is a bit hard to read and might be improved by first introducing Tetrahymena and its distinctive cellular biology and then moving to the observation that small RNA source and target loci are separated in this ciliate.

We have modified the description to “Because small RNA-producing loci are also small RNA targets in most of the studied small RNA-directed heterochromatin formation processes, it poses a challenge to separately investigate lncRNA transcription for small RNA biogenesis and that for small RNA-dependent recruitment of downstream effectors in these processes.” (Page 3, Lines 24-27). We believe this has improved overall readability of the paragraph.

Figure annotation and readability: The manuscript and figure labels are rich in abbreviation (and sometimes even abbreviations of abbreviations, e.g. na = new MAC = new macronucleus).

We agree that there are many abbreviations in this manuscript but we believe most of them are necessary to keep the text and figures concise. To increase readability, we have spelled out all “abbreviations of abbreviations” when they appear the first time in the text. In fact, “na” was used not as an abbreviation but as a mark in the figures. We have modified the corresponding figure legends to make this point clearer. Also, to make the abbreviation “TDSD” more generalizable, we modified the manuscript to used it as “target-directed small RNA degradation” instead of “target-directed scnRNA degradation”.

Also Figures 4, 5 – the addition of the protein name after α-HA, -GST or -His would make the interpretation of blots easier.

Because anti-GST is detecting both GST alone and GST-Ema2, in Figure 4B, we had indicated the names of the proteins next to the blots. These might be less visible due to the busy arrangement of the panels in the previous manuscript. We have made extra space to make these labeles more visible. For Figure 4C, Figure 5B and Figure 5C, we have followed the reviewer’s suggestion and changed the labels to show the proteins detected.

In Figure 4, it is unclear how the protein quantification was made (leading the "reduced to ~50% in the EMA2 KO" statement). Please clarify.

The total signal intensities of HA-Smt3 in triplicated experiments were analyzed by western blotting and quantified. We now have included the data as a part of Figure 4C in the revised manuscript and explained the quantification procedure in the figure lagend and Materials and Method.

In some places, the current manuscript refers to implicit knowledge that some non-specialists may not take for granted. For example, dsRNA formation is important for scnRNA production, motivating detection using the J2 antibody. Editing for non-expert readability could help reach a broader readership.

In this study, we used the J2 antibody not because dsRNA formation is important for the scnRNA production but because it allows us to cytologically detect lncRNAs in the parental MAC. We have modified the related sentence (Page 10, Lines 17-20) in the revised manuscript to improve readability. We have also added a discussion about single vs double-strand nature of lncRNA in the parental MAC (Page 10, Lines 30-34) as mentioned in our reply for the first comment of Reviewer 1.

Also, on Page 7, bottom, it would be helpful to briefly explain to the reader how SUMOylation works to motivate the conclusion from the Ubc9 interaction.

We have added a brief explanation for the actions of E1 and E2 enzymes in SUMOylation in the revised text (Page 8, Line 6-7).

Referees cross-commenting

My report (rev #2) closely aligns with that of rev #3. While all reports are positive, rev #1 suggests several lines of additional work, such as the characterization of lncRNA expression in the new MAC (major concern 3) and a search for other SUMO E3 ligase (major concern 4). While several interesting ideas are brought up here, I see such added investigations as non-essential for the current paper. I would encourage to focus revision work on the substantiation of the already included experiments.

The lncRNA expression in the new MAC in the C-KR mutant has been analyzed and included in Figure EV9. We have included some discussion regarding other SUMO E3 ligases and reserved their functional investigations for our future studies as Reviewer #2 and #3 suggested.

Reviewer #2 (Significance (Required)):

Significance

Overall, the presented work is well-structured, well-executed experimentally and carefully interpreted. The manuscript in most places (see minor comments) is clear and easy to follow for the expected broad readership in the fundamental biology of small RNAs and programmed DNA elimination. The main weakness of the paper is the proposed mechanistic connection from the Ema2 KO phenotype to Spt6 SUMOylation function in TDSD. The authors, however, have a very balanced description of this aspect in the discussion. In addition, there are some important technical questions to address regarding protein quantification by western blotting.

The work presented elucidates the crucial role of SUMO E3 ligase Ema2 in the TDSD pathway for scnRNAs in Tetrahymena. This advance is significant as TDSD is the foundation for the specificity of programmed DNA elimination in Tetrahymena and as it is currently not well understood mechanistically.

This work will be of interest to a broad readership for two reasons: (i) it advances our understanding of programmed DNA elimination in Tetrahymena, which is a major mechanistic model system for eukaryotic programmed DNA elimination. And (ii) it makes mechanistic connections to small RNA-mediated transcriptional silencing in yeast and fruit flies with possible general implications for these processes across eukaryotes.

In sum, the paper presents interesting new findings about small RNA biology and DNA elimination and was a pleasure to read.

The reviewers' declared field of expertise: small RNAs, chromatin, transcription

Reviewer #3

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

This study presents novel data and evidence for a critical involvement of protein SUMOylation in the process of noncoding RNA transcription during the process of conjugation in Tetrahymena. Loss of the critical SUMO E3 Ligase Ema2 leads to a loss of ncRNA transcription in the parental macronucleus, ultimately leading to the lack of scanRNA traget molecules on chromatin, and as a result a loss of heterochromatin formation as well as defective target-dependent small RNA degradation.

The paper is very well written, the figures are mostly a treat, the data is well discussed and placed in context, and the claims are supported by robust data. The authors went a long way to nail the relevant target protein of Ema2 and provide on the one side compelling evidence that the transcription elongation factor Spt6 is a bona fide SUMOylation substrate for Ema2. Quite surprisingly, however, a mutant Spt6 construct that shows no sign of SUMOylation in cells does rescue the Spt6 loss of function phenotype. While this puts the relevance of Spt6 SUMOylation in the process slightly into question, the authors provide a compelling discussion as to how SUMOylation still might be essential for proper Spt6 function in stimulating ncRNA transcription. All in all, this is a great paper that reports important data for the ciliate community, for the transcription community, and the larger small RNA community.

The following comments hopefully help to further improve the paper. I do not recommend any additional experiments.

Introduction: It is not entirely clear why the transcripts of small RNA targets are necessarily noncoding. labelling them as nascent would be sufficient in my opinion

In the described examples of small RNA-directed heterochromatin formation processes in the various eukaryotes in Introduction, the targets of small RNAs are indeed lncRNAs. Therefore, to separately discuss small RNA targets from mRNA, we keep using the term lncRNA for the former. It is unclear whether mRNAs can also be small RNA targets in the Tetrahymena DNA elimination process. We have added the following sentence in Introduction (Page 4, Line 30):

“Although mRNAs are transcribed in the parental MAC, it remains unclear if they also can induce TDSD and how mRNAs and pMAC-lncRNAs can be transcribed from overlapping locations.” Nonetheless, because EMA2 KO did not show detectable defect in the progression of conjugation processes, we believe any essential mRNA transcriptions for these processes occur in the parental MAC in EMA2 KO (which are now mentioned in Discussion [Page 13, Lines 14-20] for replying to one of Reviewer 1’s suggestions) and thus believe that the defects of EMA2 KO observed/discussed in this manuscript are due to the loss of lncRNAs. Therefore, we believe using lncRNA to label the RNAs transcribed by Ema2-directed SUMOylation is valid.

The nomenclature of methylated H3K9 might need some adjustment. Consider the abbreviation H3K9me2/3 instead of H3K9me

We followed the suggestion and H3K9me2/3 or H3K9m3 have been used in the revised manuscript.

It would be desirable if the authors could cross reference to the Paramecium field where possible given that this is a second, powerful study system in small RNA-mediated genome elimination.

We have extensively modified Introduction to describe the small RNA-directed genome rearrangement process of Tetrahymena and Paramecium as much as possible in parallel.

Main text:

"The conjugation-specific expression and the localization switch from the parental to the new MAC are reminiscent of the factors involved in DNA elimination (Mochizuki et al., 2002; Coyne et al., 1999; Kataoka & Mochizuki, 2015; Liu et al., 2007; Yao et al., 2007)." please name these other factors here.

We have added “such as the Piwi protein Twi1, which is loaded by scnRNAs, and PRC2 (Mochizuki et al. 2002; Liu et al. 2007; Noto et al. 2010)” at the end of this sentence (Page 6, Line 13).

Figure 5A: what is the author's interpretation of the finding that most identified proteins remain unchanged? are these Ema2 independent SUMOylated proteins or are these background proteins that are not SUMOylated?

As mentioned in our reply to Reviewer 2, some of the proteins detected from both WT and EMA2 KO were possibly poly-histidine-containing proteins that bound intrinsically to the nickel-NTA beads without His-Smt3 conjugation or proteins unpacifically bound to some of the bead material. Taking these possibilities into account, a control experiment with wild-type cells not expressing His-Smt3 in the same condition has now been included and any proteins that were also identified in this experiment with log2 LFQ score above 25 were excluded in the new Figure 5A. We also removed any proteins containing more than 6 consecutive histidine residues from the plot. After these filtering processes, it is now clear that Spt6 is the major SUMOylated protein detected in the wild-type (with His-Smt3 expression) condition and the LFQ intensities of other proteins (except Smt3) were ~16 or more hold less than that of Spt6. We have modified the corresponding figure and texts (Page 9, Lines 2-9) to explain this filtering procedure and the outcomes. Even after this filtering, many proteins were identified similarly between wild-type and EMA2 KO conditions. As mentioned in our reply for one of the comments by Reviewer 1, these are most likely Ema2-independent SUMOylated proteins either mediated by another SUMO E3 ligase or by E3-independent SUMOylation. We have added these points in the revised manuscript (Page 8, Lines 22-25).

"However, the cells rescued by HA-SPT6N-KR and HA-SPT6-M-KR showed severe defects in meiotic progression and mating initiation, respectively, making their SUMOylation status during conjugation uninvestigable." Why can't you investigate the SUMOylation capacity of these variants in wildtype cells?

The suggested experiment is probably a valid way to investigate the SUMOylation of HA-Spt6NKR and HA-Spt6-M-KR. However, in such experimental setting, SUMOylation of Spt6 might be blocked not by loss of SUMOylation sites but by competition between the wild-type and the mutant Spt6. Moreover, even if one of them is proved to be unSUMOylatable (we now decided to call it SUMOylation-defective mutant [please see below]), we cannot examine its effect on lncRNA transcription if it has to be co-expressed with the wild-type Spt6. Therefore, we decided not to further examine the SUMOylation of the two mutants.

"Therefore, Spt6-C-KR is an unSUMOylatable Spt6 mutant." How sure can you be about this given the dynamic range of the detection in this experiment?

Whatever the dynamic range is, it is not possible to conclude that there is zero SUMOylation on Spt6-C-KR in the experimental setting we used. So, we have decided to call it a “SUMOylation defective mutant” and modified the corresponding sentence as follows (Page 12, Line 18): “Therefore, Spt6-C-KR represents a SUMOylation-defective Spt6 mutant, exhibiting at least a reduced level of SUMOylation compared to Spt6 in the absence of Ema2 (compare Figure 8B and Figure 5B).”

Figure 1A: label the plot to make it more accessible. Axis labels are missing.

Axis labels and explanations for the stages have been added in the revised Figure 1A.

Figure 3A: can you speculate about the higher molecular weight signal in the northern blot that appears in the later time-points and that seems to be partially dependent on Ema2?

The appearance of these higher molecular weight signals correlates with the presence or absence of lncRNAs detected by the J2 antibody at 4.5 hpm (Figure 6B). However, their presence in EMA2 KO cells at 6 hpm, the time point before the development of the new MAC, does not fit well to the absence of J2 staining in the parental MAC in EMA2 KO cells. Therefore, we currently have no clear idea for the identity of the higher molecular weight signals.

Figure 3B: why are the scanRNA levels at 3h already so different between WT and mutant cells?

Lane 1 versus lanes 3 and 5?

The following sentence has been added in the revised manuscript (Page 7, Line 20):

“Because TDSD takes place concurrently with the scnRNA production (Schoeberl et al. 2012), the increased abundance of MDS-complementary scnRNAs at 3 hpm in the EMA2 KO cells compared to the wild-type cells can also be attributed to the necessity of Ema2 in TDSD.”

Figure 5: could you comment on the weak Smt3 signal that remains for Spt6 in the Ema2 KO conditions. Is this due to other SUMO-ligases or is the Ema2 KO not a full loss of function condition?

The following sentence has been added in the revised manuscript (Page 9, Line 31):

“The remaining SUMOylation observed on Spt6 in the absence of Ema2 is likely facilitated by other SUMO E3 ligases and/or E3-independent SUMOylation, as discussed earlier for the other instances of Ema2-independent SUMOylations.”

Figure 6C: are the many arrowheads not confusing? Are they needed?

We have removed most of the arrowheads from the figure and marked only the parental MACs. In addition, we have used the same labeling for all immunofluorescent staining figures.

Figure 8A: the cartoon depicting different colors for the various Lysine residues is not immediately clear to the reader. Try to make this more accessible.

We have modified the drawing to make the markings for the mutated lysine residues more visible in the revised figure.

Referees cross-commenting

I agree with the comment from reviewer #2 that additional experiments are not required at this stage. Several constructive points have been raised by all three reviewers that will strengthen this already very mature work.

Reviewer #3 (Significance (Required)):

This is a very strong experimental study that reports very interesting findings that do go beyond the ciliate community. Spt6 is a major transcription elongation factor and understanding the various functions of this factor by studying in vivo processes is highly important. The paper opens up a new research niche. The findings are very well presented and the discussion does a great job in putting the somewhat surprising results n the non SUMOylatable mutant into context.

https://doi.org/10.7554/eLife.95337.sa2

Article and author information

Author details

  1. Salman Shehzada

    Institute of Human Genetics (IGH), CNRS, University of Montpellier, Montpellier, France
    Present address
    Department of Genetic Medicine and Development, University of Geneva, Geneva, Switzerland
    Contribution
    Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4218-7622
  2. Tomoko Noto

    Institute of Human Genetics (IGH), CNRS, University of Montpellier, Montpellier, France
    Contribution
    Investigation, Methodology, Writing – original draft, Writing – review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0009-0008-4378-4913
  3. Julie Saksouk

    Institute of Human Genetics (IGH), CNRS, University of Montpellier, Montpellier, France
    Contribution
    Investigation, Project administration
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1885-1762
  4. Kazufumi Mochizuki

    Institute of Human Genetics (IGH), CNRS, University of Montpellier, Montpellier, France
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing
    For correspondence
    1. kazufumi.mochizuki@cnrs.fr
    2. kazufumi.mochizuki@igh.cnrs.fr
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7987-9852

Funding

Agence Nationale de la Recherche (ANR-10-INBS-04)

  • Kazufumi Mochizuki

Agence Nationale de la Recherche (ANR-10-LABX-12-01)

  • Kazufumi Mochizuki

Agence Nationale de la Recherche (ANR-16-ACHN-0017)

  • Kazufumi Mochizuki

Fondation pour la Recherche Médicale (FDT20210601285)

  • Salman Shehzada

Fondation pour la Recherche Médicale (EQU202203014651)

  • Kazufumi Mochizuki

Fondation ARC (ARCPJA2021060003830)

  • Kazufumi Mochizuki

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We acknowledge the MRI facility, member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-10-INBS-04), the Proteomics Platform (PPM) of BioCampus Montpellier, 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) and an 'Accueil de Chercheurs de Haut Niveau' grant (ANR-16-ACHN-0017) from the French National Research Agency, Equipes a FRM 2022 grant from the Fondation Recherche pour Médicale (FRM, EQU202203014651), an ARC 2021 PJA3 grant from the ARC Foundation (ARCPJA2021060003830) to KM, and a 'Fin de these' program fellowship from Fondation pour la Recherche Médicale (FRM, FDT20210601285) to SS.

Senior and Reviewing Editor

  1. Adèle L Marston, University of Edinburgh, United Kingdom

Version history

  1. Preprint posted: September 27, 2023 (view preprint)
  2. Received: December 14, 2023
  3. Accepted: January 3, 2024
  4. Accepted Manuscript published: January 10, 2024 (version 1)
  5. Version of Record published: January 31, 2024 (version 2)

Copyright

© 2024, Shehzada et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Salman Shehzada
  2. Tomoko Noto
  3. Julie Saksouk
  4. Kazufumi Mochizuki
(2024)
A SUMO E3 ligase promotes long non-coding RNA transcription to regulate small RNA-directed DNA elimination
eLife 13:e95337.
https://doi.org/10.7554/eLife.95337

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https://doi.org/10.7554/eLife.95337

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