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Genome-defence small RNAs exapted for epigenetic mating-type inheritance

Abstract

In the ciliate Paramecium, transposable elements and their single-copy remnants are deleted during the development of somatic macronuclei from germline micronuclei, at each sexual generation. Deletions are targeted by scnRNAs, small RNAs produced from the germ line during meiosis that first scan the maternal macronuclear genome to identify missing sequences, and then allow the zygotic macronucleus to reproduce the same deletions. Here we show that this process accounts for the maternal inheritance of mating types in Paramecium tetraurelia, a long-standing problem in epigenetics. Mating type E depends on expression of the transmembrane protein mtA, and the default type O is determined during development by scnRNA-dependent excision of the mtA promoter. In the sibling species Paramecium septaurelia, mating type O is determined by coding-sequence deletions in a different gene, mtB, which is specifically required for mtA expression. These independently evolved mechanisms suggest frequent exaptation of the scnRNA pathway to regulate cellular genes and mediate transgenerational epigenetic inheritance of essential phenotypic polymorphisms.

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Figure 1: Structure and expression of mtA and related proteins.
Figure 2: Structure of mtA in the MIC and in the MACs of E and O cells.
Figure 3: Molecular analysis of expression mutants.
Figure 4: Genes required for excision of the mtA promoter in O cells.
Figure 5: mtB rearrangements determine mating types in P. septaurelia.

Accession codes

Primary accessions

European Nucleotide Archive

GenBank/EMBL/DDBJ

Gene Expression Omnibus

Data deposits

Microarray data have been deposited at the Gene Expression Omnibus database49 under accession number GSE43436. RNA-seq data (transcriptomes of mtBO and mtCO mutants) have been deposited in the European Nucleotide Archive (EBI) under accession number ERP002291. Small RNA sequences have been deposited at the EBI under accession number ERP001812. The mtA, mtB and mtC sequences of all strains and species studied have been deposited at GenBank under accession codes KJ748544KJ748569.

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Acknowledgements

We thank S. Malinsky, C. Ciaudo and M.-A. Félix for critical reading of the manuscript, and S. Marker and all other laboratory members for continuous support and discussions. This work was supported by the ‘Investissements d’Avenir’ program ANR-10-LABX-54 MEMO LIFE/ANR-11-IDEX-0001-02 Paris Sciences et Lettres* Research University and by grants ANR-08-BLAN-0233 ‘ParaDice’ and ANR-12-BSV6-0017 ‘INFERNO’ to E.M., L.S. and S.D., an ‘Equipe FRM’ grant to E.M., grants ANR-2010-BLAN-1603 ‘GENOMAC’ and CNRS ATIP-Avenir to S.D., and National Science Foundation grant MCB-1050161 to M. Lynch (JFG). D.P.S. was supported by Ph.D. fellowships from the Erasmus Mundus program and from the Ligue Nationale Contre le Cancer. M.L.-A. was supported by Ph.D. fellowships from the Ministère de l’Enseignement Supérieur et de la Recherche and from the Fondation de la Recherche Médicale. A.P. was supported by grant RFBR 13-04-01683a. Some strains used in this study are maintained at the Centre of Core Facilities ‘Culture Collection of Microorganisms’ in St Petersburg State University. The sequencing of the mtBO and mtCO MAC genomes benefited from the facilities and expertise of the high-throughput sequencing platform of IMAGIF (Centre de Recherche de Gif, http://www.imagif.cnrs.fr). The mtBO and mtCO transcriptomes were sequenced at the Genomic Paris Centre - IBENS platform, member of ‘France Gènomique’ (ANR10-INBS-09-08). This study was carried out in the context of the CNRS-supported European Research Group ‘Paramecium Genome Dynamics and Evolution’ and the European COST Action BM1102.

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Authors

Contributions

D.P.S. did almost all of the experimental work presented here and contributed to the design of experiments. B.S. characterized mRNAs and contributed to silencing experiments and northern blot analyses. G.G. contributed to gene sequencing, plasmid construction, PCR analyses and cell line maintenance. J.-F.G. did the microarray analysis, and A.A.-F. the confocal analysis of mtA–GFP fusions. A.A., K.L. and J.-M.A. carried out the deep sequencing of small RNAs, and C.B. that of the mtBO and mtCO transcriptomes; O.A. and L.S. did the bioinformatic analyses. K.B., M.L.-A., V.T. and S.D. showed the role of scnRNA pathway genes in mtA promoter excision. S.B. did the mtA promoter dsRNA feeding experiment. A.P. contributed to the analysis of the mtAO mutant and provided P. octaurelia and septaurelia strains. M.P. contributed to the analysis of the mtBO mutant and prepared samples from the P. octaurelia cross, which was carried out by E.P. E.M. conceived the study and wrote the paper.

Corresponding author

Correspondence to Eric Meyer.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 The 195-bp segment contains the mtA promoter and ensures maternal inheritance of its own retention.

a, Plasmids used for microinjection into the MAC of O cells. The thick grey lines on either side of inserts represent vector sequences. Plasmid pmtA-E contains the E MAC version with entire intergenic regions; pmtA-ES lacks the downstream unannotated gene. pmtA-IES lacks intergenic sequences upstream of the 195-bp segment. pIES contains only the 195-bp segment. The PCRs used for testing transformation are shown. b, Transformation of O cells with pmtA-E or pmtA-ES. PCR1 amplifies the promoter-containing form from either plasmid and the promoter-excised form from the endogenous mtA gene; relative amounts provide an indication of plasmid copy numbers. The mating types of injected clones and of their mass post-autogamous progeny are indicated. C1 and C2, uninjected controls; pE, control PCR on pmtA-E. c, Transformation of O cells with pmtA-IES. The plasmid-specific PCR2 (1,533 bp) identifies transformed clones. The O type of clones 9 and 10 could be due to transgene silencing. d, Transformation of O cells with pIES. Injected clones were tested with a duplex PCR: PCR4 amplifies a 450-bp product from the plasmid and PCR3 a 1,035-bp product from the endogenous mtA gene, hardly detectable in high-copy transformants. C1 and C2, uninjected controls; pD, control duplex PCR on an equimolar mix of pIES and pmtA-E. PCR1 revealed that the 195-bp segment was retained in the mass progeny of transformed clones; the selfer phenotype (S) of the mass progeny from clone 4 probably reflects heterogeneity among individual post-autogamous clones.

Extended Data Figure 2 Genetic analysis of mutations affecting mating-type expression.

a, Mendelian segregation of a pair of alleles. In both conjugation and autogamy, the two MICs undergo meiosis, but only one haploid product is retained in each cell; an additional mitosis then produces two identical gametic nuclei. During conjugation of genetically different cells (m/m and +/+), reciprocal exchange of one gametic nucleus, followed by karyogamy, therefore results in genetically identical zygotic nuclei in the two exconjugants. The drawing shows the heterozygous MICs and MACs that develop from copies of the zygotic nuclei in each of the F1 cells. During autogamy, the two identical gametic nuclei fuse together, resulting in entirely homozygous zygotes; post-autogamous F2 progeny of heterozygotes have a 50% probability to keep each of the parental alleles. b, Maternal (cytoplasmic) inheritance of mating types. Coloured Os and Es around the cells indicate the mating type expressed by each vegetative clone; the MACs are coloured to symbolize their mating-type determination states (O, blue; E, orange). Because little cytoplasm is exchanged during conjugation, the parental MACs independently condition zygotic MACs for the same mating types, resulting in cytoplasmic inheritance: the F1 derived from the O parent is determined for O in 94% of cases, and the F1 derived from the E parent is E in 98% of cases66. The frequency of mating-type reversal is much lower at autogamy: <1/50,000 in the O-to-E direction, and 1/3,000 in the E-to-O direction67. c, Cross of O-determined expression mutants mtAO, mtBO and mtCO to wild-type E cells. F2 mutant homozygotes formed in the E cytoplasmic lineage express mating type O as a result of the mutation, but remain determined for E (orange MAC). d, Cross of O-expressing, E-determined mutants mtAO, mtBO and mtCO (as produced in c) to wild-type E cells. E determination of the mutant parent is revealed by E determination and expression of the derived F1, which has received the wild-type allele. In this cross all cells are determined for E, and the expressed mating type simply depends on genotype. e, mtA promoter retention in the cross shown in c, illustrated here for a cross of the mtAO mutant with wild-type E cells. Top panel: PCR analysis (PCR5) of two sets of 4 mtAO/mtA+ F1 karyonides (after fertilization, two new MACs develop in each cell from copies of the zygotic nucleus, and then segregate to daughter cells, called karyonides, at the first cellular division; each pair of conjugants thus gives rise to 4 F1 karyonidal clones). Each set contained 2 O clones that had excised the mtA promoter, and probably derived from the mutant O parent, and two E clones that retained it, and probably derived from the wild-type E parent (mating types are indicated below each lane). Bottom panels: after autogamy of two mtAO/mtA+ F1 heterozygotes of mating type E (clone 4 in each set), 12 F2 homozygous progeny were isolated for each, grown and tested for mating-type expression and for mtA promoter retention using PCR6 (Supplementary Table 6), which amplifies products of 665 bp and 470 bp from the promoter-containing and promoter-excised versions, respectively. All clones retained the promoter, although a fraction of them (14 of 23) expressed mating type O, as expected for mtAO homozygotes. ND, not determined. f, mtA promoter retention in F1 heterozygotes from the cross shown in d, illustrated by a typical set of 4 mtBO/mtB+ F1 karyonides from the cross of an E-determined, O-expressing mtBO homozygote (as produced in c) to wild-type E cells. PCR5 showed that the mtA promoter was now retained in F1 karyonides from both parents; all were of mating type E.

Extended Data Figure 3 Complementation of the mtBO and mtCO phenotypes with the wild-type alleles of GSPATG00026812001 and GSPATG00009074001, respectively.

a, Structure of the MIC and MAC versions of the mtB gene, and of the GFP fusion transgene used for complementation. The coding sequence (open arrow) is shown with the complete upstream and downstream intergenic regions. The MIC version contains two IESs (black boxes). Plasmid pmtB51-NGFP contains the MAC version with complete intergenic regions, and the EGFP coding sequence was fused at the 5′ end of the mtB coding sequence. Thick grey lines on either side represent plasmid vector sequences. b, PCR analysis and mating types of E-determined mtBO mutant clones transformed with pmtB51-NGFP. PCR7 (top panel) amplifies products of 1,148 bp from the plasmid, and of 419 bp from the endogenous mtB gene (Supplementary Table 6). The relative abundance of the two products gives an indication of plasmid copy number in each clone. C1 and C2, uninjected control clones. Mating types are indicated below each lane. Clones containing detectable plasmid amounts expressed mating type E, indicating that the GFP fusion protein is functional; the selfing phenotype (S) of clone 11 may be due to some cells having lost the plasmid. PCR1 (bottom panel) confirmed that all clones retained the mtA promoter. c, Structure of the MIC and MAC versions of the mtC gene, and of the plasmid used for complementation. The coding sequence (open arrow) is shown with the complete upstream and downstream intergenic regions. The MIC version contains one IES (black box). Plasmid pmtC51 contains the MAC version with 349 bp and 98 bp of upstream and downstream intergenic sequences, respectively. The plasmid-specific PCR8 amplifies a 419-bp product (Supplementary Table 6). d, PCR analysis and mating types of E-determined mtCO mutant clones transformed with pmtC51. PCR8 (top panel) shows that all positive clones expressed mating type E. C1, C2 and C3, uninjected control clones. PCR1 (bottom panel) confirmed that all clones retained the mtA promoter.

Extended Data Figure 4 Deep sequencing of scnRNAs from an early conjugation time point (early meiosis).

A total of 39,041,474 small-RNA sequences of 25 nucleotides in length were obtained by Illumina sequencing of four libraries previously constructed from gel-purified molecules migrating at 23, 24, 25, or 26 nucleotides (from the 7hND7 total RNA sample12). a, 25-nucleotide reads were first mapped (no mismatch allowed) on possible contaminant sequences (genomes of bacteria commonly found in cultures, P. tetraurelia mitochondrial genome, P. tetraurelia rDNA and other non-coding RNAs). The remainder was then mapped on known MIC sequences (the ‘MAC+IES’ genome, and 9 individual copies of the Sardine transposon3). To determine whether any of the remaining reads could correspond to IES excision junctions or to spliced transcripts, they were then mapped on the MAC genome and on the genome-wide set of spliced transcripts. Very few hits were found and these did not show the characteristic 5′-UNG signature of scnRNAs (see d), suggesting that these molecules represent longer forms of endogenous siRNAs and/or could be mapped because of IES or intron annotation errors. Of the remaining unmapped reads (49%), close to one-half could be mapped on ‘PGM contigs’, a 25-Mb preliminary assembly of MIC-specific sequences that are not collinear to MAC chromosomes3 and are thus likely to represent bona fide scnRNAs. b, Statistics about the coverage of the ‘MAC+IES’ genome (17,786,284 reads). The average coverage is similar for exons, introns and intergenic regions, but is 2-fold higher for IES sequences. This may mean either that scnRNAs are initially produced in higher amounts from IESs, or that active degradation of scnRNAs homologous to MAC sequences is already under way at this early stage. c, Mapping of 25-nucleotide reads on the MIC version of the mtA gene region. Coding sequences of the corrected mtA gene model (PTETG5300016001, after correction of assembly indels and re-annotation) and of the short gene downstream (PTETG5300018001) are shown as red boxes interrupted by introns and IESs. The yellow box represents the 195-bp segment of the mtA promoter that is excised in O cells; the 4 IESs are shown as green boxes. 25-nucleotide reads mapping on the top or bottom strands of the region are colour-coded to indicate the number of times each one was sequenced: blue, one read; green, 2–9 reads; red, ≥10 reads. d, Compositional profiles (nucleotide frequency on the left, and deviation from randomness on the right) of reads mapping to the MAC+IES genome, to the MAC genome, or to ‘PGM contigs’. For each set, logos are shown for all reads (left), or for the non-redundant subset only (right). For the ‘MAC+IES’ and ‘PGM contigs’ sets, the logos computed from all reads clearly show the 5′-UNG signature typical of scnRNA guide strands, as is the case for the major subset of reads starting with U (5′U), while the minor subset of sequences not starting with U (5′A/C/G), which may represent the steady-state amount of passenger strands, shows the complementary signature CNA at positions 21–23. Deviation from randomness is greater when computed from all reads than when computed from the non-redundant subset only, indicating that molecules with the signature are intrinsically produced in higher amounts, or are more stable. The small set of reads that mapped only to the MAC genome (putative IES excision junctions) does not show a clear 5′-UNG signature, suggesting that most of those are not scnRNAs. The same is true of the small number of reads mapping to exon–exon junctions (not shown).

Extended Data Figure 5 Northern blot analysis of mtA-promoter scnRNAs during autogamy of O or E cells.

Mass cultures were allowed to starve, and RNA samples were extracted during exponential growth (Exp) and then at different times (0–20 h) after the appearance of the first meiotic cells. Cells become committed for autogamy at a fixed point of the cell cycle, so that the best synchrony that can theoretically be achieved is the duration of one cell cycle; in these experiments, the time between the first and the last cells to begin meiosis was 12 h. a, Proportions of cells in different cytological stages at each time point, as determined by DAPI staining. Veg, vegetative cells; Mei, meiosis (crescent stage, meiosis I, meiosis II); Frg, cells with fragmented old MAC but new MACs not yet clearly visible; Dev, cells with two clearly visible developing new MACs; Kar, cells after the karyonidal division, with only one developing new MAC. b, Northern blot analysis of mRNAs for early (PTIWI09, NOWA2), middle (PGM), or late (PTIWI10) genes. The same blots were hybridized successively with the 4 probes. c, Northern blot analysis of small RNAs. The top panels show hybridization with the 195-bp mtA-promoter probe, revealing accumulation of 25-nucleotide scnRNAs slightly later than expression of the meiosis-specific genes PTIWI09 and NOWA2. As a control, the same blots were rehybridized with an oligonucleotide probe specific for a cluster of 23-nucleotide endogenous siRNAs on scaffold 2212,61, which are abundantly produced at all stages of the life cycle (bottom panels). Quantification of the mtA-promoter scnRNA signal and normalization with the siRNA signal did not reveal any significant difference in their amount or timing between the two mating types (not shown). Previous studies showed that the double-strand breaks that initiate IES excision in the new MACs start being detectable before the maximum of expression of the putative endonuclease PGM5,68 (no later than 10 h in these time courses). A PCR analysis of post-autogamous DNA samples confirmed that the mtA promoter was fully excised in mating type O, and fully maintained in mating type E (not shown).

Extended Data Figure 6 Phylogenetic tree of different strains from most P. aurelia species based on sequence polymorphisms in three nuclear genes.

This figure is modified with permission from figure 2 of Catania et al. Genetic diversity in the Paramecium aurelia species complex Mol. Biol. Evol. 2009, 26, 421–431 (ref. 32). Wrong species assignment of some strains studied here are corrected in red. Strain V5-13 was probably mis-assigned to P. septaurelia through the same error as for strain GFg-1 (see Extended Data Fig. 7). Species showing maternal inheritance or random determination of mating types are highlighted in red and blue, respectively.

Extended Data Figure 7 Strain GFg-1 belongs to the same species as strain 138, that is, P. octaurelia.

GFg-1 is among a set of strains that were originally assigned to P. septaurelia on the basis of conjugation tests with strain 38, a reference strain for that species. However, the stock of strain 38 used in these tests was not 38, but instead some P. octaurelia strain, as shown by the comparison of mtA sequences with those from the original strain 38 obtained from ATCC (ATCC number 30575) and those from strain 138, a reference strain for P. octaurelia. a, Scheme of the cross GFg-1 (O) × 138 (E). The mating types of parents were determined by cross-agglutination with P. tetraurelia tester lines. The green and blue arrows beside each cell represent the mtA gene, colour-coded to indicate the GFg-1 and 138 alleles; the box at the 5′ end symbolizes retention of the mtA promoter in the MAC genome on the E side of the cross. b, Molecular characterization of two pairs of F1 heterozygotes, and of some F2 clones obtained by autogamy of these F1 heterozygotes. The parental origin of each F1 clone was ascertained by a sequence polymorphism in the mitochondrial COXII gene (PCR9, Supplementary Table 6). PCR amplification and sequencing of a segment of the mtA gene encompassing the promoter (PCR10, Supplementary Table 6) showed that the two F1 clones deriving from the GFg-1 parent were heterozygotes, and that the mtA promoter was precisely excised from both alleles (see Supplementary Data 1a). ND, not determined. Analysis of 6 viable F2 clones obtained by autogamy of the F1 clones deriving from the 138 parent showed that 4 of them were homozygous for the GFg-1 allele, whereas the other two were homozygous for the 138 allele; the mtA promoter was retained in all cases. The evidence for successful genetic exchange between strains GFg-1 and 138 and for viable recombinant F2 progeny demonstrates that these strains belong to the same species; that is, P. octaurelia.

Extended Data Figure 8 Genetic and molecular analysis of the cross between strains 227 and 38 of P. septaurelia.

a, Maternal inheritance of mating types in the wild-type strain 227. b, Strain 38 is genetically restricted to O expression, but constitutively determined for E. The mutation identified in strain 38 is of particular interest because it affects both the expression and the inheritance of mating types, which suggests that it lies in a gene that controls mating-type determination through an alternative rearrangement. Indeed, known P. tetraurelia mutations fall in two distinct categories. mtAO, mtBO and mtCO prevent expression of type E but have no effect on the rearrangement that determines mating types or on its maternal inheritance, whereas mtFE lies in a trans-acting factor required for a subset of rearrangements during MAC development but has no effect on the expression of mating types during sexual reactivity. The only type of mutation that can be envisioned to affect both expression and determination/inheritance would be a mutation preventing expression of a functional protein required for E expression, and at the same time preventing in cis (by destroying a potential Pgm cleavage site) the rearrangement that normally inactivates this gene in the MAC of wild-type O cells. The mtXIII allele of strain 38 restricts cells to O expression in a recessive manner, but also has a maternal effect that enforces constitutive E determination in sexual progeny. Notably, elegant experiments showed the latter effect to be dominant20: the sexual progeny of a cell carrying at least one mtXIII allele can never be determined for O or transmit O determination. c, Sequencing of the mtB38 allele revealed features that may account for both effects. A frameshift mutation makes it a pseudogene, explaining the genetic restriction to O expression. In addition, a 6-bp deletion removes one of the IES-like boundaries used in the mtB227 allele for coding-sequence deletions in O clones. Given the requirements of IES excision in P. tetraurelia and in sibling species36, this can be predicted to make the same deletions impossible in the mtB38 allele, which would explain the constitutive E determination effect. The full-length mtB38 pseudogene in the MAC of 38 cells would indeed be expected, after a cross to 227, to protect the highly similar zygotic mtB227 allele against coding-sequence deletions in the derived F1, through titration of homologous scnRNAs. d, Molecular analysis of the 38 × 227 cross. To verify this maternal effect, we crossed an E-expressing 227 clone (pmtB51-transformed clone T, same as in Fig. 5b; C, uninjected control) with strain 38, and F1 heterozygotes were tested for mating types and for mtA expression by northern blotting. As expected, F1 heterozygotes deriving from the 38 parent (1b and 2b, as determined by sequencing of a mitochondrial polymorphism) were E and expressed mtA, indicating that the incoming mtB227 allele had been rearranged into a functional, full-length form in the MAC. After autogamy of F1 clone 2b, 24 independent F2 homozygotes were isolated and tested for mating types. Consistent with the Mendelian segregation of mtB alleles, 12 were O and 12 were E (Supplementary Table 5); northern blot analysis of 3 clones of each type showed that only E clones expressed mtA. e, All F2 clones maintained the full-length mtB gene in the MAC. PCR11 (Supplementary Table 6) amplifies an 888-bp fragment from the MAC version of mtB38, and an 893-bp fragment from the full-length MAC version of mtB227. C1 and C2, control PCR11 on two O clones of strain 227, showing the 806-bp and 744-bp fragments resulting from the two alternative coding-sequence deletions. f, Mating types co-segregate with mtB alleles among F2 homozygotes. Digestion of the PCR products with AluI distinguishes the 38 and 227 alleles. mtA and mtC alleles segregated independently (Supplementary Table 5).

Extended Data Figure 9 Transformation of P. septaurelia 227 O cells with the full-length mtB227 gene induces heritable E expression.

a, Plasmid pmtB227, containing the full-length E MAC form of mtB227, was microinjected into the MACs of O cells of strain 227, and 12 injected clones were tested by PCR11. b, This PCR amplifies an 893-bp product from the transgene, and products of 744 and 806 bp from the two internally deleted MAC forms of the endogenous mtB227. C1 and C2, control uninjected clones. Clones 4, 8, 9 and 11 contained high copy numbers of the transgene, so that the PCR products from the endogenous gene are not detectable. Because only O tester lines were available, the mating types of injected clones could be ascertained only for E-expressing clones, as indicated below each lane. This was the case of the 4 high-copy clones; other clones were presumably O, although it was impossible to make sure that the cells were sexually reactive (indicated by ‘-’). Injected clones were then taken through autogamy, and the mass progenies were again tested for mating types. In this case, the mass progenies from clones 9 and 11, which proved to be pure E, were used as E testers to carry out a full test for some of the other progenies. Of the other two transformed clones expressing E, clone 4 gave rise to a selfing progeny (S), probably a mix of O and E clones, whereas clone 8 gave rise to a pure O progeny.

Extended Data Figure 10 A general model for mating-type determination in P. aurelia species.

In the three species examined, mating type E depends on expression of the mtA protein during sexual reactivity. mtA transcription in turn requires the mtB and mtC gene products (the requirement for mtC in P. septaurelia, and for both genes in P. octaurelia, remains to be verified). In P. tetraurelia and P. octaurelia, mating type O is determined during MAC development by excision of the mtA promoter as an IES, preventing expression of the gene. In P. septaurelia, mating type O is determined by the excision of segments of the mtB coding sequence as IESs, which similarly prevents mtA expression.

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This file contains Supplementary Tables 1-6 and Supplementary Data sets 1-2. (PDF 862 kb)

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Singh, D., Saudemont, B., Guglielmi, G. et al. Genome-defence small RNAs exapted for epigenetic mating-type inheritance. Nature 509, 447–452 (2014). https://doi.org/10.1038/nature13318

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