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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Primary accessions

European Nucleotide Archive


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.


  1. 1.

    Paramecium aurelia. in Handbook of Genetics (ed. ) 469–594 (Plenum, 1974)

  2. 2.

    & DNA elimination in ciliates: transposon domestication and genome surveillance. Annu. Rev. Genet. 45, 227–246 (2011)

  3. 3.

    et al. The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences. PLoS Genet. 8, e1002984 (2012)

  4. 4.

    Large-scale genome remodelling by the developmentally programmed elimination of germ line sequences in the ciliate Paramecium. Res. Microbiol. 155, 399–408 (2004)

  5. 5.

    et al. PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia. Genes Dev. 23, 2478–2483 (2009)

  6. 6.

    & Consensus inverted terminal repeat sequence of Paramecium IESs: resemblance to termini of Tc1-related and Euplotes Tec transposons. Nucleic Acids Res. 23, 2006–2013 (1995)

  7. 7.

    et al. Transposon invasion of the Paramecium germline genome countered by a domesticated PiggyBac transposase and the NHEJ pathway. Int. J. Evol. Biol. 2012, 436196 (2012)

  8. 8.

    , & Epigenetics of ciliates. Cold Spring Harb. Perspect. Biol. 5, a017764 (2013)

  9. 9.

    , & RNA-guided DNA rearrangements in ciliates: is the best genome defence a good offence? Biol. Cell 104, 309–325 (2012)

  10. 10.

    , & Developmental genome rearrangements in ciliates: a natural genomic subtraction mediated by non-coding transcripts. Trends Genet. 25, 344–350 (2009)

  11. 11.

    , , , & Functional specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling. Nucleic Acids Res. 39, 4249–4264 (2011)

  12. 12.

    et al. Silencing-associated and meiosis-specific small RNA pathways in Paramecium tetraurelia. Nucleic Acids Res. 37, 903–915 (2009)

  13. 13.

    , , & Maternal noncoding transcripts antagonize the targeting of DNA elimination by scanRNAs in Paramecium tetraurelia. Genes Dev. 22, 1501–1512 (2008)

  14. 14.

    , & Epigenetic self-regulation of developmental excision of an internal eliminated sequence on Paramecium tetraurelia. Genes Dev. 9, 2065–2077 (1995)

  15. 15.

    , & Homology-dependent maternal inhibition of developmental excision of internal eliminated sequences in Paramecium tetraurelia. Mol. Cell. Biol. 18, 7075–7085 (1998)

  16. 16.

    , , & RNA-mediated programming of developmental genome rearrangements in Paramecium tetraurelia. Mol. Cell. Biol. 24, 7370–7379 (2004)

  17. 17.

    Sex, sex inheritance and sex determination in Paramecium aurelia. Proc. Natl Acad. Sci. USA 23, 378–385 (1937)

  18. 18.

    Mating type mutations in variety 1 of Paramecium aurelia, and their bearing upon the problem of mating type determination. Genetics 40, 321–330 (1955)

  19. 19.

    Mutational analysis of mating type inheritance in Syngen 4 of Paramecium aurelia. Genetics 74, 63–80 (1973)

  20. 20.

    The genetic control of mating type differentiation in Paramecium. Genetics 48, 815–834 (1963)

  21. 21.

    Genetics of cellular differentiation: stable nuclear differentiation in eucaryotic unicells. Annu. Rev. Genet. 11, 349–367 (1977)

  22. 22.

    Recent advances in the genetics of Paramecium and Euplotes. Adv. Genet. 1, 263–358 (1947)

  23. 23.

    Mating type inheritance at conjugation in variety 4 of Paramecium aurelia. J. Protozool. 4, 89–95 (1957)

  24. 24.

    Patterns of nucleo-cytoplasmic integration in Paramecium. Caryologia 6 (suppl.). 307–325 (1954)

  25. 25.

    & A Mendelian mutation affecting mating-type determination also affects developmental genomic rearrangements in Paramecium tetraurelia. Genetics 143, 191–202 (1996)

  26. 26.

    & Non-mendelian inheritance and homology-dependent effects in ciliates. Adv. Genet. 46, 305–337 (2002)

  27. 27.

    , & Nowa1p and Nowa2p: novel putative RNA binding proteins involved in trans-nuclear crosstalk in Paramecium tetraurelia. Curr. Biol. 15, 1616–1628 (2005)

  28. 28.

    , , & Functional diversification of Dicer-like proteins and small RNAs required for genome sculpting. Dev. Cell 28, 174–188 (2014)

  29. 29.

    et al. Study of an RNA helicase implicates small RNA-noncoding RNA interactions in programmed DNA elimination in Tetrahymena. Genes Dev. 22, 2228–2241 (2008)

  30. 30.

    & Conjugation-specific small RNAs in Tetrahymena have predicted properties of scan (scn) RNAs involved in genome rearrangement. Genes Dev. 18, 2068–2073 (2004)

  31. 31.

    , , & Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena. Genes Dev. 26, 1729–1742 (2012)

  32. 32.

    , , , & Genetic diversity in the Paramecium aurelia species complex. Mol. Biol. Evol. 26, 421–431 (2009)

  33. 33.

    & On the nature of species: insights from Paramecium and other ciliates. Genetica 139, 677–684 (2011)

  34. 34.

    & Rapid diversification of mating systems in ciliates. Biol. J. Linn. Soc. 98, 187–197 (2009)

  35. 35.

    Interspecies crosses in Paramecium aurelia (syngen 4 by syngen 8). J. Protozool. 21, 152–159 (1974)

  36. 36.

    , , & Spliced DNA sequences in the Paramecium germline: their properties and evolutionary potential. Genome Biol. Evol. 5, 1200–1211 (2013)

  37. 37.

    , , & Odd mating-type substances may work as precursor molecules of even mating-type substances in Paramecium caudatum. J. Eukaryot. Microbiol. 48, 683–689 (2001)

  38. 38.

    et al. Selecting one of several mating types through gene segment joining and deletion in Tetrahymena thermophila. PLoS Biol. 11, e1001518 (2013)

  39. 39.

    & A small-RNA perspective on gametogenesis, fertilization, and early zygotic development. Science 330, 617–622 (2010)

  40. 40.

    & Small RNAs as guardians of the genome. Cell 136, 656–668 (2009)

  41. 41.

    & Keeping the soma free of transposons: programmed DNA elimination in ciliates. J. Biol. Chem. 286, 37045–37052 (2011)

  42. 42.

    , , & PIWI-interacting small RNAs: the vanguard of genome defence. Nature Rev. Mol. Cell Biol. 12, 246–258 (2011)

  43. 43.

    et al. Adaptation to P element transposon invasion in Drosophila melanogaster. Cell 147, 1551–1563 (2011)

  44. 44.

    & RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nature Rev. Genet. 14, 100–112 (2013)

  45. 45.

    & Understanding transgenerational epigenetic inheritance via the gametes in mammals. Nature Rev. Genet. 13, 153–162 (2012)

  46. 46.

    & PIWI-interacting RNAs: from generation to transgenerational epigenetics. Nature Rev. Genet. 14, 523–534 (2013)

  47. 47.

    et al. A transposon-induced epigenetic change leads to sex determination in melon. Nature 461, 1135–1138 (2009)

  48. 48.

    et al. Analysis of sequence variability in the macronuclear DNA of Paramecium tetraurelia: a somatic view of the germline. Genome Res. 18, 585–596 (2008)

  49. 49.

    , & Gene expression omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 30, 207–210 (2002)

  50. 50.

    et al. Mass culture of Paramecium tetraurelia. Cold Spring Harb. Protoc. 2010, (2010)

  51. 51.

    et al. Maintaining clonal Paramecium tetraurelia cell lines of controlled age through daily reisolation. Cold Spring Harb. Protoc. 2010, (2010)

  52. 52.

    & Improved northern blot method for enhanced detection of small RNA. Nature Protocols 3, 1077–1084 (2008)

  53. 53.

    et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4, 249–264 (2003)

  54. 54.

    & Normalization of cDNA microarray data. Methods 31, 265–273 (2003)

  55. 55.

    et al. Gene expression in a paleopolyploid: a transcriptome resource for the ciliate Paramecium tetraurelia. BMC Genom. 11, 547 (2010)

  56. 56.

    et al. Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 36, W465–W469 (2008)

  57. 57.

    & PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem. Sci. 24, 34–35 (1999)

  58. 58.

    , & An HMM posterior decoder for sequence feature prediction that includes homology information. Bioinformatics 21 (suppl. 1). 251–257 (2005)

  59. 59.

    et al. DNA microinjection into the macronucleus of Paramecium. Cold Spring Harb. Protoc. 2010, (2010)

  60. 60.

    et al. Silencing specific Paramecium tetraurelia genes by feeding double-stranded RNA. Cold Spring Harb. Protoc. 2010, (2010)

  61. 61.

    , , & Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia. Nucleic Acids Res. 38, 4092–4107 (2010)

  62. 62.

    et al. Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia. Nature 444, 171–178 (2006)

  63. 63.

    & Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009)

  64. 64.

    et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009)

  65. 65.

    & Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010)

  66. 66.

    , , , & Genetic analysis of mating type differentiation in Paramecium tetraurelia. II. Role of the micronuclei in mating-type determination. Genetics 94, 951–959 (1980)

  67. 67.

    Genetic analysis of mating-type differentiation in Paramecium tetraurelia. Genetics 87, 633–653 (1977)

  68. 68.

    et al. Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining. PLoS Genet. 7, e1002049 (2011)

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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.

Author information

Author notes

    • Baptiste Saudemont
    • , Jean-François Goût
    •  & Khaled Bouhouche

    Present addresses: Laboratoire de Biochimie, Unité Mixte de Recherche 8231, École Supérieure de Physique et de Chimie Industrielles, 75231 Paris, France (B.S.); Department of Biology, Indiana University, Bloomington, Indiana 47405, USA (J.-F.G.); INRA, UMR 1061 Unité de Génétique Moléculaire Animale, Université de Limoges, IFR 145, Faculté des Sciences et Techniques, 87060 Limoges, France (K.B.).


  1. Ecole Normale Supérieure, Institut de Biologie de l’ENS, IBENS; Inserm, U1024; CNRS, UMR 8197 Paris F-75005, France

    • Deepankar Pratap Singh
    • , Baptiste Saudemont
    • , Gérard Guglielmi
    • , Simran Bhullar
    • , Khaled Bouhouche
    • , Véronique Tanty
    • , Corinne Blugeon
    •  & Eric Meyer
  2. Sorbonne Universités, UPMC Univ., IFD, 4 place Jussieu, 75252 Paris cedex 05, France

    • Deepankar Pratap Singh
    • , Baptiste Saudemont
    •  & Maoussi Lhuillier-Akakpo
  3. CNRS UPR3404 Centre de Génétique Moléculaire, Gif-sur-Yvette F-91198, and Université Paris-Sud, Département de Biologie, Orsay F-91405, France

    • Olivier Arnaiz
    • , Anne Aubusson-Fleury
    •  & Linda Sperling
  4. CNRS UMR5558, Laboratoire de Biométrie et Biologie Evolutive, Université de Lyon, 43 boulevard du 11 Novembre 1918, Villeurbanne F-69622, France

    • Jean-François Goût
  5. Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31-016 Krakow, Poland

    • Malgorzata Prajer
    •  & Ewa Przybòs
  6. Department of Microbiology, Faculty of Biology, St Petersburg State University, Saint Petersburg 199034, Russia

    • Alexey Potekhin
  7. Institut Jacques Monod, CNRS, UMR 7592, Université Paris Diderot, Sorbonne Paris Cité, Paris F-75205, France

    • Maoussi Lhuillier-Akakpo
    •  & Sandra Duharcourt
  8. Commissariat à l’Energie Atomique (CEA), Institut de Génomique (IG), Genoscope, 2 rue Gaston Crémieux, BP5706, 91057 Evry, France

    • Adriana Alberti
    • , Karine Labadie
    •  & Jean-Marc Aury


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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Eric Meyer.

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