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TOR targets an RNA processing network to regulate facultative heterochromatin, developmental gene expression and cell proliferation

Nature Cell Biology volume 23, pages 243–256 (2021)Cite this article

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Abstract

Cell proliferation and differentiation require signalling pathways that enforce appropriate and timely gene expression. We find that Tor2, the catalytic subunit of the TORC1 complex in fission yeast, targets a conserved nuclear RNA elimination network, particularly the serine and proline-rich protein Pir1, to control gene expression through RNA decay and facultative heterochromatin assembly. Phosphorylation by Tor2 protects Pir1 from degradation by the ubiquitin-proteasome system involving the polyubiquitin Ubi4 stress-response protein and the Cul4–Ddb1 E3 ligase. This pathway suppresses widespread and untimely gene expression and is critical for sustaining cell proliferation. Moreover, we find that the dynamic nature of Tor2-mediated control of RNA elimination machinery defines gene expression patterns that coordinate fundamental chromosomal events during gametogenesis, such as meiotic double-strand-break formation and chromosome segregation. These findings have important implications for understanding how the TOR signalling pathway reprogrammes gene expression patterns and contributes to diseases such as cancer.

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Fig. 1: Tor2 stabilizes Pir1, which is required for facultative heterochromatin assembly and gametogenic gene silencing.
Fig. 2: Tor2 phosphorylates Pir1 to confer stability.
Fig. 3: Ubi4 and Cul4–Ddb1 are required for Pir1 degradation.
Fig. 4: Pir1 stabilization sustains cell proliferation under suboptimal conditions.
Fig. 5: Pir1 depletion disengages the RNA elimination machinery during meiosis.
Fig. 6: Depletion of Pir1 is crucial for DSB formation and proper meiotic progression.
Fig. 7: Artificial stabilization of Pir1 affects the chromosome dynamics during meiosis.
Fig. 8: Tor2 control of the RNA elimination machinery coordinates developmental gene expression and facilitates proper chromosome segregation during gametogenesis.

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Data availability

ChIP-chip and RNA-seq datasets are available for the data presented in Figs. 1a,e,i–k, 2f,g, 3a,h–l, 4b,c,e–h, 6b–e, 8a and Extended Data Figs. 1a–e, 3e,g, 4a,b, 5a,d, 6d, 7b,c, 8a,b. These data have been deposited in the Gene Expression Omnibus under the accession number GSE142488. For mass spectrometry, the UniProt S. pombe database (https://www.uniprot.org/proteomes/UP000002485) was used for the protein search. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

All codes used in this study are publicly available.

References

  1. Perrimon, N., Pitsouli, C. & Shilo, B. Z. Signaling mechanisms controlling cell fate and embryonic patterning. Cold Spring Harb. Perspect. Biol. 4, a005975 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Cardenas, M. E., Cutler, N. S., Lorenz, M. C., Di Como, C. J. & Heitman, J. The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev. 13, 3271–3279 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lopez-Maury, L., Marguerat, S. & Bahler, J. Tuning gene expression to changing environments: from rapid responses to evolutionary adaptation. Nat. Rev. Genet. 9, 583–593 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Saxton, R. A. & Sabatini, D. M. mTOR signaling in growth, metabolism, and disease. Cell 169, 361–371 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Wullschleger, S., Loewith, R. & Hall, M. N. TOR signaling in growth and metabolism. Cell 124, 471–484 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Nicetto, D. & Zaret, K. S. Role of H3K9me3 heterochromatin in cell identity establishment and maintenance. Curr. Opin. Genet. Dev. 55, 1–10 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jenuwein, T. & Allis, C. D. Translating the histone code. Science 293, 1074–1080 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Kilchert, C., Wittmann, S. & Vasiljeva, L. The regulation and functions of the nuclear RNA exosome complex. Nat. Rev. Mol. Cell Biol. 17, 227–239 (2016).

    Article  CAS  PubMed  Google Scholar 

  9. Schmid, M. & Jensen, T. H. Controlling nuclear RNA levels. Nat. Rev. Genet. 19, 518–529 (2018).

    Article  CAS  PubMed  Google Scholar 

  10. Otsubo, Y. & Yamamoto, M. Signaling pathways for fission yeast sexual differentiation at a glance. J. Cell Sci. 125, 2789–2793 (2012).

    Article  CAS  PubMed  Google Scholar 

  11. Reyes-Turcu, F. E. & Grewal, S. I. Different means, same end-heterochromatin formation by RNAi and RNAi-independent RNA processing factors in fission yeast. Curr. Opin. Genet. Dev. 22, 156–163 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Yamamoto, M. The selective elimination of messenger RNA underlies the mitosis–meiosis switch in fission yeast. Proc. Jpn. Acad. B 86, 788–797 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Mata, J., Lyne, R., Burns, G. & Bahler, J. The transcriptional program of meiosis and sporulation in fission yeast. Nat. Genet. 32, 143–147 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Harigaya, Y. et al. Selective elimination of messenger RNA prevents an incidence of untimely meiosis. Nature 442, 45–50 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Zofall, M. et al. RNA elimination machinery targeting meiotic mRNAs promotes facultative heterochromatin formation. Science 335, 96–100 (2012).

    Article  CAS  PubMed  Google Scholar 

  16. Tashiro, S., Asano, T., Kanoh, J. & Ishikawa, F. Transcription-induced chromatin association of RNA surveillance factors mediates facultative heterochromatin formation in fission yeast. Genes Cells 18, 327–339 (2013).

    Article  CAS  PubMed  Google Scholar 

  17. Gallagher, P. S. et al. Iron homeostasis regulates facultative heterochromatin assembly in adaptive genome control. Nat. Struct. Mol. Biol. 25, 372–383 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sugiyama, T. & Sugioka-Sugiyama, R. Red1 promotes the elimination of meiosis-specific mRNAs in vegetatively growing fission yeast. EMBO J. 30, 1027–1039 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Lee, N. N. et al. Mtr4-like protein coordinates nuclear RNA processing for heterochromatin assembly and for telomere maintenance. Cell 155, 1061–1074 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Egan, E. D., Braun, C. R., Gygi, S. P. & Moazed, D. Post-transcriptional regulation of meiotic genes by a nuclear RNA silencing complex. RNA 20, 867–881 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zhou, Y. et al. The fission yeast MTREC complex targets CUTs and unspliced pre-mRNAs to the nuclear exosome. Nat. Commun. 6, 7050 (2015).

    Article  PubMed  Google Scholar 

  22. Sugiyama, T. et al. Enhancer of rudimentary cooperates with conserved RNA-processing factors to promote meiotic mRNA decay and facultative heterochromatin assembly. Mol. Cell 61, 747–759 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Li, J. et al. Systematic analysis reveals the prevalence and principles of bypassable gene essentiality. Nat. Commun. 10, 1002 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Yamashita, A., Takayama, T., Iwata, R. & Yamamoto, M. A novel factor Iss10 regulates Mmi1-mediated selective elimination of meiotic transcripts. Nucleic Acids Res. 41, 9680–9687 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Cam, H. P. et al. Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome. Nat. Genet. 37, 809–819 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Mattout, A. et al. LSM2-8 and XRN-2 contribute to the silencing of H3K27me3-marked genes through targeted RNA decay. Nat. Cell Biol. 22, 579–590 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Whittaker, C. & Dean, C. The FLC locus: a platform for discoveries in epigenetics and adaptation. Annu. Rev. Cell Dev. Biol. 33, 555–575 (2017).

    Article  CAS  PubMed  Google Scholar 

  28. Heitman, J., Movva, N. R. & Hall, M. N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science 253, 905–909 (1991).

    Article  CAS  PubMed  Google Scholar 

  29. Alvarez, B. & Moreno, S. Fission yeast Tor2 promotes cell growth and represses cell differentiation. J. Cell Sci. 119, 4475–4485 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Matsuo, T., Otsubo, Y., Urano, J., Tamanoi, F. & Yamamoto, M. Loss of the TOR kinase Tor2 mimics nitrogen starvation and activates the sexual development pathway in fission yeast. Mol. Cell. Biol. 27, 3154–3164 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Uritani, M. et al. Fission yeast Tor2 links nitrogen signals to cell proliferation and acts downstream of the Rheb GTPase. Genes Cells 11, 1367–1379 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Weisman, R., Roitburg, I., Schonbrun, M., Harari, R. & Kupiec, M. Opposite effects of Tor1 and Tor2 on nitrogen starvation responses in fission yeast. Genetics 175, 1153–1162 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Cohen, A. et al. TOR complex 2 in fission yeast is required for chromatin-mediated gene silencing and assembly of heterochromatic domains at subtelomeres. J. Biol. Chem. 293, 8138–8150 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Okazaki, K., Okayama, H. & Niwa, O. The polyubiquitin gene is essential for meiosis in fission yeast. Exp. Cell. Res. 254, 143–152 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Horn, P. J., Bastie, J. N. & Peterson, C. L. A Rik1-associated, cullin-dependent E3 ubiquitin ligase is essential for heterochromatin formation. Genes Dev. 19, 1705–1714 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Jia, S., Kobayashi, R. & Grewal, S. I. Ubiquitin ligase component Cul4 associates with Clr4 histone methyltransferase to assemble heterochromatin. Nat. Cell Biol. 7, 1007–1013 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Braun, S. et al. The Cul4–Ddb1Cdt2 ubiquitin ligase inhibits invasion of a boundary-associated antisilencing factor into heterochromatin. Cell 144, 41–54 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Lee, J. & Zhou, P. DCAFs, the missing link of the CUL4–DDB1 ubiquitin ligase. Mol. Cell 26, 775–780 (2007).

    Article  CAS  PubMed  Google Scholar 

  39. Liu, C. et al. Transactivation of Schizosaccharomyces pombe cdt2+ stimulates a Pcu4–Ddb1–CSN ubiquitin ligase. EMBO J. 24, 3940–3951 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Otsubo, Y., Yamashita, A., Ohno, H. & Yamamoto, M. S. pombe TORC1 activates the ubiquitin-proteasomal degradation of the meiotic regulator Mei2 in cooperation with Pat1 kinase. J. Cell Sci. 127, 2639–2646 (2014).

    CAS  PubMed  Google Scholar 

  41. Cipak, L., Polakova, S., Hyppa, R. W., Smith, G. R. & Gregan, J. Synchronized fission yeast meiosis using an ATP analog-sensitive Pat1 protein kinase. Nat. Protoc. 9, 223–231 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ding, D. Q. et al. Meiosis-specific noncoding RNA mediates robust pairing of homologous chromosomes in meiosis. Science 336, 732–736 (2012).

    Article  CAS  PubMed  Google Scholar 

  43. Fowler, K. R., Gutierrez-Velasco, S., Martin-Castellanos, C. & Smith, G. R. Protein determinants of meiotic DNA break hot spots. Mol. Cell 49, 983–996 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wahls, W. P., Siegel, E. R. & Davidson, M. K. Meiotic recombination hotspots of fission yeast are directed to loci that express non-coding RNA. PLoS ONE 3, e2887 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Cromie, G. A. et al. A discrete class of intergenic DNA dictates meiotic DNA break hotspots in fission yeast. PLoS Genet. 3, e141 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Ponticelli, A. S., Sena, E. P. & Smith, G. R. Genetic and physical analysis of the M26 recombination hotspot of Schizosaccharomyces pombe. Genetics 119, 491–497 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Marston, A. L. & Amon, A. Meiosis: cell-cycle controls shuffle and deal. Nat. Rev. Mol. Cell Biol. 5, 983–997 (2004).

    Article  CAS  PubMed  Google Scholar 

  48. Chikashige, Y. et al. Telomere-led premeiotic chromosome movement in fission yeast. Science 264, 270–273 (1994).

    Article  CAS  PubMed  Google Scholar 

  49. Ding, D. Q., Yamamoto, A., Haraguchi, T. & Hiraoka, Y. Dynamics of homologous chromosome pairing during meiotic prophase in fission yeast. Dev. Cell 6, 329–341 (2004).

    Article  CAS  PubMed  Google Scholar 

  50. Li, D., Roca, M., Yuecel, R. & Lorenz, A. Immediate visualization of recombination events and chromosome segregation defects in fission yeast meiosis. Chromosoma 128, 385–396 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Folco, H. D. et al. Untimely expression of gametogenic genes in vegetative cells causes uniparental disomy. Nature 543, 126–130 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Ozkaynak, E., Finley, D., Solomon, M. J. & Varshavsky, A. The yeast ubiquitin genes: a family of natural gene fusions. EMBO J. 6, 1429–1439 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. McFarlane, R. J. & Wakeman, J. A. Meiosis-like functions in oncogenesis: a new view of cancer. Cancer Res. 77, 5712–5716 (2017).

    Article  CAS  PubMed  Google Scholar 

  54. Pal, S. K. & Quinn, D. I. Differentiating mTOR inhibitors in renal cell carcinoma. Cancer Treat. Rev. 39, 709–719 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Chen, S. et al. ZFC3H1 provides a reference to evaluate the prognosis and treat prostate adenocarcinoma: a study based on TCGA data. Preprint at https://doi.org/10.21203/rs.3.rs-23147/v1 (2020).

  56. Kohda, T. A. et al. Fission yeast autophagy induced by nitrogen starvation generates a nitrogen source that drives adaptation processes. Genes Cells 12, 155–170 (2007).

    Article  CAS  PubMed  Google Scholar 

  57. Matsuhara, H. & Yamamoto, A. Autophagy is required for efficient meiosis progression and proper meiotic chromosome segregation in fission yeast. Genes Cells 21, 65–87 (2016).

    Article  CAS  PubMed  Google Scholar 

  58. Herbert, M., Kalleas, D., Cooney, D., Lamb, M. & Lister, L. Meiosis and maternal aging: insights from aneuploid oocytes and trisomy births. Cold Spring Harb. Perspect. Biol. 7, a017970 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Jesus, T. T., Oliveira, P. F., Sousa, M., Cheng, C. Y. & Alves, M. G. Mammalian target of rapamycin (mTOR): a central regulator of male fertility? Crit. Rev. Biochem. Mol. Biol. 52, 235–253 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Jain, D. et al. ketu Mutant mice uncover an essential meiotic function for the ancient RNA helicase YTHDC2. elife 7, e30919 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  61. Soh, Y. Q. S. et al. Meioc maintains an extended meiotic prophase I in mice. PLoS Genet. 13, e1006704 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Wojtas, M. N. et al. Regulation of m6A transcripts by the 3ʹ→5ʹ RNA helicase YTHDC2 is essential for a successful meiotic program in the mammalian germline. Mol. Cell 68, 374–387 (2017).

    Article  CAS  PubMed  Google Scholar 

  63. Saitou, M. & Miyauchi, H. Gametogenesis from pluripotent stem cells. Cell Stem Cell 18, 721–735 (2016).

    Article  CAS  PubMed  Google Scholar 

  64. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Wood, V. et al. The genome sequence of Schizosaccharomyces pombe. Nature 415, 871–880 (2002).

    Article  CAS  PubMed  Google Scholar 

  66. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  67. Wang, L., Feng, Z., Wang, X., Wang, X. & Zhang, X. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 26, 136–138 (2010).

    Article  PubMed  Google Scholar 

  68. Gu, Z., Eils, R. & Schlesner, M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics 32, 2847–2849 (2016).

    Article  CAS  PubMed  Google Scholar 

  69. Boyle, E. I. et al. GO::TermFinder–open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes. Bioinformatics 20, 3710–3715 (2004).

    Article  CAS  PubMed  Google Scholar 

  70. Holla, S. et al. Positioning heterochromatin at the nuclear periphery suppresses histone turnover to promote epigenetic inheritance. Cell 180, 150–164 (2020).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M. Yamamoto, J. Gregan, L. -L. Du, T. Toda, G. Smith, A. Lorenz and the National BioResource Project (NBRP) Japan for strains and plasmids. We also thank V. Chalamcharla for constructing a strain containing the ade6-DSR allele; H. D. Folco and T. Vo for providing strains and for valuable technical help; S. Holla, G. Thillainadesan, A. Dipiazza and H. Xiao for their helpful suggestions; J. Barrowman for editing the manuscript and the members of the Laboratory of Biochemistry and Molecular Biology, in particular the Grewal laboratory, for discussions. This study used the Helix Systems and Biowulf Linux cluster at the National Institutes of Health. This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute.

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Author notes

  1. Nathan N. Lee

    Present address: Division of Renal Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA

  2. Lixia Pan

    Present address: National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA

  3. These authors contributed equally: Yi Wei, Nathan N. Lee.

Authors and Affiliations

  1. Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Yi Wei, Nathan N. Lee, Lixia Pan, Jothy Dhakshnamoorthy, Ling-Ling Sun, Martin Zofall, David Wheeler & Shiv I. S. Grewal

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Contributions

S.I.S.G., N.N.L. and Y.W. conceived the project. Y.W. and N.N.L. performed the western blots, immunofluorescence, co-IPs, ChIPs and RNA-seq. L.P. and Y.W. conducted the synchronized meiosis analyses. J.D. performed the protein purifications for mass spectrometry. Y.W., N.N.L., L.-L.S., M.Z. and L.P. constructed strains. Y.W. performed all other experiments including those exploring cell proliferation and chromosome dynamics during meiotic progression. D.W. conducted bioinformatics analyses. All authors contributed to the data interpretation. S.I.S.G. and Y.W. wrote the manuscript with input from the other authors.

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Correspondence to Shiv I. S. Grewal.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Tor2 promotes MTREC-dependent facultative heterochromatin assembly and gametogenic gene silencing.

a and b, ChIP-chip analyses of H3K9me2 at MTREC-dependent (a) and MTREC-independent (b) heterochromatin islands. c, ChIP-chip analysis of H3K9me2 distribution at cen1. d, RNA-seq analyses of pir1, red1, and mtl1 expression in the indicated strains at 30 °C. e, RNA-seq expression profiles of gametogenic gene transcripts at 30 °C. f, Composite plot showing the median expression level of a common set of 327 upregulated genes in WT and red1∆ cells grown in rich media at 30 °C. For ChIP-chip and RNA-seq, data representative of two independent experiments are presented.

Extended Data Fig. 2 Phosphorylation by Tor2/TORC1 stabilizes Pir1.

a, Immunopurified fractions from cells expressing untagged or GFP-tagged Pir1 (left) and FLAG-tagged Tor2 (right) were subjected to mass spectrometry. The total PSM and coverage for the identified proteins is shown. b, Multiple sequence alignment of S. pombe proteins Pir1 and Red1 against human ZFC3H1 was performed using Macaw sequence alignment workbench. Pir1 aligns with ZFC3H1 in their shared serine/proline rich region. Thick rectangles indicate segments of best alignment and dotted lines indicate gaps. Alignments for two conserved regions are shown in detail. The serines that are phosphorylated in Pir1 are labelled with orange triangles. S285 is in a conserved motif which is underlined in red. Mass spectrometry results are from a single experiment. c, Phosphorylation of Xpress-tagged recombinant WT Pir1 and mutant Pir1 with 12 serines (S215, S233, S237, S248, S265, S285, S299, S303, S325, S334, S434, S503) substituted with alanine by the FLAG–Tor2 complex was detected using anti-thiophosphate-ester antibody. d, Western blot analysis of GFP–Pir1 in the indicated strains at 30 °C. 12 SD, 12 serines replaced by aspartic acid. e, Western blot analysis of Red1 in the indicated strains at 30 °C. SD, serine285 replaced by aspartic acid. For c-e, two independent experiments were performed with similar results. Source data are provided.

Source data

Extended Data Fig. 3 Pir1 degradation by Ubi4 and Cul4–Ddb1 impacts gene silencing.

a, Western blot analysis of GFP–Pir1 in tor2+ cells carrying pir1-WT, pir1-SD or pir1-SA allele. Cells were cultured in rich medium at 30 °C; SD, serine285 replaced by aspartic acid; SA, serine285 replaced by alanine. b, Western blot analysis of Pir1 in strains grown at 30 °C with or without nitrogen (+N/–N). c, IF of Pir1 in the indicated strains at 30 °C. d, Serial dilutions of the indicated strains were spotted onto rich medium (YEA) and low adenine (YE) plates, and then incubated at 30 °C for 3 days. The spd1∆ was introduced to suppress the growth defect of cul4∆ and ddb1∆ mutants. e, RNA-seq expression profiles of sme2 and mei2 transcripts in the indicated strains at 30 °C. f, Serial dilutions of the indicated strains were spotted and incubated as described in d. g, RNA-seq analysis of MTREC regulon gene expression in the indicated homothallic strains at 30 °C. For a-g, data representative of two independent experiments are shown. Source data are provided.

Source data

Extended Data Fig. 4 Defects in the assembly of MTREC-dependent heterochromatin islands observed in tor2-ts6 can be suppressed by ubi4∆ or ddb1∆.

a, ChIP-chip analysis of H3K9me2 distribution at MTREC-dependent islands in the indicated strains. b, ChIP-chip analysis of H3K9me2 at MTREC-dependent heterochromatin islands in WT, tor2-ts6, tor2-ts6 ubi4∆, and tor2-ts6 ddb1∆ cells at 30 °C. Heterochromatin island numbers correspond to those described previously15. The number of plus signs (+) indicates the relative level of detected H3K9me2. The minus sign (–) denotes a lack of detectable H3K9me2. Genomic coordinates correspond to the S. pombe genome assembly v2.29. Data representative of two independent ChIP-chip experiments are shown.

Extended Data Fig. 5 Pir1 prevents untimely gene expression in suboptimal conditions.

a, Genes within the ‘meiotic coding’ and ‘non-meiotic coding’ groups in Fig. 4c were categorized separately based on GO biological process. b, Growth curves of WT and pir1∆ cells cultured in rich or minimal medium at 26 °C. Optical density (OD) at 595 nm wavelength is plotted. Data representative of two independent experiments are shown. c, Percent cell death was determined by methylene blue staining of cells grown on minimal medium at 26 °C. Two independent experiments were performed with similar results. d, Heatmap of log2 fold changes in expression in WT and pir1∆ cells grown in minimal or rich medium relative to WT cells grown in rich medium at 30 °C. Transcripts were clustered based on their function. ‘n’ indicates number of loci in each category. Data representative of two independent experiments are presented. e, Schematic depicting how fluctuations in gametogenic gene expression, which occur upon changes in growth conditions, are controlled by MTREC–Pir1. f, Serial dilutions of the indicated strains were spotted onto rich medium and incubated at the indicated temperature for 3-4 days. Two independent experiments were performed with similar results. Source data are provided.

Source data

Extended Data Fig. 6 Synchronized meiosis system.

a, Flowchart of pat1-as2 induced synchronized meiosis. b, Progression of meiosis was monitored by fluorescence microscopy to determine the number of nuclei per cell in samples collected at the indicated time points after the addition of 1-NM-PP1. Two independent experiments were performed with similar results. c, RT–qPCR analysis of meiotic gene expression. Samples were collected at the indicated time points. Two independent experiments were performed with similar results. d, Heatmap of log2 fold changes in expression relative to vegetative cells for synchronized meiotic cells, as determined by RNA-seq analyses. Clusters are grouped by expression pattern and only transcripts with peak log2 fold changes that were ≥1.5 were assigned to a group. Data representative of two independent experiments are shown. Source data are provided.

Source data

Extended Data Fig. 7 Temporal control of gene expression by Pir1 and MTREC during meiotic progression.

a, The MTREC regulon genes grouped according to their function in meiosis. The expression peak was determined from RNA-seq analysis of the synchronized meiosis system. See also Supplementary Table 7. b, RNA-seq analysis of MTREC regulon gene expression in the indicated strains. Data representative of two independent experiments are shown. c, ChIP-chip analysis of meiotic DSBs (Rec12–DNA linkages) across chromosome 1 and 3 in cells expressing WT Pir1 or Pir1-SD protein. Data representative of two independent ChIP-chip experiments are shown.

Extended Data Fig. 8 Tor2 targets RNA elimination machinery to sustain cell proliferation and gametogenesis.

a, Line plots showing the mean log2 fold change in expression of MTREC regulon transcripts in WT, pir1Δ, and red1Δ mutants. b, Heatmap of log2 fold changes in expression of additional MTREC regulon genes relative to WT vegetative cells in WT, pir1∆, and red1∆. Data representative of two independent experiments are shown. Source data are provided.

Source data

Supplementary information

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Supplementary Tables 1–9

Supplementary Table 1. List of the loci that were upregulated in both pir1∆ and tor2-ts6 mutants cultured in rich (YEA) medium at 30 °C. Supplementary Table 2. List of the factors identified in the Pir1 and Tor2 purifications. Supplementary Table 3. List of the phospho-serine sites identified in Pir1. Supplementary Table 4. List of the loci that were upregulated in WT cells cultured in minimal (EMM) medium at 18 °C and in tor2-ts6 cells cultured at 30 °C. Supplementary Table 5. List of the loci that were upregulated in WT, pir1-SD and pir1-SA cells cultured in minimal (EMM) medium at 18 °C. Supplementary Table 6. List of the loci that were upregulated in pir1∆ cells cultured in minimal (EMM) or rich (YEA) medium at 30 °C. Supplementary Table 7. List of MTREC regulon genes. Supplementary Table 8. List of the strains used in this study. Supplementary Table 9. List of the oligonucleotides used in this study.

Supplementary Video 1

Time-lapse images obtained at 10-min intervals over 8 h of meiosis progression in WT cells.

Supplementary Video 2

Time-lapse images obtained at 10-min intervals over 10 h of meiosis progression in Pir1-SD cells.

Supplementary Video 3

Time-lapse images obtained at 10-min intervals over 10 h of meiosis progression in Pir1-SD cells.

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Wei, Y., Lee, N.N., Pan, L. et al. TOR targets an RNA processing network to regulate facultative heterochromatin, developmental gene expression and cell proliferation. Nat Cell Biol 23, 243–256 (2021). https://doi.org/10.1038/s41556-021-00631-y

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