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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.

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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.

Corresponding author

Correspondence to Shiv I. S. Grewal.

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The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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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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