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A conserved mechanism of TOR-dependent RCK-mediated mRNA degradation regulates autophagy

Nature Cell Biology volume 17, pages 930–942 (2015)Cite this article

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Abstract

Autophagy is an essential eukaryotic pathway requiring tight regulation to maintain homeostasis and preclude disease. Using yeast and mammalian cells, we report a conserved mechanism of autophagy regulation by RNA helicase RCK family members in association with the decapping enzyme Dcp2. Under nutrient-replete conditions, Dcp2 undergoes TOR-dependent phosphorylation and associates with RCK members to form a complex with autophagy-related (ATG) mRNA transcripts, leading to decapping, degradation and autophagy suppression. Simultaneous with the induction of ATG mRNA synthesis, starvation reverses the process, facilitating ATG mRNA accumulation and autophagy induction. This conserved post-transcriptional mechanism modulates fungal virulence and the mammalian inflammasome, the latter providing mechanistic insight into autoimmunity reported in a patient with a PIK3CD/p110δ gain-of-function mutation. We propose a dynamic model wherein RCK family members, in conjunction with Dcp2, function in controlling ATG mRNA stability to govern autophagy, which in turn modulates vital cellular processes affecting inflammation and microbial pathogenesis.

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Figure 1: The RCK member Dhh1 is a post-transcriptional repressor of autophagy in yeast.
Figure 2: A Vad1 RCK/p54 homologue plays a role in repression of autophagy and virulence in C. neoformans.
Figure 3: Vad1 plays a role in decapping and degradation of ATG8 in C. neoformans.
Figure 4: TOR-dependent phosphorylation of Dcp2 modulates recruitment of ATG8 transcripts to the decapping complex and autophagic body formation in C. neoformans.
Figure 5: Mammalian DDX6 is a suppressor of autophagy in mouse primary ESCs and human HeLa cells.
Figure 6: DDX6 is a suppressor of autophagy in HeLa cells.
Figure 7: The DDX6 binding partner DCP2 regulates LC3 and IL1B levels during inflammasome stimulation in macrophages and in a patient with a PIK3CD/p110δ gain-of-function mutation.
Figure 8: A TOR-mediated RCK-dependent pathway negatively regulates autophagy through mRNA decapping and degradation.

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References

  1. Feng, Y., He, D., Yao, Z. & Klionsky, D. J. The machinery of macroautophagy. Cell Res. 24, 24–41 (2014).

    Article  CAS  PubMed  Google Scholar 

  2. Kamada, Y. et al. Tor-mediated induction of autophagy via an Apg1 protein kinase complex. J. Cell Biol. 150, 1507–1513 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Yorimitsu, T., Zaman, S., Broach, J. R. & Klionsky, D. J. Protein kinase A and Sch9 cooperatively regulate induction of autophagy in Saccharomyces cerevisiae. Mol. Biol. Cell 18, 4180–4189 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Settembre, C. et al. TFEB links autophagy to lysosomal biogenesis. Science 332, 1429–1433 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Garneau, N. L., Wilusz, J. & Wilusz, C. J. The highways and byways of mRNA decay. Nat. Rev. Mol. Cell Biol. 8, 113–126 (2007).

    Article  CAS  PubMed  Google Scholar 

  6. Nagarajan, V. K., Jones, C. I., Newbury, S. F. & Green, P. J. XRN 5′ → 3′ exoribonucleases: structure, mechanisms and functions. Biochim. Biophys. Acta 1829, 590–603 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sharif, H. et al. Structural analysis of the yeast Dhh1-Pat1 complex reveals how Dhh1 engages Pat1, Edc3 and RNA in mutually exclusive interactions. Nucleic Acids Res. 41, 8377–8390 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Panepinto, J. et al. The DEAD-box RNA helicase Vad1 regulates multiple virulence-associated genes in Cryptococcus neoformans. J. Clin. Invest. 115, 632–641 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Weston, A. & Sommerville, J. Xp54 and related (DDX6-like) RNA helicases: roles in messenger RNP assembly, translation regulation and RNA degradation. Nucleic Acids Res. 34, 3082–3094 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Presnyak, V. & Coller, J. The DHH1/RCKp54 family of helicases: an ancient family of proteins that promote translational silencing. Biochim. Biophys. Acta 1829, 817–823 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Freeberg, M. A. et al. Pervasive and dynamic protein binding sites of the mRNA transcriptome in Saccharomyces cerevisiae. Genome Biol. 14, R13 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Tsukada, M. & Ohsumi, Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett. 333, 169–174 (1993).

    Article  CAS  PubMed  Google Scholar 

  13. Lucas, C. L. et al. Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110delta result in T cell senescence and human immunodeficiency. Nat. Immunol. 15, 88–97 (2014).

    Article  CAS  PubMed  Google Scholar 

  14. Shintani, T. & Klionsky, D. J. Autophagy in health and disease: a double-edged sword. Science 306, 990–995 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bartholomew, C. R. et al. Ume6 transcription factor is part of a signaling cascade that regulates autophagy. Proc. Natl Acad. Sci. USA 109, 11206–11210 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hu, G. et al. PI3K signaling of autophagy is required for starvation tolerance and virulence of Cryptococcus neoformans. J. Clin. Invest. 118, 1186–1197 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Park, B. J. et al. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23, 525–530 (2009).

    Article  PubMed  Google Scholar 

  18. Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445–544 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Feldmesser, M., Kress, Y., Novikoff, P. & Casadevall, A. Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection. Infect. Immun. 68, 4225–4237 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Goeres, D. C. et al. Components of the Arabidopsis mRNA decapping complex are required for early seedling development. Plant Cell 19, 1549–1564 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Dunckley, T. & Parker, R. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. EMBO J. 18, 5411–5422 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Huber, A. et al. Characterization of the rapamycin-sensitive phosphoproteome reveals that Sch9 is a central coordinator of protein synthesis. Genes Dev. 23, 1929–1943 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Coller, J. & Parker, R. General translational repression by activators of mRNA decapping. Cell 122, 875–886 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Teixeira, D., Sheth, U., Valencia-Sanchez, M. A., Brengues, M. & Parker, R. Processing bodies require RNA for assembly and contain nontranslating mRNAs. RNA 11, 371–382 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ashe, M. P., De Long, S. K. & Sachs, A. B. Glucose depletion rapidly inhibits translation initiation in yeast. Mol. Biol. Cell 11, 833–848 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Gray, R. S. et al. The planar cell polarity effector Fuz is essential for targeted membrane trafficking, ciliogenesis and mouse embryonic development. Nat. Cell Biol. 11, 1225–1232 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hsu, P. P. et al. The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling. Science 332, 1317–1322 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Deretic, V. Autophagy: an emerging immunological paradigm. J. Immunol. 189, 15–20 (2012).

    Article  CAS  PubMed  Google Scholar 

  29. Levine, B., Mizushima, N. & Virgin, H. W. Autophagy in immunity and inflammation. Nature 469, 323–335 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Shi, C. S. et al. Activation of autophagy by inflammatory signals limits IL-1β production by targeting ubiquitinated inflammasomes for destruction. Nat. Immunol. 13, 255–263 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Nissan, T., Rajyaguru, P., She, M., Song, H. & Parker, R. Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms. Mol. Cell 39, 773–783 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Yang, Z., Geng, J., Yen, W-L., Wang, K. & Klionsky, D. J. Positive or negative roles of different cyclin-dependent kinase Pho85-cyclin complexes orchestrate induction of autophagy in Saccharomyces cerevisiae. Mol. Cell 38, 250–264 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Chan, T. F., Bertram, P. G., Ai, W. & Zheng, X. F. Regulation of APG14 expression by the GATA-type transcription factor Gln3p. J. Biol. Chem. 276, 6463–6467 (2001).

    Article  CAS  PubMed  Google Scholar 

  34. Fullgrabe, J., Klionsky, D. J. & Joseph, B. The return of the nucleus: transcriptional and epigenetic control of autophagy. Nat. Rev. Mol. Cell Biol. 15, 65–74 (2014).

    Article  PubMed  Google Scholar 

  35. Shulman, G. I., Ladenson, P. W., Wolfe, M. H., Ridgway, E. C. & Wolfe, R. R. Substrate cycling between gluconeogenesis and glycolysis in euthyroid, hypothyroid, and hyperthyroid man. J. Clin. Invest. 76, 757–764 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Yoon, J. H., Choi, E. J. & Parker, R. Dcp2 phosphorylation by Ste20 modulates stress granule assembly and mRNA decay in Saccharomyces cerevisiae. J. Cell Biol. 189, 813–827 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hogan, D. J., Riordan, D. P., Gerber, A. P., Herschlag, D. & Brown, P. O. Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system. PLoS Biol. 6, e255 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Riordan, D. P., Herschlag, D. & Brown, P. O. Identification of RNA recognition elements in the Saccharomyces cerevisiae transcriptome. Nucleic Acids Res. 39, 1501–1509 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Hay, N. & Sonenberg, N. Upstream and downstream of mTOR. Genes Dev. 18, 1926–1945 (2004).

    Article  CAS  PubMed  Google Scholar 

  40. Thoreen, C. C. et al. A unifying model for mTORC1-mediated regulation of mRNA translation. Nature 485, 109–113 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Hardwick, J., Kuruvilla, F., Tong, J., Shamji, A. & Schreiber, S. Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins. Proc. Natl Acad. Sci. USA 96, 14866–14870 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Romero-Santacreu, L., Moreno, J., Perez-Ortin, J. E. & Alepuz, P. Specific and global regulation of mRNA stability during osmotic stress in Saccharomyces cerevisiae. RNA 15, 1110–1120 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Osterholzer, J. J. et al. Role of dendritic cells and alveolar macrophages in regulating early host defense against pulmonary infection with Cryptococcus neoformans. Infect. Immun. 77, 3749–3758 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Fan, W., Kraus, P., Boily, M. & Heitman, J. Cryptococcus neoformans gene expression during murine macrophage infection. Eukaryot. Cell 4, 1420–1433 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Schroder, K. & Tschopp, J. The inflammasomes. Cell 140, 821–832 (2010).

    Article  CAS  PubMed  Google Scholar 

  46. Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

    Article  CAS  PubMed  Google Scholar 

  47. Teste, M. A., Duquenne, M., Francois, J. M. & Parrou, J. L. Validation of reference genes for quantitative expression analysis by real-time RT–PCR in Saccharomyces cerevisiae. BMC Mol. Biol. 10, 99 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Vandesompele, J. et al. Accurate normalization of real-time quantitative RT–PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research0034-research0034.11 (2002).

    Article  Google Scholar 

  49. Zhu, X., Gibbons, J., Zhang, S. & Williamson, P. R. Copper-mediated reversal of defective laccase in a Deltavph1 avirulent mutant of Cryptococcus neoformans. Mol. Microbiol. 47, 1007–1014 (2003).

    Article  CAS  PubMed  Google Scholar 

  50. Panepinto, J. C. et al. Overexpression of TUF1 restores respiratory growth and fluconazole sensitivity to a Cryptococcus neoformans vad1Δ mutant. Microbiology 156, 2558–2565 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Liu, X., Hu, G., Panepinto, J. & Williamson, P. Role of a VPS41 homolog in starvation response and virulence of Cryptococcus neoformans. Mol. Microbiol. 61, 1132–1146 (2006).

    Article  CAS  PubMed  Google Scholar 

  52. Rice, P., Longden, I. & Bleasby, A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277 (2000).

    Article  CAS  PubMed  Google Scholar 

  53. Blom, N., Sicheritz-Ponten, T., Gupta, R., Gammeltoft, S. & Brunak, S. Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 4, 1633–1649 (2004).

    Article  CAS  PubMed  Google Scholar 

  54. Iakoucheva, L. M. et al. The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res. 32, 1037–1049 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. de Castro, E. et al. ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res. 34, W362–W365 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Wong, Y. H. et al. KinasePhos 2.0: a web server for identifying protein kinase-specific phosphorylation sites based on sequences and coupling patterns. Nucleic Acids Res. 35, W588–W594 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Casadevall, A. & Perfect, J. Cryptococcus neoformans (ASM Press, 1998).

    Book  Google Scholar 

  58. Takizawa, P. A. & Vale, R. D. The myosin motor, Myo4p, binds Ash1 mRNA via the adapter protein, She3p. Proc. Natl Acad. Sci. USA 97, 5273–5278 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Zenklusen, D., Larson, D. R. & Singer, R. H. Single-RNA counting reveals alternative modes of gene expression in yeast. Nat. Struct. Mol. Biol. 15, 1263–1271 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Inacio, J. & da Luz Martins, M. Microscopic detection of yeasts using fluorescence in situ hybridization. Methods Mol. Biol. 968, 71–82 (2013).

    Article  CAS  PubMed  Google Scholar 

  61. Blewett, N. H. & Goldstrohm, A. C. A eukaryotic translation initiation factor 4E-binding protein promotes mRNA decapping and is required for PUF repression. Mol. Cell. Biol. 32, 4181–4194 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Schlumpberger, M. et al. AUT1, a gene essential for autophagocytosis in the yeast Saccharomyces cerevisiae. J. Bacteriol. 179, 1068–1076 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Salas, S. D., Bennett, J. E., Kwon-Chung, K. J., Perfect, J. R. & Williamson, P. R. Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J. Exp. Med. 184, 377–386 (1996).

    Article  CAS  PubMed  Google Scholar 

  64. Overbeek, R., Fonstein, M., D’Souza, M., Pusch, G. D. & Maltsev, N. The use of gene clusters to infer functional coupling. Proc. Natl Acad. Sci. USA 96, 2896–2901 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Kabeya, Y. et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 19, 5720–5728 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Li, Y., Song, M. & Kiledjian, M. Differential utilization of decapping enzymes in mammalian mRNA decay pathways. RNA 17, 419–428 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Paquette, N. et al. Serine/threonine acetylation of TGFβ-activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling. Proc. Natl Acad. Sci. USA 109, 12710–12715 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Nallamsetty, S. & Waugh, D. S. A generic protocol for the expression and purification of recombinant proteins in Escherichia coli using a combinatorial His6–maltose binding protein fusion tag. Nat. Protoc. 2, 383–391 (2007).

    Article  CAS  PubMed  Google Scholar 

  70. Ikenoue, T., Hong, S. & Inoki, K. Monitoring mammalian target of rapamycin (mTOR) activity. Methods Enzymol. 452, 165–180 (2009).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The authors thank J. Kim (University of Michigan, National Institutes of Health grant GM088565) for providing the RBP knockout library and V. Nagarajan (Genomic Technologies Section, Research Technologies Branch, NIAID, NIH) for genomic analysis. This work was financially supported, in part, by the Intramural Research Program of the NIH, NIAID, NICHD and by National Institutes of Health grant GM053396 (to D.J.K.).

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Author notes

  1. Guowu Hu, Travis McQuiston and Amélie Bernard: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

    Guowu Hu, Travis McQuiston, Yoon-Dong Park, Jin Qiu, Nannan Zhang, Scott R. Waterman, Gulbu Uzel & Peter R. Williamson

  2. Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, USA

    Amélie Bernard & Daniel J. Klionsky

  3. Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

    Ali Vural & John H. Kehrl

  4. Intramural Research Program in Genomics of Differentiation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA

    Nathan H. Blewett & Richard J. Maraia

  5. Genomic Technologies Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Maryland 20892, USA

    Timothy G. Myers

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Contributions

G.H.: experimental work, project planning, data analysis, writing; T.M.: experimental work, project planning, data analysis, writing; A.B.: experimental work, project planning, data analysis, writing; Y-D.P.: experimental work, project planning, data analysis, writing; J.Q.: experimental work, data analysis, writing; A.V.: experimental work, data analysis, writing; N.Z.: experimental work, data analysis, writing; S.R.W.: experimental work, data analysis, writing; N.H.B.: experimental work, data analysis, writing; T.G.M.: experimental work, data analysis, writing; R.J.M.: data analysis, project planning, writing; J.H.K.: data analysis, project planning, writing, editing; G.U.: experimental work, data analysis, protocol preparation, writing, editing; D.J.K.: project planning, data analysis, writing, editing; P.R.W.: project planning, data analysis, writing, editing.

Corresponding author

Correspondence to Peter R. Williamson.

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Integrated supplementary information

Supplementary Figure 7 RCK Member Dhh1 is a Post-transcriptional Repressor of Autophagy in Yeast.

(a,b) Dhh1 represses the expression of ATG genes in nutrient-replete conditions. Wild-type (WT; WLY176) and dhh1Δ cells (YAB269) were grown in rich medium (a), then shifted to nitrogen starvation for 1 h (b). Total RNA was extracted and analysed as in Fig. 1a. Error bars: standard deviation (SD). (a) n = 5 independent experiments except n = 3 for ATG9, 32,34; n = 4 for ATG2 and n = 6 for n = 5 for ATG1,7,8. (b) n = 3 independent experiments except n = 2 for ATG4 in WT cells. Student’s t-test, ∗p < 0.05,∗∗∗p < 0.001. (c–e) Mild overexpression of DHH1 modestly represses autophagy. WT (YAB281) and OE DHH1 cells (YAB282) were grown in YPGal medium (+N) until mid-log phase, then starved for nitrogen (−N) for 5 h (c,d) or 1 h (e). (c) Protein extracts were analysed by western blot. (d) Pho8Δ60 activity was normalized to that of WT cells (set to 100%). Error bars: SD, n = 4 independent experiments. Student’s t-test, ∗∗p < 0.01. (e) Total RNA was extracted and the mRNA levels were quantified by RT-qPCR. The mRNA level of ATG8 was normalized to that in WT cells in rich conditions (set to 1). Error bars: SD, n = 4 independent experiments. Student’ t-test, NS > 0.05. (f) Dcp2 represses autophagy. WT (YTS158) and dcp2-7Δ cells were grown in YPD at 24 °C until early log-phase, then shifted to 38.5 °C for 1 h and starved for 3 h at the same temperature. Pho8Δ60 activity was normalized to that of WT cells (set to 100%). Error bars: SD, n = 5 independent experiments. Student’s t-test, ∗∗∗p < 0.001. (g) Dcp2 represses the expression of Atg1 and Atg9. WT and dcp2-7Δ cells were grown in rich medium until early-log phase. Cells were shifted to 38.5 °C for 1 h then starved. Protein extracts were analysed by western blot. (h) WT (BY4742) and dcp2-7Δ cells were grown in rich medium until early log phase. Cells were then shifted to 38.5 °C for 1 h then starved for 1 h. The mRNA level of individual ATG genes was normalized to the mRNA level of the corresponding gene in WT cells (set to 1). Error bars: SD, n = 3 independent experiments.

Supplementary Figure 8 The RCK Fungal Homolog Vad1 Plays a Role in Decapping and Degradation of Autophagy-related Transcripts

(a) Northern blots of RNA from the indicated strains were hybridized with fragments of the indicated genes. (b) Wild-type fungal cells overexpressing VAD1 from an ACTIN promoter (OE-1, 2) or containing empty vector alone (EV-1, 2) were induced for autophagy by starvation for 1 h followed by northern blot analysis. (c) The indicated strains were observed by DIC for the presence of autophagic bodies (ABs) and quantified in n = 3 independent experiments of 200 cells each by DIC + / − SD. Student’s t-test, ∗∗∗p < 0.001. (d) RNA from WT (left panel) or vad1D mutant mid-log phase strains (right panel) was subjected to either northern blot (hybridized with a fragment of ATG8) and ratios of signal by densitometry to rRNA plotted over time (top panels) or ATG8 was assayed by quantitative RT–PCR normalized to actin (bottom panels) slope: WT versus vad1D p < 0.001. (e) The indicated strains expressing a GFP-Atg8 fusion protein containing either the native or heterologous 3’UTR (EF1a) under the indicated conditions were incubated with phenylmethanesulfonylfluoride for 30 min and observed by DIC microscopy for the presence of autophagic bodies (arrows). Scale bar = 2 μm. Representative image from n = 50 cells.

Supplementary Figure 9 Phosphorylation of TOR-Dependent Sites S614 and S617 Mediates Decapping and Degradation of ATG8 Transcripts, Related to Fig. 4.

(a) Yeast cells under the indicated conditions expressing either WT Dcp2 or an equivalent protein containing either an S > D mutation or S > A mutation at positions S614 and S617 were assayed for decapping of ATG8 transcripts over 30 min by a PCR assay and visualized by ethidium bromide as in Fig. 3b. (b) Decapping assay: Densitometry of n = 3 independent assays Dcp2 containing an S > D mutation or S > A mutation at a single position S614 or S617 were assayed for degradation as in Fig. 4b. Degradation (c) and Decapping (d) assays conducted on yeast cells expressing WT or Dcp2 with single mutations; decapping assay conducted as in Supplementary Fig. 3a and B. All experiments the results of n = 3 independent assays + / − SD. Student’s t-test, ∗p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001,∗∗∗∗p < 0.0001.

Supplementary Figure 10 Phosphorylation of TOR-Dependent Sites S614 and S617 Mediate Decapping of ATG5 T ranscripts, Related to Fig. 4.

(a) Yeast cells under the indicated conditions expressing either WT Dcp2 or an equivalent protein containing either the S > D mutation or S > A mutation at positions S614 and S617 were assayed for decapping of ATG5 transcripts over 30 min by a PCR assay and visualized by ethidium bromide as in Fig. 3b. (b) Densitometry of n = 3 independent decapping assays performed as in Supplementary Fig. 3a. Bars + / − SD; Student’s t-test, ∗p < 0.05,∗∗p < 0.01,∗∗∗p < 0.001.

Supplementary Figure 11 ATG8 and ATG5 Transcripts Demonstrate Robust Translational Efficiency and Induction under Starvation Conditions, related to Figs 2 and 4.

(a) Left panels: Indicated gene transcripts were localized using multiple Cy3-labelled oligonucleotide probes by single mRNA-sensitivity FISH (ATG8-Cy3, ATG5-Cy3) and P-bodies localized by a Vad1-GFP fusion protein. White arrows correspond to P-bodies, red arrows to the indicated transcripts. Right panels: Quantification of mid-log (Glucose +) or starvation conditions (Glucose −) of the indicated fluorescent transcript puncta, Vad1-GFP labelled P-bodies (Vad1) and co-localized puncta on deconvolved images of 20 cells. Student’s t-test, ∗∗∗∗ indicates p < 0.0001. Scale bar = 4 μm. (b) Sucrose gradient sedimentation polysome profiles of extracts from C. neoformans cells under mid-log or starvation conditions. Results of polysome profile quantification of the indicated mRNA are shown below the corresponding sucrose fractions. Corresponding ribonuclear protein (RNP) 40S, 60/80S and polysome fractions as indicated. Error bars indicate the standard deviation of n = 3 independent experiments + / − SD.

Supplementary Figure 12 The RCK Mammalian Homolog DDX6 is a Suppressor of Autophagy in Primary Embryonic Stem Cells, Related to Fig. 5.

(a) Schematic representation of DDX6 WT and two independent DDX6 gene trap insertion clones. (b) Decapping assay: The presence of capped LC3 transcripts was assayed in DDX6 WT, DDX6 clone #1 (DDX6+/−#1), and DDX6 clone #2 (DDX6+/−#2) by the method of Fig. 3 at the indicated times after transcriptional suppression. (c) Degradation assay: Quantification of LC3 transcripts of the indicated cells after transcriptional suppression in n = 3 independent experiments normalized to each respective time zero + / − SD. Student’s t-test, ∗p < 0.05;∗∗p < 0.01.

Supplementary Figure 13 The Mammalian Dcp2 Homolog Is Phosphorylated by MTOR, Related to Fig. 5.

(a) Schematic of the identification of phosphorylated Ser249 of DCP2 by mass spectroscopy. The peptides are represented by green lines and the phosphorylation site is indicated in purple. (b) Differential phosphorylation of DCP2 after treatment with rapamcyin assayed by targeted ion mass spectrometry. (c) SDS-PAGE of recombinant DCP2-MBP fusion protein purified by amylose-agarose affinity chromatography. (d) Ex vivo phosphorylation of DCP2 by MTOR. MTOR was immunoprecipitated from HeLa cells using an anti-MTOR antibody, washed, then incubated with 100 ng DCP2-MBP at 30 °C for the indicated times, and subjected to western blot using a rabbit DCP2-pSer249 affinity-purified antibody. Lower panel, density quantification of pSer249 in n = 3 independent experiments. Bar + / − SD. (e) DCP2-MBP phosphorylated with MTOR for 20 min as in in Supplementary Fig. 3d, then subjected to treatment (+) or no treatment (−) with calf intestinal alkaline phosphatase (CIAP) before western blot using antigen-purified anti-DCP2-pSer249 antibody.

Supplementary Table 1 S. cerevisiae strains used in this study.
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Supplementary Table 2 qPCR Primers used in this study.
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Supplementary Table 3 C. neoformans yeast strains used in this study1.
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Supplementary Table 4 Primers for C. neoformans yeast studies.
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Supplementary Table 5 FISH probes used in this study.
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Supplementary Table 6 Primers for mammalian studies.
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Hu, G., McQuiston, T., Bernard, A. et al. A conserved mechanism of TOR-dependent RCK-mediated mRNA degradation regulates autophagy. Nat Cell Biol 17, 930–942 (2015). https://doi.org/10.1038/ncb3189

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