Article | Published:

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

Nature Cell Biology volume 17, pages 930942 (2015) | Download Citation

Subjects

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , , & The machinery of macroautophagy. Cell Res. 24, 24–41 (2014).

  2. 2.

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

  3. 3.

    , , & Protein kinase A and Sch9 cooperatively regulate induction of autophagy in Saccharomyces cerevisiae. Mol. Biol. Cell 18, 4180–4189 (2007).

  4. 4.

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

  5. 5.

    , & The highways and byways of mRNA decay. Nat. Rev. Mol. Cell Biol. 8, 113–126 (2007).

  6. 6.

    , , & XRN 5′ → 3′ exoribonucleases: structure, mechanisms and functions. Biochim. Biophys. Acta 1829, 590–603 (2013).

  7. 7.

    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).

  8. 8.

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

  9. 9.

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

  10. 10.

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

  11. 11.

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

  12. 12.

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

  13. 13.

    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).

  14. 14.

    & Autophagy in health and disease: a double-edged sword. Science 306, 990–995 (2004).

  15. 15.

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

  16. 16.

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

  17. 17.

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

  18. 18.

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

  19. 19.

    , , & Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection. Infect. Immun. 68, 4225–4237 (2000).

  20. 20.

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

  21. 21.

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

  22. 22.

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

  23. 23.

    & General translational repression by activators of mRNA decapping. Cell 122, 875–886 (2005).

  24. 24.

    , , , & Processing bodies require RNA for assembly and contain nontranslating mRNAs. RNA 11, 371–382 (2005).

  25. 25.

    , & Glucose depletion rapidly inhibits translation initiation in yeast. Mol. Biol. Cell 11, 833–848 (2000).

  26. 26.

    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).

  27. 27.

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

  28. 28.

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

  29. 29.

    , & Autophagy in immunity and inflammation. Nature 469, 323–335 (2011).

  30. 30.

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

  31. 31.

    , , , & Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms. Mol. Cell 39, 773–783 (2010).

  32. 32.

    , , , & 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).

  33. 33.

    , , & Regulation of APG14 expression by the GATA-type transcription factor Gln3p. J. Biol. Chem. 276, 6463–6467 (2001).

  34. 34.

    , & The return of the nucleus: transcriptional and epigenetic control of autophagy. Nat. Rev. Mol. Cell Biol. 15, 65–74 (2014).

  35. 35.

    , , , & Substrate cycling between gluconeogenesis and glycolysis in euthyroid, hypothyroid, and hyperthyroid man. J. Clin. Invest. 76, 757–764 (1985).

  36. 36.

    , & Dcp2 phosphorylation by Ste20 modulates stress granule assembly and mRNA decay in Saccharomyces cerevisiae. J. Cell Biol. 189, 813–827 (2010).

  37. 37.

    , , , & Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system. PLoS Biol. 6, e255 (2008).

  38. 38.

    , & Identification of RNA recognition elements in the Saccharomyces cerevisiae transcriptome. Nucleic Acids Res. 39, 1501–1509 (2011).

  39. 39.

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

  40. 40.

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

  41. 41.

    , , , & 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).

  42. 42.

    , , & Specific and global regulation of mRNA stability during osmotic stress in Saccharomyces cerevisiae. RNA 15, 1110–1120 (2009).

  43. 43.

    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).

  44. 44.

    , , & Cryptococcus neoformans gene expression during murine macrophage infection. Eukaryot. Cell 4, 1420–1433 (2005).

  45. 45.

    & The inflammasomes. Cell 140, 821–832 (2010).

  46. 46.

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

  47. 47.

    , , & Validation of reference genes for quantitative expression analysis by real-time RT–PCR in Saccharomyces cerevisiae. BMC Mol. Biol. 10, 99 (2009).

  48. 48.

    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).

  49. 49.

    , , & Copper-mediated reversal of defective laccase in a Deltavph1 avirulent mutant of Cryptococcus neoformans. Mol. Microbiol. 47, 1007–1014 (2003).

  50. 50.

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

  51. 51.

    , , & Role of a VPS41 homolog in starvation response and virulence of Cryptococcus neoformans. Mol. Microbiol. 61, 1132–1146 (2006).

  52. 52.

    , & EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277 (2000).

  53. 53.

    , , , & Prediction of post-translational glycosylation and phosphorylation of proteins from the amino acid sequence. Proteomics 4, 1633–1649 (2004).

  54. 54.

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

  55. 55.

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

  56. 56.

    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).

  57. 57.

    & Cryptococcus neoformans (ASM Press, 1998).

  58. 58.

    & The myosin motor, Myo4p, binds Ash1 mRNA via the adapter protein, She3p. Proc. Natl Acad. Sci. USA 97, 5273–5278 (2000).

  59. 59.

    , & Single-RNA counting reveals alternative modes of gene expression in yeast. Nat. Struct. Mol. Biol. 15, 1263–1271 (2008).

  60. 60.

    & Microscopic detection of yeasts using fluorescence in situ hybridization. Methods Mol. Biol. 968, 71–82 (2013).

  61. 61.

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

  62. 62.

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

  63. 63.

    , , , & Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans. J. Exp. Med. 184, 377–386 (1996).

  64. 64.

    , , , & The use of gene clusters to infer functional coupling. Proc. Natl Acad. Sci. USA 96, 2896–2901 (1999).

  65. 65.

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

  66. 66.

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

  67. 67.

    , & Differential utilization of decapping enzymes in mammalian mRNA decay pathways. RNA 17, 419–428 (2011).

  68. 68.

    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).

  69. 69.

    & 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).

  70. 70.

    , & Monitoring mammalian target of rapamycin (mTOR) activity. Methods Enzymol. 452, 165–180 (2009).

Download references

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.).

Author information

Author notes

    • Guowu Hu
    • , Travis McQuiston
    •  & Amélie Bernard

    These authors contributed equally to this work.

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

Authors

  1. Search for Guowu Hu in:

  2. Search for Travis McQuiston in:

  3. Search for Amélie Bernard in:

  4. Search for Yoon-Dong Park in:

  5. Search for Jin Qiu in:

  6. Search for Ali Vural in:

  7. Search for Nannan Zhang in:

  8. Search for Scott R. Waterman in:

  9. Search for Nathan H. Blewett in:

  10. Search for Timothy G. Myers in:

  11. Search for Richard J. Maraia in:

  12. Search for John H. Kehrl in:

  13. Search for Gulbu Uzel in:

  14. Search for Daniel J. Klionsky in:

  15. Search for Peter R. Williamson in:

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Peter R. Williamson.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Information

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/ncb3189

Further reading

Newsletter Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing