Letter | Published:

A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly

Nature Cell Biology volume 10, pages 12241231 (2008) | Download Citation



Stress granules (SGs) and processing bodies (PBs) are microscopically visible ribonucleoprotein granules that cooperatively regulate the translation and decay of messenger RNA1,2,3. Using an RNA-mediated interference-based screen, we identify 101 human genes required for SG assembly, 39 genes required for PB assembly, and 31 genes required for coordinate SG and PB assembly. Although 51 genes encode proteins involved in mRNA translation, splicing and transcription, most are not obviously associated with RNA metabolism. We find that several components of the hexosamine biosynthetic pathway, which reversibly modifies proteins with O-linked N-acetylglucosamine (O-GlcNAc) in response to stress, are required for SG and PB assembly. O-GlcNAc-modified proteins are prominent components of SGs but not PBs, and include RACK1 (receptor for activated C kinase 1), prohibitin-2, glyceraldehyde-3-phosphate dehydrogenase and numerous ribosomal proteins. Our results suggest that O-GlcNAc modification of the translational machinery is required for aggregation of untranslated messenger ribonucleoproteins into SGs. The lack of enzymes of the hexosamine biosynthetic pathway in budding yeast may contribute to differences between mammalian SGs and related yeast EGP (eIF4E, 4G and Pab1 containing) bodies.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    & RNA granules. J. Cell Biol. 172, 803–808 (2006).

  2. 2.

    & Stress granules: the Tao of RNA triage. Trends. Biochem. Sci. 33, 141–150 (2008).

  3. 3.

    & Mammalian stress granules and processing bodies. Methods Enzymol. 431, 61–81 (2007).

  4. 4.

    & P bodies and the control of mRNA translation and degradation. Mol. Cell 25, 635–646 (2007).

  5. 5.

    , & P bodies: at the crossroads of post-transcriptional pathways. Nature Rev. Mol. Cell Biol. 8, 9–22 (2007).

  6. 6.

    et al. Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules. Mol. Biol. Cell 13, 195–210 (2002).

  7. 7.

    , , , & Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes. Am. J. Physiol. Cell Physiol. 284, C273–C284 (2003).

  8. 8.

    , & The deacetylase HDAC6 is a novel critical component of stress granules involved in the stress response. Genes Dev. 21, 3381–3394 (2007).

  9. 9.

    , , & P-body formation is a consequence, not the cause, of RNA-mediated gene silencing. Mol. Cell. Biol. 27, 3970–3981 (2007).

  10. 10.

    & Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805–808 (2003).

  11. 11.

    & Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1. Genes Dev. 19, 351–361 (2005).

  12. 12.

    & TTP and BRF proteins nucleate processing body formation to silence mRNAs with AU-rich elements. Genes Dev. 21, 719–735 (2007).

  13. 13.

    et al. Stress granules and processing bodies are dynamically liked sites of mRNP remodeling. J. Cell Biol. 169, 871–884 (2005).

  14. 14.

    , , & Methods Enzymol. (in the press).

  15. 15.

    , & Cytoplasmic foci are sites of mRNA decay in human cells. J. Cell Biol. 165, 31–40 (2004).

  16. 16.

    et al. Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules. J. Cell Biol. 151, 1257–1268 (2000).

  17. 17.

    & Ins (endocytosis) and outs (exocytosis) of GLUT4 trafficking. Curr. Opin. Cell Biol. 19, 466–473 (2007).

  18. 18.

    & The hexosamine signaling pathway: deciphering the 'O-GlcNAc code'. Sci. STKE 2005, re13 (2005).

  19. 19.

    Role of insulin, adipocyte hormones, and nutrient-sensing pathways in regulating fuel metabolism and energy homeostasis: a nutritional perspective of diabetes, obesity, and cancer. Sci. STKE 2006, re7 (2006).

  20. 20.

    , & O-GlcNAc cycling: how a single sugar post-translational modification is changing the way we think about signaling networks. J. Cell. Biochem. 97, 71–83 (2006).

  21. 21.

    & Cell signaling, the essential role of O-GlcNAc! Biochim. Biophys. Acta 1761, 599–617 (2006).

  22. 22.

    et al. Dynamic O-GlcNAc modification of nucleocytoplasmic proteins in response to stress. A survival response of mammalian cells. J. Biol. Chem. 279, 30133–30142 (2004).

  23. 23.

    et al. Cardioprotection by N-acetylglucosamine linkage to cellular proteins. Circulation 117, 1172–1182 (2008).

  24. 24.

    The sweet nature of cardioprotection. Am. J. Physiol. Heart Circ. Physiol. 293, H1324–H1326 (2007).

  25. 25.

    & O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress. Biochim. Biophys. Acta 1673, 13–28 (2004).

  26. 26.

    & AMP-activated protein kinase and p38 MAPK activate O-GlcNAcylation of neuronal proteins during glucose deprivation. J. Biol. Chem. 283, 13009–13020 (2008).

  27. 27.

    et al. Glucose deprivation stimulates O-GlcNAc modification of proteins through up-regulation of O-linked N-acetylglucosaminyltransferase. J. Biol. Chem. 283, 6050–6057 (2008).

  28. 28.

    et al. Sortilin is the major 110-kDa protein in GLUT4 vesicles from adipocytes. J. Biol. Chem. 273, 3582–3587 (1998).

  29. 29.

    et al. The sortilin cytoplasmic tail conveys Golgi–endosome transport and binds the VHS domain of the GGA2 sorting protein. EMBO J. 20, 2180–2190 (2001).

  30. 30.

    et al. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol. Cell Proteomics 1, 791–804 (2002).

  31. 31.

    & Inhibition of MDM2-mediated p53 ubiquitination and degradation by ribosomal protein L5. J. Biol. Chem. 279, 44475–44482 (2004).

  32. 32.

    , , & Inhibition of the ubiquitin–proteasome system induces stress granule formation. Mol. Biol. Cell 18, 2603–2618 (2007).

  33. 33.

    et al. Stress granule assembly is mediated by prion-like aggregation of TIA-1. Mol. Biol. Cell 15, 5383–5398 (2004).

  34. 34.

    & Accumulation of polyadenylated mRNA, Pab1p, eIF4E, and eIF4G with P-bodies in Saccharomyces cerevisiae. Mol. Biol. Cell 18, 2592–2602 (2007).

  35. 35.

    , , , & Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies. J. Cell Biol. 179, 65–74 (2007).

  36. 36.

    , & ARE-mRNA degradation requires the 5′-3′ decay pathway. EMBO Rep. 7, 72–77 (2006).

Download references


We thank C. Shamu and colleagues in the ICCB-L RNAi screening facilities at Harvard Medical School for their assistance. We also thank N. Ramadan at the Drosophila RNAi Screening Centre at Harvard Medical School for assistance with the automated microscope. We thank Jens Lykke-Andersen for the DCP1a antibody, and Carlos Morales for the Myc-tagged sortilin plasmid. We thank Keith Blackwell, Pavel Ivanov and Dan Schoenberg for critical review of the manuscript. This work was supported by NIH grants AI065858, AI033600 and AR0514732.

Author information

Author notes

    • Tyler Hickman
    •  & Sarah Tisdale

    These authors contributed equally to this work.


  1. Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, 1 Jimmy Fund Way, Boston, Massachusetts 02115, USA.

    • Takbum Ohn
    • , Nancy Kedersha
    • , Tyler Hickman
    • , Sarah Tisdale
    •  & Paul Anderson


  1. Search for Takbum Ohn in:

  2. Search for Nancy Kedersha in:

  3. Search for Tyler Hickman in:

  4. Search for Sarah Tisdale in:

  5. Search for Paul Anderson in:


T.O. was responsible for experimental design, data acquisition, data analysis and the preparation of figures; N.K. contributed to experimental design, data acquisition, data analysis and the preparation of figures; S.T. and T.H. contributed to data acquisition and provided technical assistance; P.A. conceived the project and supervised all experimental activities. The manuscript was written by P.A., N.K. and T.O.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Paul Anderson.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Figures S1, S2, S3, S4, S5, S6, S7, Supplementary Tables S1, S2, S3, Supplementary Methods


  1. 1.

    Supplementary Information

    Supplementary Movie 1

About this article

Publication history