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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

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Abstract

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.

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Acknowledgements

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.

Affiliations

  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

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Contributions

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.

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DOI

https://doi.org/10.1038/ncb1783