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. 2008 Oct;10(10):1224-31.
doi: 10.1038/ncb1783. Epub 2008 Sep 14.

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

Takbum Ohn  1 , Nancy Kedersha, Tyler Hickman, Sarah Tisdale, Paul Anderson
Affiliations

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

Takbum Ohn et al. Nat Cell Biol. 2008 Oct.

Abstract

Stress granules (SGs) and processing bodies (PBs) are microscopically visible ribonucleoprotein granules that cooperatively regulate the translation and decay of messenger RNA. 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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Figures

Figure 1
Figure 1
RNAi screen identifies candidate genes involved in SG and/or PB assembly. (a) Venn diagram of genes affecting SG and/or PB assembly. (b) Graphic depiction of functional classification of genes revealed with the DAVID bioinformatics tool. (c) Effect of siRNA-mediated knockdown on the arsenite-induced assembly of SGs and PBs in RDG3 cells. Representative siRNA treatments from the primary screen alter the assembly of stress granules (green, GFP–G3BP) and P-bodies (red, RFP–DCP1a). SORT1, BDP1 and RPL10 knockdowns eliminate both SGs and PBs; RAB36 prevents PB assembly but not SG assembly, eIF3S2 knockdown prevents SG assembly but not PB assembly, and PRPF18 eliminates SG assembly but induces huge PBs.
Figure 2
Figure 2
The Hexosamine Biosynthetic Pathway (HBP) is required for SG and PB assembly. (a) Schematic representation of the HBP. (b) Sortilin and OGT regulate stress-induced O-GlcNAc modifications. U2OS cells transfected with the indicated siRNAs (D0, non-targeting control siRNA) were cultured in the absence or presence of arsenite before processing for immunoblots to assess protein expression. Densitometric quantifications of relative O-GlcNAc modification levels are indicated under the O-GlcNAc panel. The asterisk denotes a non-specific band recognized by anti-sortilin antibody. Numbers at the left are molecular masses in kDa. (c) Components of the HBP regulate SG assembly. U2OS cells were treated with the indicated siRNAs, then with arsenite for the indicated times before processing for immunofluorescence microscopy and quantifying of the percentage of cells with SGs. (d) Components of the HBP regulate PB assembly. (e) O-GlcNAc-modified proteins are components of SGs. U2OS cells were treated with the indicated drugs for 40 min, then stained for the stress granule marker eIF3b (eIF3, green), O-GlcNAc (OGN, red), and the P-body marker DCP1a (DCP1, blue). Enlarged views (8.5) of boxed areas show separate channels and merged views. Yellow arrowheads indicate P-bodies; white arrows indicate stress granules. Full scans of blots in b are shown in Supplementary Information, Fig. S7.
Figure 3
Figure 3
Immunopurification and identification of arsenite-induced O-GlcNAc-modified proteins. (a) O-GlcNAc-modified proteins are specifically enriched in fractions containing 80S ribosomes and ribosome subunits in response to oxidative stress. Mock and arsenite-treated U2OS cells were lysed, fractionated over sucrose gradients (10–50%; top panels), and probed by immunoblotting with two different O-GlcNAc antibodies (middle two panels, CTD110.6; bottom two panels, RL2) to quantify O-GlcNAc-modified proteins. The indicated fractions (red boxes) were pooled, precipitated with acetone, and denatured with SDS before reprecipitation with anti-O-GlcNAc antibodies (see Methods). Numbers at the left of the blots are molecular masses in kDa. (b) Reprecipitates were separated by SDS–PAGE and stained with colloidal blue. Ten clearly distinguished bands were excised and subjected to mass spectrometry. A contaminating band is marked with an asterisk in the mouse IgG purified lane as a control. (c) Detection of biotinylated proteins from dimethylsulfoxide-treated (DMSO) U2OS cells (mock) or GlcNAz-treated (Azido-glucosamine) U2OS cells metabolically labelled with or without arsenite treatment (0.5 mM, 45 min) with the use of chemiluminescent substrate after blotting with horseradish peroxidaseconjugated streptavidin. Small aliquots (5%) of resolubilized biotinylated protein pellets were treated with SDS sample buffer and subjected to SDS– PAGE. (d) Biotinylated proteins before (input) or after (capture) pulldown with streptavidin beads were probed by immunoblotting with antibodies against RPS3, RPL13a and OGT (positive control). Full scans of blots in d are shown in Supplementary Information, Fig. S7.
Figure 4
Figure 4
Stress-induced O-GlcNAc modifications are not required for polysome disassembly. (a–d) U2OS cells were treated with the indicated siRNAs (D0 used as a control), then cultured in the absence (green lines) or presence (red lines) of arsenite (0.1 mM, 1 h) before polysome profile analysis. (e) Knockdown of HRI inhibits arsenite-induced eIF2α phosphorylation but not O-GlcNAc modification. U2OS cells treated with the indicated siRNAs were cultured in the absence or presence of arsenite before harvesting and processing for western blotting analysis with the indicated antibodies. Normalized densitometric quantification of O-GlcNAc modification levels is indicated under the respective panels.
Figure 5
Figure 5
Model of SG versus PB assembly. Left: normal termination results in an mRNP that is eligible for deadenylation and eventual recruitment to the decapping and decay machinery of a PB. Right: stress-induced polysome disassembly caused by stalled initiation results in a 48S complex that is eligible for O-GlcNAc modification and SG assembly. Some mRNPs from SGs may be translocated to PBs, whereas others may be reinitiated or stored.
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Comment in

  • Stressed out? Make some modifications!
    Hilliker A, Parker R. Hilliker A, et al. Nat Cell Biol. 2008 Oct;10(10):1129-30. doi: 10.1038/ncb1008-1129. Nat Cell Biol. 2008. PMID: 18830219 Free PMC article.

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References

    1. Anderson P, Kedersha N. RNA granules. J. Cell Biol. 2006;172:803–808. - PMC - PubMed
    1. Anderson P, Kedersha N. Stress granules: the Tao of RNA triage. Trends. Biochem. Sci. 2008;33:141–150. - PubMed
    1. Kedersha N, Anderson P. Mammalian stress granules and processing bodies. Methods Enzymol. 2007;431:61–81. - PubMed
    1. Parker R, Sheth U. P bodies and the control of mRNA translation and degradation. Mol. Cell. 2007;25:635–646. - PubMed
    1. Eulalio A, Behm-Ansmant I, Izaurralde E. P bodies: at the crossroads of post-transcriptional pathways. Nature Rev. Mol. Cell Biol. 2007;8:9–22. - PubMed

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