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. 2007 Apr;13(4):493-502.
doi: 10.1261/rna.355807. Epub 2007 Feb 16.

Microtubule disruption stimulates P-body formation

Thomas J Sweet  1 , Brooke Boyer, Wenqian Hu, Kristian E Baker, Jeff Coller
Affiliations

Microtubule disruption stimulates P-body formation

Thomas J Sweet et al. RNA. 2007 Apr.

Abstract

Processing bodies (P-bodies) are subcellular ribonucleoprotein (RNP) granules that have been hypothesized to be sites of mRNA degradation, mRNA translational control, and/or mRNA storage. Importantly, P-bodies are conserved from yeast to mammals and contain a common set of evolutionarily conserved protein constituents. P-bodies are dynamic structures and their formation appears to fluctuate in correlation with alterations in mRNA metabolism. Despite these observations, little is understood about how P-body structures are formed within the cell. In this study, we demonstrate a relationship between P-bodies and microtubules in the budding yeast, Saccharomyces cerevisiae. First, we demonstrate that disruption of microtubules by treatment with the drug benomyl leads to aggregation of P-body components. Consistent with this finding, we also demonstrate that disruption of microtubules by a temperature-sensitive allele of the major alpha tubulin, TUB1 (tub1-724) stimulates P-body formation. Second, we find that the alpha-tubulin protein Tub1 colocalizes with P-bodies upon microtubule destabilization. Third, we determine that a putative tubulin tyrosine ligase, encoded by YBR094W, is a protein component of P-bodies, providing additional evidence for a physical connection between P-bodies and microtubules. Finally, we establish that P-bodies formed by microtubule destabilization fail to correlate with global changes in the stability of mRNA or in general mRNA translation. These findings demonstrate that the aggregation of P-body components is linked to the intracellular microtubule network, and, further, that P-bodies formed by disruption of microtubules aggregate independent of broad alterations in either mRNA decay or mRNA translation.

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Figures

FIGURE 1.
FIGURE 1.
Both benomyl and tub1-724-mediated microtubule disruption leads to the formation of Dcp2p-containing foci. Cultured cells encoding a chromosomal copy of TUB1-GFP transformed with DCP2-RFP encoded on a plasmid were treated with DMSO alone (A) or benomyl (B), followed by imaging of the cells by fluorescence microscopy. The two sets of panels per treatment represent different images from the same preparation. (C) TUB1 and tub1-724 cell cultures were transformed with DCP2-GFP encoded on a plasmid, and were grown at either the permissive temperature (30°C) or the restrictive temperature (15°C), followed immediately by imaging of the cells by fluorescence microscopy.
FIGURE 2.
FIGURE 2.
Foci formed within cells as a consequence of microtubule-destabilizing conditions are P-bodies. Wild-type cells were transformed with plasmids encoding DHH1-GFP and DCP2-RFP, and cultures of these cells were treated with DMSO alone (A) or benomyl (B), and then cells were imaged by fluorescence microscopy. The MFA2-pG-MS2 reporter used to detect the intermediate of mRNA decay is diagrammed in C. The poly(G) tract provides an efficient block to Xrn1p-catalyzed 5′–3′ exonucleolytic digestion of the mRNA. The MS2-binding sites are bound by MS2-GFP, thus allowing visualization of the reporter. Wild-type cells were transformed with MFA2-pG-MS2, MS2-GFP, and DCP2-RFP (all encoded on plasmids), and cultures of these cells were treated with DMSO alone (D) or benomyl (E), and then the cells were imaged by fluorescence microscopy.
FIGURE 3.
FIGURE 3.
Ybr094wp (Pby1p) localizes in P-bodies and plays no obvious role in 5′–3′ mRNA decay, NMD, or decay of EDC1 mRNA. (A) Cells encoding a chromosomal copy of YBR094W-GFP were transformed with DCP2-RFP encoded on a plasmid, and cultures were treated with medium either containing or lacking glucose for 10 min, at which time DAPI was added directly to the culture medium. Cells were then imaged by fluorescence microscopy. Half-lives of MFA2pG mRNA (B) and PGK1pG mRNA (C) were determined in cultures of wild-type or pby1Δ cells by transcriptional shut-off by glucose repression. RNA was isolated at various time points after repression (indicated in the figure) and analyzed by Northern blot. Steady-state levels of CYH2 pre-mRNA and EDC1 mRNA were determined in cultures of wild-type, pby1Δ, and xrn1Δ cells by RNA isolation followed by Northern blot analysis (D).
FIGURE 4.
FIGURE 4.
Benomyl treatment of cells does not alter mRNA decay or global translation. rpb1-1 cells were treated with either benomyl (A) or DMSO alone (B), and the half-life of MFA2 mRNA was determined by transcriptional shut-off by temperature shift. RNA was isolated at various time points after temperature shift (indicated in the figure) and analyzed by Northern blot. Cells containing a chromosomal copy of TUB1-GFP were treated with either benomyl (C) or DMSO alone (D), and sucrose gradient fractionation followed by polysome analysis was carried out as described in the Materials and Methods. Curves represent a continuous plot of absorbance at 254 nm (y-axis) versus position in the sucrose gradient (x-axis). 40S and 60S ribosomal subunit peaks as well as 80S monosome peaks, polysome peaks, and RNP peaks are labeled on the curves.
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