Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies - PubMed (original) (raw)

Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies

Nathaniel P Hoyle et al. J Cell Biol. 2007.

Abstract

Cytoplasmic RNA granules serve key functions in the control of messenger RNA (mRNA) fate in eukaryotic cells. For instance, in yeast, severe stress induces mRNA relocalization to sites of degradation or storage called processing bodies (P-bodies). In this study, we show that the translation repression associated with glucose starvation causes the key translational mediators of mRNA recognition, eIF4E, eIF4G, and Pab1p, to resediment away from ribosomal fractions. These mediators then accumulate in P-bodies and in previously unrecognized cytoplasmic bodies, which we define as EGP-bodies. Our kinetic studies highlight the fundamental difference between EGP- and P-bodies and reflect the complex dynamics surrounding reconfiguration of the mRNA pool under stress conditions. An absence of key mRNA decay factors from EGP-bodies points toward an mRNA storage function for these bodies. Overall, this study highlights new potential control points in both the regulation of mRNA fate and the global control of translation initiation.

PubMed Disclaimer

Figures

Figure 1.

Glucose starvation caused the resedimentation of specific translation factors. (A and B) Sucrose density gradient analysis on extracts from strain yMK36 grown in YPD (A) or SCD (B) and resuspended in YPD (+glucose), YP (−glucose; A), SCD (+amino acids), or SC (−amino acids; B) for 30 min. Immunoblots on gradient fractions probed using antibodies against the indicated proteins are shown below the traces.

Figure 2.

Glucose starvation induces cytoplasmic granules of eIF4E, eIF4G1, eIF4G2, and Pab1p. (A) Confocal microscopic images of yMK strains 885 (eIF4E-GFP), 1172 (eIF4G1-GFP), 1214 (eIF4G2-GFP), 1185 (Pab1p-GFP), 1170 (eIF4AI-GFP), 883 (eIF2α-GFP), or 881 (eIF3b-GFP) in which exponential cells were preincubated for 30 min in SCD (complete), SCD lacking glucose (−glucose), or SCD lacking amino acids (−AAs). (B) Epifluorescent microscopic images of glucose-starved cells of yMK strains 1299 (eIF4G1-GFP and eIF4E-RFP), 1304 (eIF4G1-GFP and Pab1p-RFP), and 1305 (eIF4E-GFP and Pab1p-RFP). Bars, 5 μm.

Figure 3.

mRNA colocalizes with the translation initiation factor granules. (A) Immunoblots on affinity purifications of extracts from glucose-starved (−glucose) or unstarved (+glucose) cultures. 7-methyl-GTP-Sepharose (left) and poly(A)-Sepharose (right) input (I) and pellet (P) fractions were probed using specific antibodies. (B) Epifluorescent microscopic images of yMK1307 (eIF4E-RFP; top) or yMK1366 (Dcp1p-RFP; bottom) starved for glucose and coexpressing PGK1-U1A mRNA (pPS2037) and U1A-GFP protein (pRP1187) to allow analysis of PGK1 mRNA localization. Controls expressing either the PGK1-U1A mRNA or U1A-GFP protein are shown. The arrow indicates a granule bearing both Dcp1p and PGK1 mRNA, whereas the arrowhead indicates a granule harboring just the PGK1 mRNA. Bars, 5 μm.

Figure 4.

EGP-bodies represent a distinct subpopulation of mRNP foci. (A) Epifluorescent microscopic images of glucose-starved yMK strains 1302 (Dcp1p-GFP and eIF4G1-RFP), 1303 (Dcp1p-GFP and eIF4E-RFP), and 1344 (Dcp1-GFP and Pab1p-RFP). Arrowheads identify bodies containing only the translation initiation factor, whereas arrows indicate bodies where Dcp1p colocalizes with the translation initiation factor. (B) Area-proportional Venn diagrams derived from the quantitation of three biological replicates (>50 fluorescent foci each) for the glucose-starved strains yMK1299, 1304, 1305, 1302, 1303, 1344, and 1359. Errors are ±1 SD. (C) Bar chart depicting a count of granules per cell for strains bearing the indicated GFP-tagged factor. 12-image merged z-stacks generated from >75 cells starved for glucose were counted manually using ImageJ. Error bars indicate ±1 SD. Bar, 5 μm.

Figure 5.

EGP-bodies form independently of P-bodies. (A) Epifluorescent real-time 2D deconvolved projections generated from continuous z-sweep acquisition of a representative glucose-starved cell of strain yMK1303 (Dcp1p-GFP and eIF4E-RFP). Exponential cells were washed and resuspended in SC and visualized at 5-min time points for 70 min. The arrowheads identify an emergent body that contains only eIF4E and has never contained Dcp1p. The arrows indicate a body in which eIF4E is recruited to an existing Dcp1p focus. (B) A bar chart showing proportions of granules per cell from images of 10 cells, generated as described in A. Granules were categorized as those containing only Dcp1-GFP, those containing both Dcp1p-GFP and eIF4E-RFP (colocalizing), and those granules exclusively containing eIF4E-RFP, which arise independently of Dcp1p-GFP foci. Error bars are ±1 SEM. Bar, 5 μm.

Figure 6.

A model for mRNA sorting in yeast. A closed loop mRNP enters the translational pool via interaction with the 43S complex. Glucose starvation inhibits this process, leaving the mRNP two potential fates: redistribution to P-bodies, in which decapping facilitates mRNA degradation, or transfer to EGP-bodies, in which a lack of decapping factors dictates mRNA storage.

References

1. Altmann, M., N. Schmitz, C. Berset, and H. Trachsel. 1997. A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E. EMBO J. 16:1114–1121. - PMC - PubMed
1. Anderson, P., and N. Kedersha. 2006. RNA granules. J. Cell Biol. 172:803–808. - PMC - PubMed
1. Andrei, M.A., D. Ingelfinger, R. Heintzmann, T. Achsel, R. Rivera-Pomar, and R. Luhrmann. 2005. A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies. RNA. 11:717–727. - PMC - PubMed
1. Ashe, M.P., S.K. De Long, and A.B. Sachs. 2000. Glucose depletion rapidly inhibits translation initiation in yeast. Mol. Biol. Cell. 11:833–848. - PMC - PubMed
1. Ashe, M.P., J.W. Slaven, S.K. De Long, S. Ibrahimo, and A.B. Sachs. 2001. A novel eIF2B-dependent mechanism of translational control in yeast as a response to fusel alcohols. EMBO J. 20:6464–6474. - PMC - PubMed

Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies - PubMed (original) (raw)