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First published online 8 July 2008
doi: 10.1242/jcs.024976

Journal of Cell Science 121, 2463-2472 (2008)
Published by The Company of Biologists 2008
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A role for Q/N-rich aggregation-prone regions in P-body localization

Martin A. M. Reijns*, Ross D. Alexander, Michael P. Spiller{ddagger} and Jean D. Beggs§

Wellcome Trust Centre for Cell Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK


Figure 1
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  		Fig. 1 Lsm4p has an N-rich C-terminal region. (A) The N-terminus of Lsm4p (aa 1-92) contains the Sm domain; the C-terminus (aa 93-187; boxed) contains N-rich (or Q-rich) stretches of variable lengths. Scer, S. cerevisiae; Spar, S. paradoxus; Sbay, S. bayanus; Smik, S. mikatae; Scas, S. castellii; Skud, S. kudriavzevii; Sklu, S. kluyveri. (B) Lsm4 protein alignment of the budding yeast (yLsm4p) and the human (hLsm4p) protein. Sequences were aligned using ClustalW. Q residues are highlighted in grey, N residues in black and RG (arginine-glycine) repeats are underlined.

 

Figure 2
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  		Fig. 2 Overproduction of Lsm4p or its C-terminus leads to aggregation in cytoplasmic foci. (A) GFP-Lsm4 (pMPSLsm4), GFP-Lsm4C (pMPSlsm4D2) and GFP-Lsm4{Delta}C (pMPSLsm4D1) were overexpressed from the MET25 promoter in BY4741 cells grown in SD-Ura-Met. Localization was examined in cells during log-phase growth. (B) Colocalization of Lsm4p aggregates with Dcp2-RFP (pRP1155) was examined in BY4741 cells grown in SD-Ura-Leu-Met during log-phase growth or 20 minutes after hypo-osmotic shock. Dcp2-RFP (pMR171) localization was examined in PGAL-LSM4 cells expressing GFP-Lsm4{Delta}C grown in SD-Ura-His-Met (to prevent competition between GFP-Lsm4{Delta}C and endogenous Lsm4p for incorporation into Lsm1-7p), 20 minutes after hypo-osmotic shock. (C) Localization of GFP-Lsm1 (pGFP-N-Lsm1), GFP-Lsm6 (pMPSLsm6), Lsm8-GFP (pMR83) and GFP (pGFP-N-FUS) was examined in log-phase cells overproducing Lsm4p (PGAL-LSM4 cells grown in SDGal-Ura) and in cells with normal levels of Lsm4p (PGAL-LSM4 cells with pUSS1 grown in SD-Ura-Met). Nuclear DNA stained with DAPI is shown in blue.

 

Figure 3
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  		Fig. 3 The Lsm4 C-terminus is required for efficient localization of Lsm1p to P-bodies. (A) GFP-Lsm1 (pGFP-N-Lsm1) localization 20 minutes after hypo-osmotic shock in LSM4 (MRY71) or lsm4{Delta}C (MRY73) cells. (B) Percentage of cells showing GFP-Lsm1 in foci 5 minutes or 1 hour after osmotic shock (n=100 cells per time point). (C,D) Localization of GFP-Lsm2 (pMPSLsm2) (C) and GFP-Lsm6 (pMPSLsm6) (D) in LSM4 or lsm4{Delta}C cells before and after hypo-osmotic shock (E) Dcp2-RFP (pMR159) localization in log phase LSM4 and lsm4{Delta}C cells grown in SD-His and 20 minutes after osmotic shock. All experiments in this figure were performed with strains expressing non-tagged Lsm4p or Lsm4{Delta}Cp from the native LSM4 promoter.

 

Figure 4
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  		Fig. 4 The Lsm4 C-terminus is required for efficient mRNA degradation, but not splicing. (A) Degradation of PGK1pGmini reporter transcript in LSM4 (MRY71) or lsm4{Delta}C (MRY73) strains grown in SDGal-Ura after addition of glucose to 4% (w/v). scR1 RNA was used as a loading control. The asterisk indicates a stable degradation fragment. (B) PGK1pGmini transcript levels over time as a percentage of the level in LSM4 cells at t=0; averages of three northern blots with vertical bars indicating standard deviations. (C) Linearized degradation curves [–ln(PGK1t/PGK1t=0)] against time showing averages of qRT-PCR data of six RT repeats of two independent biological replicates; vertical bars indicate standard errors; half-lives indicated are based on the linearized qRT-PCR data (D) Northern blot detecting pre-U3 RNA and U3 RNA in LSM4 and lsm4{Delta}C strains grown in YPDA, and in a PGAL-LSM8 strain (MPS7) before and after 12 hours of growth on glucose.

 

Figure 5
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  		Fig. 5 Other P-body components contain Q/N-rich regions. (A) Alignment of the N-terminal region of S. cerevisiae Ccr4p with homologues from closely related Saccharomyces species (see Fig. 1 legend for abbreviations). The Q/N-rich region is boxed, with Q residues highlighted in grey, N residues highlighted in black and P residues in red. (B) Schematic representation of P-body components with areas rich in Q and/or N residues indicated in white (approximately to scale; Not1p is broken to fit; lengths are indicated in numbers of aa). (C) Q, N and P residues were counted in aa 80-mers of Ccr4p, Pop2p, Not1p and Dhh1p starting at position 1, shifting ten aa at a time.

 

Figure 6
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  		Fig. 6 The Q/N-rich region from Ccr4p aggregates in cytoplasmic foci and responds to stress. (A) Localization of GFP-tagged Q/N-rich regions of Ccr4p (aa 1-229; pMR202), Pop2p (aa 1-156; pMR203) and Dhh1p (aa 427-506; pMR204) before and after hypo-osmotic shock (B) GFP-Ccr4(1-229) aggregates localize to the cytoplasm as shown in these fixed cells with DAPI stained nuclear DNA (C) The majority of GFP-Ccr4(1-229) aggregates does not colocalize with Dcp2-RFP (pRP1155) foci after osmotic shock.

 

Figure 7
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  		Fig. 7 Q/N-rich regions from Ccr4p, Pop2p and Dhh1p contribute to efficient accumulation of these proteins in P-bodies. (B) Localization of GFP-tagged full-length Ccr4p (pMR212), Pop2p (pMR214), Dhh1p (pMR210) or truncated versions of these proteins (Ccr4{Delta}N(148-837) from pMR218, Pop2{Delta}N(147-433) from pMR215, Dhh1{Delta}C(1-427) from pMR211) before and after osmotic shock. All GFP-fusions were expressed in BY4741 cells and localization was examined in cells during normal growth (normal) or 20-40 minutes after osmotic shock (stress). (B) Localization of GFP-tagged Ccr4{Delta}N(148-837) and Ccr4{Delta}N2 (aa 250-837; pMR213) in fixed cells with DAPI-stained nuclear DNA (C) Localization of C-terminally GFP-tagged full-length Pop2p (pMR216) or Pop2{Delta}Np (pMR217); DAPI-stained nuclear DNA in blue. (D) Localization of GFP-Ccr4, GFP-Ccr4{Delta}N and Dcp2-RFP in ccr4{Delta} cells (Y10387) 30 minutes after hypo-osmotic shock (E) Anti-GFP western blot analysis of full-length and truncated Lsm4, Ccr4, Pop2 and Dhh1 proteins. Curiously, GFP-Ccr4 (122 kDa) migrates faster than GFP-Ccr4{Delta}N (106 kDa), but slower than GFP-Ccr4{Delta}N2 (95 kDa; Fig. 7E), and all three GFP-Ccr4 proteins migrate faster than their predicted molecular weights. The presence or absence of the highly polar N-terminal region of Ccr4p causes a change in the effective charge of the entire protein (predicted charges at pH 7 are –5.8, –4.0 and –5.5 respectively) and might affect protein conformation resulting in unusual migration during SDS-PAGE. Nop1p was used as a loading control.

 

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