Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae

Abstract

Processing bodies (P-bodies) are cytoplasmic RNA granules that contain translationally repressed messenger ribonucleoproteins (mRNPs) and messenger RNA (mRNA) decay factors. The physical interactions that form the individual mRNPs within P-bodies and how those mRNPs assemble into larger P-bodies are unresolved. We identify direct protein interactions that could contribute to the formation of an mRNP complex that consists of core P-body components. Additionally, we demonstrate that the formation of P-bodies that are visible by light microscopy occurs either through Edc3p, which acts as a scaffold and cross-bridging protein, or via the "prionlike" domain in Lsm4p. Analysis of cells defective in P-body formation indicates that the concentration of translationally repressed mRNPs and decay factors into microscopically visible P-bodies is not necessary for basal control of translation repression and mRNA decay. These results suggest a stepwise model for P-body assembly with the initial formation of a core mRNA-protein complex that then aggregates through multiple specific mechanisms.

Figure 1.

Edc3p is important for P-body assembly. (A) Localization of Dhh1GFP, Pat1GFP, Lsm1GFP, Dcp1GFP, Dcp2GFP, and Xrn1GFP in wild-type and edc3Δ strains during exponential growth in YP with (+glu) or without (−glu) glucose for 10 min. (B) Localization of Dhh1GFP, Dcp2GFP, and Xrn1GFP in dcp1Δ or dcp1Δ edc3Δ strains or Dhh1GFP, Dcp2GFP, and Pat1GFP in xrn1Δ or xrn1Δ edc3Δ strains during exponential growth in YP containing glucose. Bars, 3 μm.

Figure 2.

When cells are unable to respire, Edc3p is required for P-body assembly, but not for translation repression, in response to glucose deprivation. (A) Localization of Dcp2GFP or Dhh1GFP in respiratory-competent or -deficient wild-type or edc3Δ strains during exponential growth in YP containing glucose (+glu, +aeration), deprived of glucose for 10 min while being aerated (−glu, +aeration), or deprived of glucose for 10 min while concentrated in a microcentrifuge tube (−glu, −aeration). Bar, 3 μm. (B) Polysome profiles, A254 traces of sucrose density gradients, were obtained from respiratory-deficient wild-type or edc3Δ strains grown under standard growth conditions (+glu) or deprived of glucose for 10 min while being aerated (−glu).

Figure 3.

Edc3p interacts with itself, Dcp2p, and Dhh1p through distinct domains. (A) Summary of two-hybrid protein interaction assays between full-length or the indicated deleted versions of Edc3p and full-length Dcp1p or Edc3p, the conserved region of Dcp2p (amino acids 1–300), the catalytic domain of Dcp2p (amino acids 102–300), or a C-terminal fragment of Dhh1p (amino acids 250–461). +++, ++, and + indicate relative positive interaction based on β-galactosidase assays. − indicates no detectable interaction. (B) An in vitro binding assay between purified His-Dcp2(1–300)-Flag or His-Dcp2(102–300)-Flag and E. coli lysate containing the His-tagged Lsm domain of Edc3p in the presence of 225 mM NaCl. Anti-Flag affinity resin was used to pull down (PD) the Flag-tagged Dcp2 proteins and anti-His antibody used in Western blots (WB) to detect the His-tagged Lsm domain. Total (T) lanes represent 1/8 of the material loaded in the pellet (P) lanes. (C) An in vitro binding assay between purified His-Dcp2(1–300)-Flag or His-Dcp2(102–300)-Flag and a purified His-tagged FDF domain of Edc3p in the presence of 100 or 225 mM NaCl. Total lanes represent 1/8 of the material loaded in the pellet lanes. (D) An in vitro binding assay between purified GST, GST-tagged Dhh1(46–461) or Dhh1(250–461), and an E. coli lysate containing a His-tagged Lsm domain of Edc3p. Glutathione-Sepharose was used to pull down the GST-tagged Dhh1 proteins and anti-His antibody was used in Western blots to detect the His-tagged Lsm domain. Total lanes represent 1/8 of the material loaded in the pellet lanes. (E) An in vitro binding assay between purified GST, GST-tagged Dhh1(46–461) or Dhh1(250–461), and a purified His-tagged FDF domain of Edc3p. Total lanes represent 1/8 of the material loaded in the pellet lanes. (F) An in vitro binding assay between purified GST or GST-tagged Dhh1(46–461) and His-Dcp2(1–300)-Flag or His-Dcp2(102–300)-Flag. Glutathione-Sepharose was used to pull down the GST-tagged Dhh1 protein and anti-His antibody used in Western blots to detect the His-tagged Dcp2 proteins. Total lanes represent 1/8 of the material loaded in the pellet lanes.

Figure 4.

The Lsm and Yjef-N domains are required for Edc3p to function in P-body assembly. Localization of Dcp2GFP in a respiratory-deficient edc3Δ strain expressing from a plasmid Flag-tagged full-length Edc3p, no Edc3p, or versions of Flag-tagged Edc3p in which the Lsm, FDF, or Yjef-N domains were deleted during exponential growth in SC containing glucose (+glu) or after being deprived of glucose for 10 min under aeration (−glu). Bar, 3 μm.

Figure 5.

In the absence of Edc3p, the prionlike domain of Lsm4p is required for P-body aggregation, but not for translation repression, in response to glucose deprivation. (A) Localization of Dcp2GFP in wild-type, edc3Δ, lsm4ΔC (deletion of C terminus of Lsm4 including the Q/N-rich prionlike domain), or lsm4ΔC edc3Δ strains, or an lsm4Δ edc3Δ strain carrying a plasmid in which the C-terminal Q/N-rich domain of Lsm4p has been replaced with the prion domain of Rnq1p during exponential growth in YP containing glucose (+glu) or after being deprived of glucose for 10 min while being aerated (−glu). (B) Localization of MFA2P-U1A mRNA in wild-type or lsm4ΔC edc3Δ strains deprived of glucose for 10 min while being aerated. The MFA2P-U1A mRNA was detected by coexpressing U1A fused with GFP, which binds to the U1A binding sites within the 3′ UTR of the mRNA. Note that a different contrast range was used than the range in A because of the intense fluorescence signal of the RNA foci in the wild-type cells. Bars, 3 μm. (C) Polysome profiles, A254 traces of sucrose density gradients, obtained from wild-type or lsm4ΔC edc3Δ strains grown under standard growth conditions (+glu) or deprived of glucose for 10 min while being aerated (−glu).

Figure 6.

P-body aggregation does not substantially affect mRNA decay. (A) Decay analysis of MFA2pG mRNA in wild-type, edc3Δ, lsm4ΔC, or lsm4ΔC edc3Δ strains. Time points after transcriptional repression are indicated above each lane. The half-life values shown on the right are in minutes and represent the mean and SD based on three experiments. (B) Decay analysis of MFA2pG mRNA in edc3Δ or control strains carrying the temperature-sensitive allele dcp2-7 performed at 24°C, where dcp2-7 is partially active. The dcp2-7 edc3Δ strain contained plasmids expressing no Edc3p, Flag-tagged full-length Edc3p, or versions of Flag-tagged Edc3p in which the Lsm, FDF, or Yjef-N domains were deleted. The half-life values are in minutes and represent the mean and SD based on four experiments. (C) Growth of the dcp2-7 ski3Δ control strain or the dcp2-7 ski3Δ edc3Δ strain expressing no Edc3p, Flag-tagged full-length Edc3p, or versions of Flag-tagged Edc3p in which the Lsm, FDF, or Yjef-N domains were deleted on synthetic medium at 21 or 34°C.

Figure 7.

Model for P-body assembly. First, two subcomplexes of conserved “core” proteins assemble onto mRNA through a series of direct interactions between their components and with the mRNA. One of these subcomplexes is composed of Dcp1p, Dcp2p, Dhh1p, and Edc3p, although Dcp2, Dhh1p, and Edc3p may or may not bind simultaneously with each other. The other subcomplex consists of Pat1p, Lsm1-7p, and Xrn1p. Individual core P-body mRNPs could then potentially aggregate into larger assemblies through interactions dependent on the Edc3p YjeF-N domain and/or the Lsm4p Q/N-rich prionlike domain.