Identification and analysis of the interaction between Edc3 and Dcp2 in Saccharomyces cerevisiae

Abstract

Cap hydrolysis is a critical control point in the life of eukaryotic mRNAs and is catalyzed by the evolutionarily conserved Dcp1-Dcp2 complex. In Saccharomyces cerevisiae, decapping is modulated by several factors, including the Lsm family protein Edc3, which directly binds to Dcp2. We show that Edc3 binding to Dcp2 is mediated by a short peptide sequence located C terminal to the catalytic domain of Dcp2. This sequence is required for Edc3 to stimulate decapping activity of Dcp2 in vitro, for Dcp2 to efficiently accumulate in P-bodies, and for efficient degradation of the RPS28B mRNA, whose decay is enhanced by Edc3. In contrast, degradation of YRA1 pre-mRNA, another Edc3-regulated transcript, occurs independently from this region, suggesting that the effect of Edc3 on YRA1 is independent of its interaction with Dcp2. Deletion of the sequence also results in a subtle but significant defect in turnover of the MFA2pG reporter transcript, which is not affected by deletion of EDC3, suggesting that the region affects some other aspect of Dcp2 function in addition to binding Edc3. These results raise a model for Dcp2 recruitment to specific mRNAs where regions outside the catalytic core promote the formation of different complexes involved in mRNA decapping.

FIG. 1.

Region 248-300 of Dcp2 mediates its accumulation in P-bodies. (A) Domain architecture of S. cerevisiae Dcp2. The N-terminal globular domain is shown in gray. The Nudix domain is shown in black. Region 248-300 of Dcp2 is shown in light gray. (B) Localization of truncated Dcp2 proteins. Wild-type cells (yRP840) expressing the full-length (FL) or truncated Dcp2 protein (1-300, 1-247, 243-970, or 327-970) C-terminally tagged with GFP from plasmid pRP1891, pRP1892, pRP1893, pRP1894, or pRP1895 were used. Cells were cultured to mid-log phase and shifted to media lacking glucose for 10 min. A single focal plane image is shown. Bar, 5 μm. (C) Levels of the truncated Dcp2 proteins. The same strains as those described in the legend to panel B were cultured to mid-log phase and shifted to media lacking glucose for 10 min. pRP250 was used as a control vector (no GFP). Anti-GFP antibody was used in Western blot to detect GFP-tagged proteins. Anti-Pgk1 antibody was used to detect the endogenous Pgk1 protein as a loading control. Arrowheads indicate GFP-tagged proteins. Asterisks indicate nonspecific bands.

FIG. 2.

Region 248-300 of Dcp2 is required for Edc3 binding in vitro. (A) Coomassie blue-stained SDS-PAGE showing purified recombinant proteins used in the pulldown assay. (B) In vitro pulldown (PD) assay between purified GST-tagged Lsm-FDF domain of Edc3 and His-tagged Dcp2 (amino acids 1 to 300) or His-Dcp2(1-247). Glutathione Sepharose was used to pull down either the GST-tagged Lsm-FDF of Edc3 or the GST portion alone as a control. Anti-His and anti-GST antibody were used in a Western blot (WB) to detect His-Dcp2 proteins and GST-tagged proteins, respectively. The amounts corresponding to the same volume in the binding reaction were loaded for all samples: T, total; S, supernatant; and P, pellet. The asterisk indicates partial degradation products of GST-Lsm-FDF.

FIG. 3.

Region 246-315 of Dcp2 is required for decapping stimulation by Edc3 in vitro. (A) Time courses of the fraction of m⁷GDP released by decapping by recombinant purified GB1-Dcp1-His-Dcp2(1-245) and GB1-Dcp1-His-Dcp2(1-315) with and without recombinant purified His-Edc3 measured under single-turnover conditions using the 343-nt MFA2 RNA substrate. (B) Bar graphs of the observed first-order rate constants (k_obs) fitted to the time courses depicted in panel A. Error bars represent the standard error.

FIG. 4.

Mutations in region 248-300 of Dcp2 impair interaction between Dcp2 and Edc3. (A) Conservation of the region outside the catalytic core of Dcp2 across the seven Dcp2 fungal homologs (Saccharomyces cerevisiae, Candida glabrata, Ashbya gossypii, Kluveromyces waltii, Debaryomyces hansenii, Pichia stipitis, and Candida albicans). The numbers refer to residues of S. cerevisiae Dcp2. (B) Localization of Dcp2(1-300) with mutations. A wild-type (WT) strain (yRP840) expressing C-terminal GFP-tagged Dcp2(1-300) with or without the mutation (L255A K256A, R271A L273A, L255A K256A R271A L273A, E252A, D268A or E252A D268A) from plasmid pRP1892, pRP1896, pRP1906, pRP1907, pRP1908, pRP1909, or pRP1910 was cultured as described in the legend to Fig. 1B. A single focal image is shown. Bar, 5 μm. (C) Level of Dcp2 protein with the L255A K256A mutation. Wild-type cells (yRP840) expressing C-terminal GFP-tagged Dcp2(1-300) with or without the mutation from plasmid pRP1892 or pRP1896 were cultured to mid-log phase and shifted to media lacking glucose for 10 min. pRP250 was used as a control vector (no GFP). Anti-GFP antibody was used in the Western blot (WB) to detect GFP-tagged proteins. Anti-Pgk1 antibody was used to detect the endogenous Pgk1 protein as a loading control. Arrowheads indicate GFP-tagged proteins. Asterisks indicate nonspecific bands. (D) Coomassie blue-stained SDS-PAGE showing purified recombinant proteins used in the pulldown (PD) assay (left panel). An in vitro pulldown assay between purified GST-tagged Lsm-FDF domain of Edc3 and His-tagged Dcp2 (amino acids 1 to 300) or His-Dcp2(1-300) L255A K256A (right panel) is shown. The asterisk indicates partial degradation products of GST-Lsm-FDF.

FIG. 5.

Region 248-300 of Dcp2 is required for efficient degradation of RPS28B mRNA but not for degradation of YRA1 pre-mRNA. (A) The RPS28B mRNA was analyzed in strain yRP1346 (dcp2Δ) expressing the full-length (FL) or truncated Dcp2 protein (1-300, 1-247, or 1-227) from plasmid pRP1207, pRP1453, pRP1450, or pRP1449. pRP10 was used as a control vector. Cells were grown in SC medium containing 2% galactose at 30°C. The RPS28B level normalized to the SCR1 RNA level in each strain relative to the normalized mRNA level in the strain expressing full-length Dcp2 is indicated below each lane. The average and standard deviation of values obtained with three independent transformants are shown. (B) RPS28B mRNA was analyzed in yRP1346 (dcp2Δ) expressing Dcp2(1-300)-GFP or Dcp2(1-300) L255A K256A-GFP from plasmid pRP1892 or pRP1896. Cells were grown in SC medium containing 2% galactose at 30°C. The mRNA level normalized to the SCR1 RNA level relative to the normalized mRNA level in the strain expressing wild-type Dcp2 is indicated. (C) Upregulation of the RPS28B mRNA by deletion of EDC3. Cells of strain yRP1745 (edc3Δ) complemented by wild-type Edc3 expressed from plasmid pRP1432 were grown in SC medium containing 2% galactose to mid-log phase at 30°C. pRP642 was used as a control vector. The mRNA level normalized to the SCR1 RNA level relative to the normalized mRNA level in the strain expressing wild-type Edc3 is indicated. (D) Analysis of the YRA1 pre-mRNA. The same RNA samples as described for panel A were analyzed. The YRA1 pre-mRNA level normalized to the SCR1 RNA level in each strain relative to the normalized pre-mRNA level in the strain expressing full-length Dcp2 is indicated below each lane. (E) The same RNA samples as described in the legend to panel B were analyzed. The YRA1 pre-mRNA level normalized to the SCR1 RNA level in each strain relative to the normalized pre-mRNA level in the strain expressing wild-type Dcp2 is indicated below each lane. (F) The same RNA samples as described for panel C were analyzed. The YRA1 pre-mRNA level normalized to the SCR1 RNA level in each strain relative to the normalized pre-mRNA level in the strain expressing wild-type Edc3 is indicated below each lane.

FIG. 6.

Region 248-300 of Dcp2 promotes decapping in vivo. (A) Deletion of region 248-300 causes a subtle but consistent growth defect. The growth phenotype of yRP1346 (dcp2Δ) expressing the full-length (FL) or truncated Dcp2 protein (1-300, 1-247, or 1-227) from plasmid pRP1207, pRP1453, pRP1450, or pRP1449 was analyzed by serial dilution spotting. pRP10 was used as a control vector. Cells were incubated on SC medium containing 2% glucose for 2 days at 30°C. (B) Region 248-300 of Dcp2 promotes decapping in vivo. Decay intermediates (pG) of the MFA2pG reporter mRNA were analyzed in the same strains described in the legend to Fig. 5A. Cells were grown in SC medium containing 2% galactose at 30°C to express the MFA2pG mRNA from the GAL promoter. “FL” and “pG” represent the full-length mRNA and the poly(G) decay intermediate, respectively. The ratio of the full-length mRNA to the poly(G) decay intermediate in each strain relative to the ratio in the strain expressing full-length Dcp2 is indicated below each lane. The average and standard deviation of values obtained with three independent transformants are shown. The SCR1 RNA was detected as a loading control. (C) Deletion of EDC3 does not affect the MFA2pG mRNA. The same RNA samples as those described in the legend to Fig. 5C were analyzed. The ratio of full-length mRNA to the poly(G) decay intermediate in each strain relative to the ratio in the strain expressing wild-type Edc3 is indicated below each lane. (D) The LAKA mutation does not affect the MFA2pG mRNA. The same RNA samples as those described in the legend to Fig. 5B were analyzed. The ratio of the full-length mRNA to the poly(G) decay intermediate in each strain relative to the ratio in the strain expressing wild-type Dcp2 is indicated below each lane.

FIG. 7.

Region 248-300 of Dcp2 is dispensable for NMD. The same RNA samples as those described in the legend to Fig. 5A were analyzed. To assess relative accumulation, the CYH2 pre-mRNA/mRNA ratio was normalized to the ratio in the dcp2Δ strain expressing the full-length Dcp2 protein. Values represent the average and standard deviation of fold changes obtained with three independent transformants.