The non-conserved C-terminal 81 residues of Dhh1p are required for its interaction with Rbp1p. (A) Schematic representation of the Dhh1p protein domain structure and C-terminal truncated variants used in yeast two hybrid and in vitro pull-down assays. (B) Dhh1p interacts with Rbp1p through C-terminal 81 residues in yeast two-hybrid assay. YEM1α cells expressing LexA- and Gal4AD-fusion proteins as indicated were used to perform β-galactosidase reporter assay. Immunoblotting shows the expression level of indicated proteins. (C) Dhh1p directly binds Rbp1p. In vitro pull-down assay between purified GST-tagged Rbp1p-CF and His-tagged Dhh1p or Dhh1p-dC81. Anti-His and anti-GST antibodies were used to detect indicated fusion proteins in western blotting. The same amounts of fusion proteins in binding reaction were loaded as input controls.

Dhh1p-dC81 complements the temperature-sensitive phonotype of dhh1Δ strain and efficiently accumulates to P-bodies. (A) Dhh1p-dC81 complements the growth defect of dhh1Δ mutant strain. BY4741 wild-type or dhh1Δ strain expressing Dhh1p, Dhh1p-dC81, or Dhh1p-dC106 from CEN or 2 µ plasmid was grown to log phase, serially diluted and spotted on two plates, which were then separately incubated at 30°C or 37°C for 2 days. (B) Isometric expression level of various Dhh1p mutants. Immunoblotting confirmed the protein expression of various Dhh1p mutants in panel A. (C) Dhh1p-dC81-GFP localizes to P-bodies under glucose deprivation. BY4741 wild-type cells chromosomally expressing C-terminal tagged GFP-fusion Dhh1p, Dhh1p-dC81 or Dhh1p-dC106 with the P-bodies marker Dcp2p-mCherry were grown in YPD medium to log phase and then shifted to medium lacking glucose for 20 min. Co-localization of fluorescence fusion proteins was quantitated as described in ‘Materials and Methods’ section (D) Isometric expression level of various GFP-fusion Dhh1p mutants. Immunoblotting confirms the protein expression of GFP-fusion Dhh1p, Dhh1p-dC81 and Dhh1p-dC106.

Mammalian DDX6 fused with the non-conserved C-terminal 85 residues of Dhh1p gains function to confer specific regulation for Rbp1p-mediated porin mRNA. (A) Schematic representation of the Dhh1p, DDX6 and DDX6 chimera protein domain structure. Portion of DDX6 chimera protein, DDX6-C85, derived from Dhh1p are shown. (B) DDX6 and its chimera protein can complement the growth defect of dhh1Δ strain. BY4741dhh1Δ strain expressing Dhh1p, DDX6 or DDX6-C85 was used to perform growth assays as described in Figure 4A. Protein expression level was shown by western blotting. (C) Steady-state POR1 mRNA levels in dhh1Δ strain expressing DDX6 or DDX6-C85. BY4741dhh1Δ strain expressing DDX6 or DDX6-C85 from 2-µ plasmid was grown to log phase and total RNA were extracted and analyzed. The levels of the mRNAs were quantitated as described in ‘Materials and Methods’ section. (D) DDX6 chimera protein DDX6-C85, but not DDX6, interacts with Rbp1p in yeast two-hybrid assay. YEM1α cells expressing LexA- and Gal4AD-fusion proteins as indicated were used to perform β-galactosidase reporter assays. Immunoblotting shows the expression level of indicated proteins. (E) DDX6-C85 associates with POR1 mRNA in vivo through interaction with Rbp1p. BY4741dhh1Δ strain expressing Dhh1p-2HA, DDX6-2HA or DDX6-C85-2HA were used to perform immunoprecipitation and RT–PCR as described in ‘Materials and Methods’ section. The level of RT–PCR products of POR1 mRNA was quantitated as described in ‘Materials and Methods’ section.

Rbp1p requires Dhh1p to elicit porin mRNA decay. (A) The steady-state POR1 mRNA levels in dhh1Δ, pat1Δ, lsm1Δ, and xrn1Δ mutant cells overexpressing Rbp1p. BY4741 wild-type or indicated mutant strains transformed with pVT101U or pVT101U-HA-RBP1 plasmid were grown to log phase. Total RNA of these cells was extracted and analyzed by northern blotting. (B) Steady-state levels of POR1 mRNA in wild-type, rbp1Δ, dhh1Δ, and rbp1Δ dhh1Δ mutant cells. BY4741 strains were grown to log phase. Total RNA samples were isolated and analyzed by northern blot. (C) Effect of Dhh1p overexpression on POR1 mRNA steady-state level. BY4741 wild-type strain were transformed with pVT101U plus YEplac181, pVT101U plus YEplac181-HA-RBP1, pVT101U-DHH1 plus YEplac181, or pVT101U-DHH1 plus YEplac181-HA-RBP1 and grown to log phase. Total RNA samples were isolated and analyzed by northern blot. Total proteins were precipitated by TCA and analyzed by western blotting. (D) POR1 mRNA turnover in dhh1Δ strain overexpressing Rbp1p. YTC345 wild-type or dhh1Δ strain transformed with either pVT101U, or pVT101U-HA-RBP1 plasmid was grown to log phase at 25°C and then shifted to 37°C. Total RNA was extracted at each indicated time point after temperature shift and analyzed. t_1/2 indicated the half-life of POR1 mRNA. Graphical representation of the POR1 mRNA decay kinetics is shown. The levels of the mRNAs in (B–D) were quantitated as described in ‘Materials and Methods’ section. Mean values ± SD are shown.

The C-terminal 81 residues of Dhh1p are required for Rbp1p-mediated POR1 mRNA decay. (A) Steady-state POR1 mRNA levels in dhh1Δ strain expressing Dhh1p, Dhh1p-dC81, or Dhh1p-dC106. BY4741dhh1Δ strain carrying YCplac111, YCplac111-DHH1, YCplac111-DHH1-dC81, or YCplac111-DHH1-dC106 was grown to log phase and total RNA were extracted and analyzed by northern blotting. Total proteins were precipitated by TCA and analyzed by western blotting. (B) Turnover of POR1 mRNA in dhh1Δ strain expressing Dhh1p or Dhh1p-dC81. YTC345dhh1Δ strain carrying YCplac111, YCplac111-DHH1, or YCplac111-DHH1-dC81 was grown to log phase at 25°C and then shifted to 37°C. Total RNA was extracted at each indicated time point after temperature shift and analyzed. t_1/2 indicated the half-life of POR1 mRNA. Graphical representation of the POR1 mRNA decay kinetics is shown. The levels of the mRNAs were quantitated as described in ‘Materials and Methods’ section. Mean values ± SD are shown.

Dhh1p associated with POR1 mRNA in vivo requires its interaction with Rbp1p. (A) Dhh1p association with POR1 mRNA depends on the presence of Rbp1p. Dhh1p chromosomally tagged with three HA epitopes in wild-type or rbp1Δ strain was immunoprecipitated and used to perform RT–PCR as described in ‘Materials and Methods’ section. (B) Dhh1p associates with POR1 mRNA through C-terminal 81 amino acids. Dhh1p or Dhh1-dC81 chromosomally tagged with three HA epitopes was immunoprecipitated and used to perform RT–PCR as described in ‘Materials and Methods’ section. (C) Dhh1p association with POR1 mRNA in rbp1Δ mutant was rescued by Rbp1p, but not Rbp1p-dNMP or Rbp1p-rrm1. DHH1-3HA rbp1Δ strains carrying pVT101U, pVT101U-RBP1, pVT101U-RBP1-dNMP, or pVT101U-RBP1-rrm1 were grown and then used to perform immunoprecipitation and RT–PCR as described in ‘Materials and Methods’ section. (D) Rbp1p specifically associates with POR1 mRNA independent of Dhh1p. Rbp1p chromosomally tagged with three HA epitopes in wild-type or dhh1Δ strain was immunoprecipitated and used to perform RT–PCR as described in ‘Materials and Methods’ section. The presence of Dhh1p or Rbp1p was shown in western blots. The level of the RT–PCR products of POR1 mRNA was quantitated as described in ‘Materials and Methods’ section. Mean values ± SD are shown.