Possible models for the function of Stm1. (A) Stm1 physically recruits Dhh1 to the translating ribosomes, and this allows Dhh1 to repress translation. (B) Stm1 stalls ribosomes at some stage of translation and represses translation, and this triggers the function of Dhh1.

Stm1 is a high-copy suppressor of pat1Δ^ts. Stm1 was overexpressed using a 2μ overexpression plasmid in wild-type (yRP840) and pat1Δ (yRP1372) strain backgrounds and the growth was monitored under the conditions mentioned. Overexpression of Stm1 allowed the pat1Δ strains to survive at nonpermissive temperatures.

Stm1 has genetic interactions with the general translation repressors Dhh1 and Pat1. Stm1 was overexpressed using a 2μ plasmid in wild type (yRP2065) and dhh1Δ (yRP2066) (A) and dhh1Δ pat1Δ (yRP2323) (B) double-deletion strain backgrounds. Growth was monitored over a period of 3 days. Overexpression of Stm1 has a deleterious effect on growth in dhh1Δ and dhh1Δ pat1Δ strain backgrounds. (C) Dhh1 was overexpressed using GAL10 promoter in wild-type (yRP2065) and stm1Δ (yRP1437) strains. Growth was monitored over a period of 3 days. Deletion of STM1 prevented the overexpression lethality of Dhh1.

Stm1 promotes the accumulation of Dhh1 in P-bodies. (A) Wild-type (yRP2065) and stm1Δ (yRP1437) strains were cotransformed with GFP-tagged plasmid of Dhh1 and mCherry-tagged plasmid of Edc3, and the localization of these proteins was observed by microscopy under glucose depletion or high cell densities (see materials and methods). Deletion of STM1 affects the accumulation of Dhh1 in P-bodies under high cell densities. (B) Whole-cell extracts were prepared from wild-type (yRP2065) and stm1Δ (yRP1437) strains carrying Dhh1-GFP plasmid and Western analysis was performed using anti-GFP antibody (Covance). Equal amounts of total protein were loaded on the gel.

Overexpression of Stm1 prolongs the half-life of EDC1 mRNA in a dhh1Δ strain. The decay of MFA2pG, PGK1pG, and EDC1pG reporters following transcriptional repression in dhh1Δ (yRP2066) strain overexpressing Stm1 or empty vector under a galactose promoter is shown. Time points indicated represent minutes after transcriptional repression. SCR1 was used as a loading control. MFA2pG, PGK1pG, and EDC1pG transcripts were detected using oligonucleotide probes oRP140, oRP141, and oRP1211, respectively, while SCR1 was detected using oRP100. A graphical representation of the data is shown by plotting the percentage of mRNA remaining as a function of time. Best-fit lines were determined by exponential curve fitting using the graphing software Deltagraph. Experiments were done in triplicate and error bars denote the standard deviation.

Stm1 deletion stabilizes EDC1 and COX17 mRNAs. (A) Decay of the reporters MFA2pG and PGK1pG following transcriptional repression in wild-type (yRP2065) and stm1Δ (yRP1437) strains. MFA2pG and PGK1pG were detected using oligonucleotide probes oRP140 and oRP141, respectively. SCR1 was detected using oRP100. (B) The decay of the EDC1pG reporter following transcriptional repression in wild-type (yRP2065), stm1Δ (yRP1437), and dhh1Δ (yRP2066) strains. Oligonucleotide probes oRP1211 and oRP100 were used to detect EDC1 and SCR1 transcripts, respectively. (C) The decay of the COX17 mRNA following transcriptional repression in wild-type (yRP2065), stm1Δ (yRP1437), and dhh1Δ (yRP2066) strains. Oligonucleotide probes oRP1427 and oRP100 were used to detect COX17 and SCR1 transcripts, respectively. (A–C) Time points represent minutes after transcriptional repression. SCR1 was used as a loading control. Each decay experiment was done in triplicate. A graphical representation of the data is shown by plotting the percentage of mRNA remaining as a function of time. Best-fit lines were determined by exponential curve fitting using the graphing software Deltagraph. Error bars denote the calculated standard deviation.

Overexpression of Stm1 does not affect the half-life of mRNAs in a pat1Δ strain. The decay of MFA2pG, PGK1pG, and EDC1pG reporters following transcriptional repression in the pat1Δ (yRP1372) strain overexpressing Stm1 or empty vector under a GAL10 promoter is shown. Time points indicated represent minutes after transcriptional repression. SCR1 was used as a loading control. MFA2pG, PGK1pG, and EDC1pG transcripts were detected using oligonucleotide probes oRP140, oRP141, and oRP1211, respectively, while SCR1 was detected using oRP100. A graphical representation of the data is shown by plotting the percentage of mRNA remaining as a function of time. Best-fit lines were determined by exponential curve fitting using the graphing software Deltagraph. Experiments were done in triplicate and error bars denote the standard deviation.