Mutational analysis of Stm1. (A) Schematic of the Stm1 protein, its known motifs, and the positions of the deletions generated in this study. (*) The mutations that showed in vivo phenotypes. (B) Stm1 is a highly conserved protein within the fungal genomes. Shown is the protein sequence alignment of Stm1 between closely related Saccharomyces species. (Red boxes) Locations of the mutations that displayed in vivo phenotypes.

Stm1 functions as a translation repressor. (A) Mutational analysis of Stm1. In vivo phenotypes of overexpressing the relevant STM1 mutations in pat1Δ and dhh1Δ strains. (B) Western analysis showing the expression levels of the deletion mutants of STM1 using a rabbit antibody generated against Stm1 produced in E. coli. (C) Dose-dependent repression of translation by GST Stm1 in vitro in yeast extracts. (D) The percentage luciferase mRNA remaining during the reaction at 0, 20, and 50 min normalized to 25S rRNA levels. The amount of mRNA remaining during the time course of reaction in the presence of buffer (open triangle), GST (open square), and GST Stm1 (open circle) is also plotted using a logarithmic scale. (E) Comparison of translation repression abilities of wild-type (solid black bar) and two mutant Stm1Δ67-74 (dotted bar) and Δ240–244 (grid bar) proteins in vitro. (F) Stm1 can repress translation in vitro independent of the tag used. Shown is the effect of GST (solid black bar) and His-tagged Stm1 (diamond bar) on the translation of luciferase mRNA.

Stm1's translation repression activity is independent of Cap or poly(A) tail or eEF3. (A) Shown is the effect of GST (gray bars) or GST-Stm1 (black bars) on the translation of luciferase mRNA with or without methyl G7 cap and/or poly(A) tail. (B) Stm1 is able to repress translation in reticulocyte lysates system where eEF3 is absent. Dose-dependent repression of translation by Stm1 in vitro in yeast extracts (solid black bars) and reticulocyte lysates (dashed bars).

Stm1 can stall 80S ribosome on mRNAs. (A) Sedimentation of radiolabeled MFA2 mRNA in sucrose gradients, when translation reactions are assembled with GST (blue curve), GST-Stm1 (green curve), GMP-PNP (purple curve), or cycloheximide (pink curve). The location of the 48S, 80S, and Stm1 complexes is indicated in the figures. (B) Comparison of 80S complex formation between GST (gray curve), GST-Stm1 (dark green curve), and GST-Stm1Δ67-74 (light green curve) recombinant proteins. (C) Stm1 complex formation depends on 60S subunit joining. Shown is the sedimentation of radiolabeled MFA2 mRNA in sucrose gradients, when translation reactions are assembled with GST-Stm1 alone (green curve) or GST-Stm1 in combination with GMP-PNP (purple curve). (D) STM1 shows genetic interaction with DOM34. Stm1 was overexpressed using a 2μ plasmid in wild-type and dom34Δ strains. Growth at 16°C was monitored over a period of 5 d.