BackgroundSaccharomyces cerevisiae myosin type II-deficient (myo1Δ) strains remain viable and divide, despite the absence of a cytokinetic ring, by activation of the PKC1-dependent cell wall integrity pathway (CWIP). Since the myo1Δ transcriptional fingerprint is a subset of the CWIP fingerprint, the myo1Δ strain may provide a simplified paradigm for cell wall stress survival.ResultsTo explore the post-transcriptional regulation of the myo1Δ stress response, 1,301 differentially regulated ribosome-bound mRNAs were identified by microarray analysis of which 204 were co-regulated by transcription and translation. Four categories of mRNA were significantly affected - protein biosynthesis, metabolism, carbohydrate metabolism, and unknown functions. Nine genes of the 20 CWIP fingerprint genes were post-transcriptionally regulated. Down and up regulation of selected ribosomal protein and cell wall biosynthesis mRNAs was validated by their distribution in polysomes from wild type and myo1Δ strains. Western blot analysis revealed accumulation of the phosphorylated form of eukaryotic translation initiation factor 2 (eIF2α-P) and a reduction in the steady state levels of the translation initiation factor eIF4Gp in myo1Δ strains. Deletion of GCN2 in myo1Δ abolished eIF2αp phosphorylation, and showed a severe growth defect. The presence of P-bodies in myo1Δ strains suggests that the process of mRNA sequestration is active, however, the three representative down regulated RP mRNAs, RPS8A, RPL3 and RPL7B were present at equivalent levels in Dcp2p-mCh-positive immunoprecipitated fractions from myo1Δ and wild type cells. These same RP mRNAs were also selectively co-precipitated with eIF2α-P in myo1Δ strains.ConclusionsQuantitative analysis of ribosome-associated mRNAs and their polyribosome distributions suggests selective regulation of mRNA translation efficiency in myo1Δ strains. Inhibition of translation initiation factor eIF2α (eIF2α-P) in these strains was by Gcn2p-dependent phosphorylation. The increase in the levels of eIF2α-P; the genetic interaction between GCN2 and MYO1; and the reduced levels of eIF4Gp suggest that other signaling pathways, in addition to the CWIP, may be important for myo1Δ strain survival. Selective co-immunoprecipitation of RP mRNAs with eIF2α-P in myo1Δ strains suggests a novel mode of translational regulation. These results indicate that post-transcriptional control is important in the myo1Δ stress response and possibly other stresses in yeast.

Previous analysis of global mRNA expression in Saccharomyces cerevisiae myosin type II deficient strains (myo1Δ) revealed 547 genes that are regulated at the transcriptional level. The objective of this study is to explore the post‐transcriptional regulation of mRNAs in myo1Δ. To assess the mode of regulation of ribosomal protein (RP) mRNAs, we analyzed the polysomal distribution of three representative RP mRNAs in sucrose gradient fractions of myo1Δ and wild‐type (wt) cells. Our results showed that RPS8A, RPL7B and RPL3 mRNAs were recruited from heavy to lighter polyribosome fractions in myo1Δ. Furthermore, western blot analysis revealed accumulation of the phosphorylated form of eukaryotic initiation factor 2 (eIF2α‐P) in myo1Δ and RPS8A and RPL7B mRNAs were co‐precipitated with immunoprecipitated eIF2α‐P, suggesting a direct association between eIF2α‐P and translationally regulated RP mRNAs. Treatment of myo1Δ with rapamycin did not increase the levels of eIF2α‐P yet, a gcn2Δmyo1Δ strain exhibited a synthetic growth defect. Reduced steady state levels of the initiation factor eIF4G was also observed supporting regulation of translation by TOR pathway. These findings support the conclusion that post‐transcriptional control by translation inhibition is a key component of the stress response in yeast. Supported by NIGMS/NIAID (5SC1AI081658‐02), NCRR‐RCMI(G12RR03051) and MBRS‐RISE (R25GM061838).