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Loc1 and Puf6, which are localized predominantly to the nucleus, are required for the localization and translational repression of the ASH1 mRNA in the yeast, Saccharomyces cerevisiae . During its transport to the daughter cell, the ASH1 mRNA is translationally repressed via associations with She2, Loc1, and Puf6. Here, we investigated the roles of Loc1 and Puf6 in the translation of mRNAs other than that encoding ASH1 . In loc1 or puf6 deletion strains, expression of the mating-specific transcription factor, Ste12, was significantly increased at the post-transcriptional level. These phenotypes required the 5’ untranslated region (UTR) of STE12 , which carries the putative Puf6-binding sequences. The RNA helicase, Dhh1, which is a known positive regulator for the translation of STE12 mRNA, was found to be functionally connected with Loc1 and Puf6 in the context of Ste12 expression. Our results collectively show that the phosphorylation of the N-terminal Thr16 residue of Dhh1 affects the protein interactions of Dhh1 with Loc1 or Puf6, and consequently regulates Ste12 expression.

Dhh1 and Dhh1 homologues (RCK/p54/DDX6) are members of the DEAD-box protein family of RNA helicases. These proteins display conserved sequence motifs for ATPase and RNA binding activities. Dhh1 is a component of the P-bodies (processing bodies) of mRNA granules and functions as an mRNA decapping activator in Saccharomyces cerevisiae. Dhh1 also contributes to gene-specific regulation during yeast mating. The dhh1 deletion mutation results in a significant decrease in the expression of Ste12, a mating-specific transcription factor, showing severe mating defects. Here, we introduced amino-acid substitution mutations in the ATPase and RNA binding domains of Dhh1 and also constructed a deletion of 79 amino acids at the Q/P-rich C-terminal region. The mutations in ATPase A and B motif (K96R, D195A) and C-terminus deletion showed reduced levels of mating efficiency as well as Ste12 protein expression. The Q/P-rich C-terminal region of Dhh1 was dispensable for growth at nonpermissive temperature 37°C but appeared to play an important role in regulating the Ste12 protein expression and mating processes. The P-body accumulation induced by treatment with α-mating factor required ATPase, RNA-binding and the Q/P-rich C-terminal domains of Dhh1.

Translation initiation factor eIF4E forms eIF4E-eIF4G complex at the 5' cap of mRNA. This interaction can be inhibited by the family of 4E-binding proteins (4E-BP). In yeast Saccharomyces cerevisiae, two 4E-BPs, Caf20 and Eap1, compete with eIF4G for binding to eIF4E via the shared conserved interaction motif. In order to investigate the roles of Caf20 in gene-specific translational regulation and the formation of mRNA granules (P-bodies), we introduced substitution mutations, caf20-Y4A or caf20-L9A, in the eIF4E-binding motif for CAF20. Overexpression of the wild-type CAF20 showed an increased protein level of Ste12 transcription factor as well as highly developed P-body formation. However, 4E-binding site mutations of CAF20 led to a reduced number of P-body foci and decreased levels of Ste12 protein. The phenotypes of the caf20 deletion mutation were also analyzed, and we suggest that Caf20 plays a critical role in Ste12 protein expression and in the control of P-body formation.

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