Translation of ASH1 mRNA is repressed by Puf6p-Fun12p/eIF5B interaction and released by CK2 phosphorylation.
Translational repression during mRNA transport is essential for spatial restriction of protein production. In the yeast Saccharomyces cerevisae, silencing of ASH1 mRNA before it is localized to the bud cortex in late anaphase is critical for asymmetric segregation of Ash1p to the daughter cell nucleus. Puf6p, an ASH1 mRNA-binding protein, has been implicated in this process as a translational repressor, but the underlying mechanism is unknown. Here, we used yeast extract-based in vitro translation assays, which recapitulate translation and phosphorylation, to characterize the mechanism of Puf6p-mediated translational regulation. We report that Puf6p interferes with the conversion of the 48S complex to the 80S complex during initiation, and this repression by Puf6p is mediated through the general translation factor eIF5B (Fun12p in S. cerevisiae). Puf6p interacts with Fun12p via the PUF domain, and this interaction is RNA-dependent and essential for translational repression by Puf6p. This repression is relieved by phosphorylation of the N-terminal region of Puf6p mediated by protein kinase CK2 (casein kinase II). Inhibition of phosphorylation at Ser31, Ser34, and Ser35 of Puf6p increases its translational repression and results in ASH1 mRNA delocalization. Our results indicate that Puf6p suppresses the translation initiation of ASH1 mRNA via interaction with Fun12p during its transport, and this repression can be released by CK2 phosphorylation in the N-terminal region of Puf6p when the mRNA reaches the bud tip.
Deng
Yingfeng
Y
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA;
Singer
Robert H
RH
Gu
Wei
W
D047390:Casein Kinase II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D010766:Phosphorylation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
C098733:ASH1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors; C451460:FUN12 protein, S cerevisiae; C487800:Puf6 protein, S cerevisiae; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; C451459:eukaryotic initiation factor-5B; D047390:Casein Kinase II