Characterization of PUF6 in regulated translation of localized RNA
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RNA localization is a fundamental mechanism of cell fate determination. ASH1 mRNA is a cell fate determinant in Saccharomyces cerevisiae. It is localized at the bud tip during mitosis, which restricts Ash1p to the daughter cell, allowing only the mother cell to switch mating type. The entwined pathways of RNA transport and translation are the two key determinants in the spatiotemporal control of the polarity of ASH1 mRNA and protein. ASH1 mRNA is assumed to be translationally dormant during transport and translated once localized. However it remains unclear how translational control is coordinated with RNA localization. To identify regulatory factors functioning in the linkage of these processes, a biochemical purification of ASH1 mRNP was performed. Puf6p, a PUF family member, was identified by this approach. Our studies reveal that Puf6p is an important regulator of ASH1 mRNA localization and translation. Loss of Puf6p decreases ASH1 mRNA localization and Ashlp asymmetric distribution. It also results in an increase of ASH1 protein levels with no effect on the abundance of the ASH1 mRNA. Puf6p binds to the consensus PUF protein binding site in the 3'UTR of ASH1 mRNA. Yeast extract-based in vitro translation assays suggest that Puf6p blocks 60S joining to repress translation initiation. One target of the block by Puf6p is Fun12p, the yeast eIF5B, a general translation factor assisting 60S joining. Fun12p is specifically required for ASH1 mRNA translation. Puf6p interacts with Fun12p through the PUF domain. However, both the PUF domain and the NH2 terminal region are necessary for Puf6p repressor activity. The PUF domain binds to RNA and the NH2 terminal region is involved in regulating Puf6p activity. CK2, a protein kinase, phosphorylates the NH2 terminal region of Puf6p and releases translational repression by Puf6p through decreasing the RNA binding activity of Puf6p. The double staining of ASH1 mRNA and protein suggests that translational control is required for both localized ASH1 mRNAs and those in transit. This study demonstrates that Puf6p regulates ASH1 mRNA localization as a translational repressor and suggests how Puf6p is regulated for the spatiotemporal expression of cell fate determinant ASH1..