Abstract
Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport. The budding yeast Saccharomyces cerevisiae is well suited to studying these mechanisms because many of its transcripts are transported from the mother cell to the budding daughter cell. Here, we investigated the translational control of ASH1 mRNA after transport and localization. We show that although ASH1 transcripts were translated after they reached the bud tip, some mRNAs were bound by the RNA-binding protein Puf6 and were non-polysomal. We also found that the DEAD-box helicase Dhh1 complexed with the untranslated ASH1 mRNA and Puf6. Loss of Dhh1 affected local translation of ASH1 mRNA and resulted in delocalization of ASH1 transcript in the bud. Forcibly shifting the non-polysomal ASH1 mRNA into polysomes was associated with Dhh1 dissociation. We further demonstrated that Dhh1 is not recruited to ASH1 mRNA co-transcriptionally, suggesting that it could bind to ASH1 mRNA within the cytoplasm. Of note, Dhh1 bound to the 5'-UTR of ASH1 mRNA and inhibited its translation in vitro These results suggest that after localization to the bud tip, a portion of the localized ASH1 mRNA becomes translationally inactive because of binding of Dhh1 and Puf6 to the 5'- and 3'-UTRs of ASH1 mRNA.
Highlights
Local translation of specific mRNAs is regulated by dynamic changes in their subcellular localization, and these changes are due to complex mechanisms controlling cytoplasmic mRNA transport
We have reported previously that binding of Puf6 to the 3Ј-UTR of ASH1 mRNA repressed its translation during transport, and this repression could be released by CK2 phosphorylation in the N-terminal region of Puf6 when the mRNA was localized to the bud tip [12, 13]
Our new observations showed that translation occurred after localization, a fraction of ASH1 mRNA was still associated with Puf6 and was translationally inactive
Summary
It has been reported previously that the average time to transcribe a yeast gene is about 25–50 s/kb (14 –17), and the transport time for an ASH1 mRNA reporter (from mother to the bud tip) is about 128 s [18]. In CHX-treated cells, the binding capability of Puf to the transcripts and Dhh was dramatically reduced (supplemental Fig. S4A) This suggests that in addition to ASH1 mRNA, Dhh could be involved in the translation of other bud-localized mRNAs. Because Puf binds to the 3Ј-UTR of the ASH1 mRNA [12] and Dhh co-precipitated with Puf and ASH1 mRNA (Fig. 3), we tested whether the two proteins could interact with each other. When yeast extracts and the non-polysomal fraction 3 (Fig. 2) were treated with RNase A and RNase One, Dhh was not able to co-precipitate with Puf (Fig. 6B, bottom panels), indicating that association of the two proteins was mediated by ASH1 or perhaps the other mRNAs. We prepared yeast strains expressing ASH1 WT mRNA (5ЈUTR-ASH1ORF-3ЈUTR) and ASH1 mutant (ADH2-5ЈUTR-ASH1ORF-3ЈUTR), in which the 5Ј-UTR of ASH1 mRNA was replaced by the 5Ј-UTR of ADH2 mRNA (Fig. 6C, top panels), and performed co-IP assays using Dhh antibodies. Binding of Dhh to the 5Ј-UTR of ASH1 mRNA conferred repression of translation
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