Translation of ASH1 mRNA is repressed by Puf6p–Fun12p/eIF5B interaction and released by CK2 phosphorylation

Abstract

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). RNA localization is a fundamental mechanism to restrict protein expression to a specific region in the cell, vital to the establishment of cellular polarity and determination of cell fate (St Johnston 2005;Corral-Debrinski 2007;Du et al. 2007). In budding yeast Saccharomyces cerevisiae, ASH1 mRNA localization is required for mating-type switching. The ASH1 transcripts are localized at the bud cortex in late anaphase, which confines the Ash1 protein to the daughter cell nucleus (Long et al. 1997;Takizawa et al. 1997). ASH1 mRNA localization is achieved by active transport along actin bundles (Long et al. 1997;Takizawa et al. 1997) by a core localization complex (the "locasome") consisting of proteins She1/ Myo4, She2, and She3 (Chartrand et al. 2001;Kwon and Schnapp 2001;Darzacq et al. 2003). She2p is the primary RNA-binding protein that recognizes four cis localization elements (E1, E2A, E2B, and E3) on the ASH1 transcript (Chartrand et al. 1999). She2p recruits Myo4p, a type V myosin, to the ASH1 mRNA via the adaptor protein She3p (Bohl et al. 2000;Long et al. 2000;. ASH1 mRNA localization in budding yeast serves as a model to study RNA transport and localization in mammals and other species (Darzacq et al. 2003;St Johnston 2005). Puf6p interacts withTo achieve spatial and temporal regulation of ASH1 expression, translational repression is coordinated during RNA transport to prevent premature protein synthesis. Both cis-and trans-factors have been shown to play critical roles in translational repression during ASH1 mRNA transport. There are four elements in the coding region of ASH1 mRNA that have been proposed to slow down translation during mRNA transport and prevent premature translation of ASH1 (Chartrand et al. 1999(Chartrand et al. , 2002. Two RNA-binding proteins, Khd1p and Puf6p, have been identified that are required for the localization and translation of ASH1 mRNA (Irie et al. 2002;Gu et al. 2004;Paquin et al. 2007). Release of translational repression is needed once ASH1 mRNA localizes and has been implicated in proper ASH1 mRNA anchoring at the bud

Pumilio Puf domain RNA-binding proteins in Arabidopsis

“…Nucleolar localized Puf proteins have also been reported in Saccharomyces cerevisae (ScPuf6 and ScNop9), 10,11,30 and Trypanosoma brucei (TbPUF7). 29 Most of the nucleolar Puf proteins are associated with rRNA processing and ribosome biogenesis.…”

“…8,9 Similarly, yeast Puf6p represses the translation of Ash1 mRNA during localization to the distal tip of the budding cell. 10,11 Translational repression of Ash1 mRNA by Puf6p is inhibited by eIF-5B, 11 which is responsible for the conversion of 48S to the 60S ribosomal subunit. 12 This suggests that RNA transport is tightly linked to ribosome maturation and assembly.…”

Nuclear Shuttling of She2p Couples ASH1 mRNA Localization to its Translational Repression by Recruiting Loc1p and Puf6p

“…Two other nuclear RNA-binding proteins are known to play a role in ASH1 mRNA localization and Ash1p sorting: Puf6p and Loc1p. Puf6p is predominantly nucleolar, binds the localization element E3 in the 3ЈUTR of ASH1 and is involved in the translational repression of this transcript (Gu et al, 2004;Deng et al, 2008). Its deletion results in defects in both ASH1 mRNA localization and Ash1p asymmetric distribution.…”

“…She2p also interacts with the transport machinery, constituted of the type V myosin Myo4p, via the bridging protein She3p (Bohl et al, 2000;Long et al, 2000;Takizawa and Vale, 2000). During its transport to the bud tip, the ASH1 mRNA is translationally repressed by the RNA-binding proteins Khd1p (Irie et al, 2002) and Puf6p (Gu et al, 2004), and phosphorylation of these factors at the bud tip promotes the local synthesis of the Ash1 protein (Paquin et al, 2007;Deng et al, 2008).…”

The cytoplasmic fate of an mRNP is determined cotranscriptionally: exception or rule?: Figure 1.

“…Furthermore, when the N terminus of She3p, an adapter of She2 interaction with Myo4p, was fused with She2p to prevent its nuclear import, ASH1 mRNAs could localize to the bud tip as in wild-type cells, but, remarkably, failed to redistribute Ash1p asymmetrically (Du et al 2008). While in the nucleus, She2p also recruits the Loc1p and Puf6p proteins that are involved in the translational repression of the ASH1 transcript (Deng et al 2008;Shen et al 2009). Alhough cytoplasmic association of ASH1 mRNA with She2p does occur (and these mRNAs can localize to the bud tip with a reduced efficiency), nuclear mRNA processing mediated by the activity of nuclear She2p appears to be critical for coordination of cellular localization and appropriate Ash1p translation.…”