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

Département de Biochimie, Université de Montréal, Canada.
Molecular biology of the cell (Impact Factor: 4.47). 03/2009; 20(8):2265-75. DOI: 10.1091/mbc.E08-11-1151
Source: PubMed


The transport and localization of mRNAs results in the asymmetric synthesis of specific proteins. In yeast, the nucleocytoplasmic shuttling protein She2 binds the ASH1 mRNA and targets it for localization at the bud tip by recruiting the She3p-Myo4p complex. Although the cytoplasmic role of She2p in mRNA localization is well characterized, its nuclear function is still unclear. Here, we show that She2p contains a nonclassical nuclear localization signal (NLS) that is essential for its nuclear import via the importin alpha Srp1p. Exclusion of She2p from the nucleus by mutagenesis of its NLS leads to defective ASH1 mRNA localization and Ash1p sorting. Interestingly, these phenotypes mimic knockouts of LOC1 and PUF6, which encode for nuclear RNA-binding proteins that bind the ASH1 mRNA and control its translation. We find that She2p interacts with both Loc1p and Puf6p and that excluding She2p from the nucleus decreases this interaction. Absence of nuclear She2p disrupts the binding of Loc1p and Puf6p to the ASH1 mRNA, suggesting that nuclear import of She2p is necessary to recruit both factors to the ASH1 transcript. This study reveals that a direct coupling between localization and translation regulation factors in the nucleus is required for proper cytoplasmic localization of mRNAs.

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    • "Puf6 and Loc1 are mainly nucleolar proteins, suggesting that their interaction with ASH1 mRNA might take place in the nucleus. Indeed, it was shown that presence of She2 in the nucleus is essential for the association of Puf6 and Loc1 with ASH1 mRNA and for the translational repression of ASH1 (12,13). Both Puf6 and Loc1 are thought to be recruited post-transcriptionally during a nucleolar transit of the ASH1 mRNA mediated by She2 (12). "
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    ABSTRACT: Messenger RNA (mRNA) localization is coupled to the translational repression of transcripts during their transport. It is still unknown if this coupling depends on physical interactions between translational control and mRNA localization machineries, and how these interactions are established at the molecular level. In yeast, localization of transcripts like ASH1 to the bud depends on the RNA-binding protein She2. During its transport, ASH1 mRNA translation is repressed by Puf6. Herein, we report that She2 recruits Puf6 on ASH1 co-transcriptionally. The recruitment of Puf6 depends on prior co-transcriptional loading of Loc1, an exclusively nuclear protein. These proteins form a ternary complex, in which Loc1 bridges Puf6 to She2, that binds the ASH1 3′UTR. Using a genome-wide ChIP-chip approach, we identified over 40 novel targets of Puf6, including several bud-localized mRNAs. Interestingly, the co-transcriptional recruitment of Puf6 on genes coding for these bud-localized mRNAs is also She2- and Loc1-dependent. Our results suggest a coordinated assembly of localization and translational control machineries on localized mRNAs during transcription, and underline the importance of co-transcriptional events in establishing the cytoplasmic fate of mRNAs.
    Full-text · Article · Jul 2014 · Nucleic Acids Research
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    • "It has become clear that many mRNAs are earmarked for specific compartments from their birth in the nucleus, as all major nuclear events—transcription, pre-mRNA splicing and nuclear export—deposit specific components to an mRNP, which affect its localization (Erickson and Lykke-Andersen, 2011). In budding yeast, restricted localization of Ash1 mRNAs to the bud tip of the daughter cell ensures its expression there, and the disruption of Ash1 mRNA interaction with She2p, an RBP that shuttles between the nucleus and cytoplasm, specifically in the nucleus results in diffuse localization of Ash1 mRNA throughout the whole bud and impaired sorting of Ash1 protein (Shen et al., 2009). In addition, the nuclear pre-mRNA cap-binding complex protein CBP80 is bound to dendritically localized mRNPs, indicating that dendritically targeted mRNPs are assembled in the nucleus and transported in a translationally repressed form (di Penta et al., 2009). "
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    ABSTRACT: The subcellular position of a protein is a key determinant of its function. Mounting evidence indicates that RNA localization, where specific mRNAs are transported subcellularly and subsequently translated in response to localized signals, is an evolutionarily conserved mechanism to control protein localization. On-site synthesis confers novel signaling properties to a protein and helps to maintain local proteome homeostasis. Local translation plays particularly important roles in distal neuronal compartments, and dysregulated RNA localization and translation cause defects in neuronal wiring and survival. Here, we discuss key findings in this area and possible implications of this adaptable and swift mechanism for spatial control of gene function.
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    • "Thus, yeast cells display polarity only during specific stages of their life cycle [1]. Recent developments in elucidating the roles of the cytoskeleton during polarity establishment have been reported [1,53,54] but, in addition to the cytoskeleton, the nuclear transport machinery is relevant, since it plays a key role in the correct nucleocytoplasmic transport of asymmetrically distributed mRNAs [55]. However, signal transition between the cytoplasm and the nucleus in yeast seems less complex than in other cell types. "
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