title: Nucleocytoplasmic shuttling of Ssd1 defines the destiny of its bound mRNAs.
abstract: Mechanisms that control mRNA metabolism are critical for cell function, development and stress response. The Saccharomyces cerevisiae mRNA-binding protein Ssd1 has been implicated in mRNA processing, ageing, stress response and maintenance of cell integrity. Ssd1 is a substrate of the LATS/NDR tumour suppressor orthologue Cbk1 kinase. Previous data indicate that Ssd1 localizes to the cytoplasm; however, biochemical interactions suggest that Ssd1 at least transiently localizes to the nucleus. We therefore explored whether nuclear localization is important for Ssd1 cytoplasmic functions. We identified a functional NLS in the N-terminal domain of Ssd1. An Ssd1-derived NLS-GFP fusion protein and several C-terminally truncated Ssd1 proteins, which presumably lack nuclear export sequences, accumulate in the nucleus. Alanine substitution of the Ssd1 NLS prevents Ssd1 nuclear entry, mRNA binding and disrupts Srl1 mRNA localization. Moreover, Ssd1-NLS mutations abolish Ssd1 toxicity in the absence of Cbk1 phosphorylation and cause Ssd1 to localize prominently to cytoplasmic puncta. These data indicate that nuclear shuttling is critical for Ssd1 mRNA binding and Ssd1-mRNA localization in the cytoplasm. Collectively these data support the model that Ssd1 functions analogously to hnRNPs, which bind mRNA co-transcriptionally, are exported to the cytoplasm and target mRNAs to sites of localized translation and P-bodies.
authors.affiliation: Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Kurischko
authors.forename: Venkata K
authors.initials: VK
authors.lastname: Kuravi
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Herbert
authors.forename: Francis C
authors.initials: FC
authors.lastname: Luca
mesh_terms: D021581:Active Transport, Cell Nucleus; D019677:Artificial Gene Fusion; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D019913:Nuclear Localization Signals; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion
chemical_list: D050505:Mutant Proteins; D019913:Nuclear Localization Signals; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C485219:Ssd1 protein, S cerevisiae; D049452:Green Fluorescent Proteins