Nucleocytoplasmic shuttling of Ssd1 defines the destiny of its bound mRNAs

Abstract

SUMMARY Mechanisms that control mRNA metabolism are critical for cell function, development and stress response. The S. cerevisiae mRNA-binding protein Ssd1 has been implicated in mRNA processing, aging, stress response and maintenance of cell integrity. Ssd1 is a substrate of the LATS/NDR tumor 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.

Mitotic Exit and Separation of Mother and Daughter Cells

“…Ssd1 can localize to cytoplasmic granules known as P-bodies, which are associated with translational suppression (Balagopal and Parker 2009;Jansen et al 2009;Kurischko et al 2011a), but Ssd1 can clearly carry out its translational repression function in cells lacking these structures. Ssd1 likely assembles a translationally silent mRNP in the nucleus: it associates with unspliced and centromeric mRNAs, interacts with the phosphorylated Cterminal tail of RNA polymerase II, and requires a sequence that can function as an NLS (Phatnani et al 2004;Jansen et al 2006;Hogan et al 2008;Kurischko et al 2011b). The mechanisms and precise function of Ssd1's control of translation remain poorly understood, but since RAM network proteins concentrate at the bud neck during cell separation, the Ssd1 circuit could allow the cytokinesis site to exert posttranscriptional control over the expression of proteins needed at different stages of the process (Nelson et al 2003;Jansen et al 2006;Kurischko et al 2011a).…”

The RAM Network in Pathogenic Fungi

“…The RAM network has been most extensively studied in S. cerevisiae and has been implicated in the regulation of a number of cellular processes, including mother-daughter cell separation (3,18,65,69), daughter cell-specific gene transcription (12,81), maintenance of cell wall integrity (18,50,77), cell cycle progression (4,7,70), mating projection formation and mating efficiency (3,5,65,81), polarized growth (18,65,69,81), and stress signaling (47). As detailed in this review, many of the functions of the RAM pathway characterized in S. cerevisiae are conserved in other model and pathogenic fungi.…”

“…However, the physiologic substrates of ScCbk1 are only beginning to be identified. To date, only two ScCbk1 substrates have been well characterized: the zinc finger transcription factor ScAce2 (8,15) and the mRNA binding protein ScSsd1 (38,49,50). There is additional evidence suggesting that ScCbk1 may also regulate several other proteins, including the mRNA-binding protein ScPuf5/Mpt5 (5), the RAB GEF ScSec2 (51), and the cell wall integrity (CWI) pathway MAPKK ScBck2 (47).…”

“…ScCbk1 binds to and phosphorylates ScSsd1, thereby influencing the subcellular distribution and translation of ScSsd1-mRNA complexes (37,48,49). ScCbk1 inhibition causes ScSsd1-mRNA complexes to localize to P bodies and stress granules (49,50), where the translation of the ScSsd1-targeted mRNAs is repressed. ScCbk1 inhibition also decreases the amount of ScSsd1-associated mRNA bound to polysomes (37).…”

“…Recent data also suggest that ScCbk1 directly regulates ScSsd1 localization (49,50). For example, an S. cerevisiae ssd1 mutant lacking multiple ScCbk1 phosphorylation sites constitutively localizes to P bodies and, like Sccbk1 mutants, are toxic to cells (49).…”