In N crassa a point mutation

In N. crassa, a point mutation conferring a single amino beta-Nicotinamide mononucleotide mutation (His to Arg substitution at amino acid 351 within catalytic domain VII of the kinase) of COT-1 protein resulted in the formation of a temperature sensitive strain, exhibiting severe morphological defects at temperatures higher than 32 °C (Gorovits et al., 1999, Maerz et al., 2008, Yarden et al., 1992). These are mainly characterized by a greatly reduced growth rate and excessive branching, the outcome of which is the formation of a highly restricted and dense colony. Changes in the presence of different forms of the kinase have also been observed as a consequence of the point mutation (Feldman et al., 2013, Gorovits et al., 1999, Ziv et al., 2013). Prominent changes in such mutants also include increased cell wall and septum thicknesses (Gorovits et al., 2000, Herold and Yarden, 2017). Several extragenic cot-1 (ts) suppressors have been identified. Among them, cytoplasmic dynein, dynactin, and nuclear distribution mutants of N. crassa, as well as an additional gene gul-1 (NCU01197), have been shown to have genetic interactions with cot-1 (Bruno et al., 1996, Seiler et al., 2006, Terenzi and Reissig, 1967). A mutation ingul-1partially suppresses the severity of thecot-1(ts) phenotype and is accompanied by altered gene expression of the cell wall remodeling machinery and, subsequently, cell wall composition, (Herold and Yarden, 2017). Similarly, SSD1, which is the gul-1 homologue in Saccharomyces cerevisiae and was determined to encode a translational regulator of cell wall remodeling genes, can suppress the phenotype of the yeast cot-1 homologue, CBK1 (Hogan et al., 2008, Jansen et al., 2009, Kurischko et al., 2011a). Ssd1 has been shown to be an mRNA-binding protein whose function has an impact on maintenance of cell integrity, mRNA processing, stress signaling and cellular aging (Kurischko et al., 2011b). Ssd1 was also shown to migrate between the cytoplasm and the nucleus and has been suggested to be involved in shuttling mRNAs destined for polarized localization and translation, including mRNAs encoding cell wall proteins (Hogan et al., 2008, Jansen et al., 2009, Kurischko et al., 2011a, Kurischko et al., 2011b, Kurischko and Broach, 2017, Mitchell et al., 2013, Uesono et al., 1997). Under stress conditions, the protein-RNA complex was shown to physically interact with processing bodies (P bodies) and stress granules in the nucleus (Kurischko et al., 2011a, Richardson et al., 2012, Tarassov et al., 2008, Zhang et al., 2014). These were subsequently exported to the cytoplasm for mRNA storage or decay (Kurischko and Broach, 2017). Ssd1 function has been demonstrated to be negatively regulated via phosphorylation by Cbk1 (Jansen et al., 2009, Kurischko et al., 2011a). Cbk1 inhibition and cellular stress lead to translational repression along with the localization of Ssd1 to cytoplasmic puncta (Kurischko et al., 2011a, Kurischko et al., 2011b). One explanation for the pleiotropic nature of the cot-1 (ts) mutant is the fact that COT-1 is an upstream component in a hierarchal pathway that can effect different processes in the cell. In this study, we describe the transcriptional profile of cot-1 inactivation and demonstrate that gul-1 affects transcript abundance of multiple genes in the COT-1 pathway, including processes such as cell wall remodeling, nitrogen and amino acid metabolism as well as 298 genes of yet unknown function. Furthermore, we show that GUL-1 transport in the cell is tubulin-dependent and the extent of its association with nuclei is stress-dependent.