RESUMEN
Eukaryotic cells control their growth and morphogenesis to maintain integrity and viability. Free-living cells are further challenged by their direct interaction with the environment and in many cases maintain a resilient cell wall to stay alive under widely varying conditions. For these organisms, stringent and highly localized control of the cell wall's remodeling and expansion is crucial for cell growth and reproduction. In the budding yeast Saccharomyces cerevisiae the RNA binding protein Ssd1 helps control cell wall remodeling by repressing translation of proteins involved in wall expansion. Ssd1 is itself negatively regulated by the highly conserved Ndr/LATS protein kinase Cbk1. We sought to identify mRNA regions that confer Ssd1-mediated translational control. After validating a GFP reporter system as a readout of Ssd1 activity we found that 3' untranslated regions of the known Ssd1 targets CTS1, SIM1 and UTH1 are sufficient for Cbk1-regulated translational control. The 5' untranslated region of UTH1 also facilitated Ssd1-mediated translational control in a heterologous context. The CTS1 and SIM1 3' untranslated regions confer Ssd1 binding, and the SIM1 3' untranslated region improves Ssd1 immunoprecipitation of the endogenous SIM1 transcript. However, SIM1's 3' untranslated region is not essential for Ssd1-regulated control of the message's translation. We propose that Ssd1 regulates translation of its target message primarily through UTRs and the SIM1 message through multiple potential points of interaction, permitting fine translational control in various contexts.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación Fúngica de la Expresión Génica , Morfogénesis/genética , Biosíntesis de Proteínas , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Pared Celular/genética , Pared Celular/metabolismo , Quitinasas/genética , Quitinasas/metabolismo , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Spatial control of gene expression, at the level of both transcription and translation, is critical for cellular differentiation [1-4]. In budding yeast, the conserved Ndr/warts kinase Cbk1 localizes to the new daughter cell, where it acts as a cell fate determinant. Cbk1 both induces a daughter-specific transcriptional program and promotes morphogenesis in a less well-defined role [5-8]. Cbk1 is essential in cells expressing functional Ssd1, an RNA-binding protein of unknown function [9-11]. We show here that Cbk1 inhibits Ssd1 in vivo. Loss of this regulation dramatically slows bud expansion, leading to highly aberrant cell wall organization at the site of cell growth. Ssd1 associates with specific mRNAs, a significant number of which encode cell wall remodeling proteins. Translation of these messages is rapidly and specifically suppressed when Cbk1 is inhibited; this suppression requires Ssd1. Transcription of several of these Ssd1-associated mRNAs is also regulated by Cbk1, indicating that the kinase controls both the transcription and translation of daughter-specific mRNAs. This work suggests a novel system by which cells coordinate localized expression of genes involved in processes critical for cell growth and division.