RESUMO
mRNA localization may be an important determinant for protein localization. We describe a simple PCR-based genomic-tagging strategy (m-TAG) that uses homologous recombination to insert binding sites for the RNA-binding MS2 coat protein (MS2-CP) between the coding region and 3' untranslated region (UTR) of any yeast gene. Upon coexpression of MS2-CP fused with GFP, we demonstrate the localization of endogenous mRNAs (ASH1, SRO7, PEX3 and OXA1) in living yeast (Saccharomyces cerevisiae).
Assuntos
Reação em Cadeia da Polimerase/métodos , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas do Capsídeo/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/química , Levivirus/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Peroxinas , RNA Fúngico/análise , RNA Mensageiro/análise , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Polarized growth in the budding yeast Saccharomyces cerevisiae depends upon the asymmetric localization and enrichment of polarity and secretion factors at the membrane prior to budding. We examined how these factors (i.e., Cdc42, Sec4, and Sro7) reach the bud site and found that their respective mRNAs localize to the tip of the incipient bud prior to nuclear division. Asymmetric mRNA localization depends upon factors that facilitate ASH1 mRNA localization (e.g., the 3' untranslated region, She proteins 1 to 5, Puf6, actin cytoskeleton, and a physical association with She2). mRNA placement precedes protein enrichment and subsequent bud emergence, implying that mRNA localization contributes to polarization. Correspondingly, mRNAs encoding proteins which are not asymmetrically distributed (i.e., Snc1, Mso1, Tub1, Pex3, and Oxa1) are not polarized. Finally, mutations which affect cortical endoplasmic reticulum (ER) entry and anchoring in the bud (myo4Delta, sec3Delta, and srp101) also affect asymmetric mRNA localization. Bud-localized mRNAs, including ASH1, were found to cofractionate with ER microsomes in a She2- and Sec3-dependent manner; thus, asymmetric mRNA transport and cortical ER inheritance are connected processes in yeast.
Assuntos
Divisão Celular , Polaridade Celular/genética , Retículo Endoplasmático/metabolismo , Exocitose/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal , Transporte Biológico , Proteínas de Transporte/genética , Núcleo Celular/genética , Citoesqueleto/genética , Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Proteínas Nucleares/genética , Peroxinas , Ligação Proteica , Proteínas R-SNARE/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Anosmia affects the western world population, mostly the elderly, reaching to 5% in subjects over the age of 45 years and strongly lowering their quality of life. A smaller minority (about 0.01%) is born without a sense of smell, afflicted with congenital general anosmia (CGA). No causative genes for human CGA have been identified yet, except for some syndromic cases such as Kallman syndrome. In mice, however, deletion of any of the 3 main olfactory transduction components (guanidine triphosphate binding protein, adenylyl cyclase, and the cyclic adenosine monophosphate-gated channel) causes profound reduction of physiological responses to odorants. In an attempt to identify human CGA-related mutations, we performed whole-genome linkage analysis in affected families, but no significant linkage signals were observed, probably due to the small size of families analyzed. We further carried out direct mutation screening in the 3 main olfactory transduction genes in 64 unrelated anosmic individuals. No potentially causative mutations were identified, indicating that transduction gene variations underlie human CGA rarely and that mutations in other genes have to be identified. The screened genes were found to be under purifying selection, suggesting that they play a crucial functional role not only in olfaction but also potentially in additional pathways.
