RESUMEN
MSA1 mRNA encodes Msa1p, a protein associated with the SCB-binding factor (SBF) and MCB-binding factor (MBF) complex. Msa1p promotes the transcription of G1 phase-specific genes, and is subjected to cell cycle-dependent regulation for its abundance and subcellular localization. MSA1 mRNA and Msa1p levels oscillate in the cell cycle with peaks at the late M/early G1 phase and early G1 phase, respectively. Phosphorylation by CDK1 negatively regulates the nuclear localization of Msa1p. In the present study, we identified MSA1 mRNA as a bud tip-localized mRNA in screening using a Tag-GFP system. A fragmentation analysis revealed a sequence of ~145 bases for the bud tip localization. Endogenous MSA1 mRNA localized at the bud tip in a manner that depended on SHE2. Msa1p levels were also affected by SHE2 in cells constitutively expressing MSA1 mRNA. These results suggest the existence of a regulatory mechanism for Msa1p through the localized control of MSA1 mRNA.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuencia de Bases , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transcripción GenéticaRESUMEN
RNA localization is an important event that is essential for the polarization and differentiation of a cell. Although several methods are currently used to detect localized RNAs, a simplified detection system has not yet been developed for Schizosaccharomyces pombe. In the present study, we describe a new vector system for the visualization of localized RNAs in S. pombe using a U1A-tag-GFP system. A pREP1-U1A-tag vector plasmid to express U1A-tagged RNA and a pREP2-U1AGFP plasmid to produce a U1A-GFP fusion protein were constructed for this system. Since the U1A-GFP protein binds U1A-tagged RNA, fluorescence is observed at the location of U1A-tagged RNA in cells expressing both of these. The nucleolar localization of U3 snoRNA was successfully detected using this system, and a novel RNA localized at the DNA region of the nucleus was found by screening localized RNAs. This system will accelerate the study of localized RNAs in S. pombe.
Asunto(s)
Núcleo Celular/genética , Regulación Fúngica de la Expresión Génica , Vectores Genéticos/metabolismo , ARN de Hongos/genética , ARN Nucleolar Pequeño/genética , Schizosaccharomyces/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Expresión Génica , Genes Reporteros , Vectores Genéticos/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Imagen Óptica , ARN de Hongos/metabolismo , ARN Nucleolar Pequeño/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/ultraestructuraRESUMEN
Exons are ligated in an ordered manner without the skipping of exons in the constitutive splicing of pre-mRNAs with multiple introns. To identify factors ensuring ordered exon joining in constitutive pre-mRNA splicing, we previously screened for exon skipping mutants in Schizosaccharomyces pombe using a reporter plasmid, and characterized three exon skipping mutants named ods1 (ordered splicing 1), ods2, and ods3, the responsible genes of which encode Prp2/U2AF59, U2AF23, and SF1, respectively. They form an SF1-U2AF59-U2AF23 complex involved in recognition of the branch and 3' splice sites in pre-mRNA. In the present study, we identified a fourth ods mutant, ods4, which was isolated in an exon-skipping screen. The ods4+ gene encodes Cwf16p, which interacts with the NineTeen Complex (NTC), a complex thought to be involved in the first catalytic step of the splicing reaction. We isolated two multi-copy suppressors for the ods4-1 mutation, Srp2p, an SR protein essential for pre-mRNA splicing, and Tif213p, a translation initiation factor, in S. pombe. The overexpression of Srp2p suppressed the exon-skipping phenotype of all ods mutants, whereas Tif213p suppressed only ods4-1, which has a mutation in the translational start codon of the cwf16 gene. We also showed that the decrease in the transcriptional elongation rate induced by drug treatment suppressed exon skipping in ods4-1. We propose that Cwf16p/NTC participates in the early recognition of the branch and 3' splice sites and cooperates with the SF1-U2AF59-U2AF23 complex to maintain ordered exon joining.
