Abstract
The regulation of gene expression in eukaryotes is a complex process that is controlled at many levels. Posttranscriptional regulation is performed by RNA-binding proteins that can effect the processing, localization and decay rates of mRNAs. One such family of RNA-binding proteins is Pumilio and FBF (Puf), which are conserved across eukaryotes. In this work, we have started to functionally characterize the nine puf+ genes found in the fission yeast Schizosaccharomyces pombe through the use of fluorescence localization studies, transcriptomics, RNA immunoprecipitation microarrays (RIP-chip) and molecular genetic techniques. Deletion strains of the classical puf+ genes were created and observed to have no phenotype, while strains overexpressing these genes did. This characterization also led to the finding that the puf+ genes of S. pombe are intracellularly localized in three distinct patterns of nucleolar, cytoplasmic and granular. The localizations were observed under heat shock conditions and only the cytoplasmic Pufs relocalized to foci in the cell that appeared to correspond to a stress granule marker. It was also observed that the Puf gene pfr1+ is involved in regulating flocculation, adhesion and invasive growth as the deletion strain cannot be induced to flocculate or invasively grow, while overexpression causes constitutive flocculation. Putative mRNA targets of Pfr1 regulation include pvg1+ and SPBPB7E8.01, which were identified by expression microarrays and RIP-chip analysis. These putative target genes were found to be repressors of flocculation, while the posttranscriptional regulation genes ccr4+ and ste13+were shown to be required for flocculation. The analysis of puf2 and puf4 using expression microarrays and RIP-chip to identify putative mRNA targets of these puf+ genes was also performed. This analysis indicated that puf2+ may be involved in regulating hexose import while puf4+ could be involved in the regulation of iron homeostasis. The results of this study have substantially contributed to our understanding of Puf function and posttranscriptional regulation in S. pombe.
