Browsing by Author "George, Iain Fraser Scotney"

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    Deducing the post-transcriptional role of the Pumilio protein Puf3 in fission yeast.
    (2019-05-29) George, Iain Fraser Scotney; Chua, Gordon; Muench, Douglas G.; Turner, Raymond Joseph; Grewal, Savraj S.; Kothe, Ute
    Pumilio family proteins (Pufs) are a highly-conserved group of post-transcriptional regulators in eukaryotes. Puf proteins contain an arc-shaped Pumilio homology domain with several coiled repeats that bind to potentially predictable sequences in the 3’-UTR of their mRNA targets. The binding of Puf proteins to mRNA targets can influence their stability, translation and cellular localization. In S. pombe, there are seven non-essential genes that encode classical Puf proteins. The biological function of these Puf proteins and their mRNA targets remain unknown. In this study, we investigated the biological role and mRNA targets of Puf3 in S. pombe. Over-expression of puf3+ with the nmt41 promoter results in reduced fitness and an enrichment of elongated cells arrested in mitosis. Phenotypic characterization of puf3 overexpression strains and genetic interaction screens identified a cell cycle regulatory role for Puf3, principally at the G2/M transition and possibly at the G1/S control point. Genetic interaction screening, fluorescence microscopy and tandem mass spectrometry analysis of immunoprecipitated Puf3-HA identified Puf3-interacting proteins in the post-transcriptional machinery of 3’-deadenylation of mRNA and localized to stress granules. Bioinformatic analysis of the Puf proteins in S. pombe identified the Puf repeat domains and these were used to enumerate the predicted Puf binding element in the transcriptome. The enumeration data and annotation data were used to identify putative mRNA targets from microarray, RNA-seq and RNA-immunoprecipitation sequencing experiments. These identified several components of the kinetochore and mitotic checkpoint as putative mRNA targets of Puf3.