The role of Pumilio-domain proteins in Trypanosoma brucei

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Proteins with Pumilio (Puf) RNA-binding domains play important roles in control of eukaryotic gene expression. Puf proteins bind specific motifs in the 3’-untranslated regions of mRNAs; usually, this results in translation inhibition and mRNA degradation, but in most cases the mechanisms are unknown. In Kinetoplastid protozoa, nearly all control of gene expression is post-transcriptional: therefore, regulated mRNA degradation is essential for parasite survival. The enzymes that degrade mRNAs in the Kinetoplastid Trypanosoma brucei are well described, but the mechanisms by which they are controlled are unknown. This application concerns the roles of two trypanosome Puf-domain proteins, PUF5 and PUF9, in trypanosome gene regulation; the information obtained is likely to provide general insights into the functions of Puf proteins in eukaryotes. Trypanosome PUF9 stabilises four mRNAs during the S phase of the cell cycle, and this requires a specific sequence motif in the mRNA 3’-untranslated region. Evidence suggests that PUF9 antagonises another, destabilising protein: the most likely candidate is PUF5. Preliminary data indicate that PUF5 promotes mRNA degradation. Analysis of PUF5 and PUF9 will include assessment of the effect of downregulation or over-expression on trypanosome growth and the transcriptome, monitoring expression and activity throughout the cell cycle, detailed determination of RNA binding specificity, and investigating how the proteins control mRNA levels.