title: Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii.
abstract: BACKGROUND
  Puf proteins act as translational regulators and affect many cellular processes in a wide range of eukaryotic organisms. Although Puf proteins have been well characterized in many model systems, little is known about the structural and functional characteristics of Puf proteins in the parasite Toxoplasma gondii.
  
  
  METHODS
  Using a combination of conventional molecular approaches, we generated endogenous TgPuf1 tagged with hemagglutinin (HA) epitope and investigated the TgPuf1 expression levels and localization in the tachyzoites and bradyzoites. We used RNA Electrophoretic Mobility Shfit Assay (EMSA) to determine whether the recombination TgPuf1 has conserverd RNA binding activity and specificity.
  
  
  RESULTS
  TgPuf1 was expressed at a significantly higher level in bradyzoites than in tachyzoites. TgPuf1 protein was predominantly localized within the cytoplasm and showed a much more granular cytoplasmic staining pattern in bradyzoites. The recombinant Puf domain of TgPuf1 showed strong binding affinity to two RNA fragments containing Puf-binding motifs from other organisms as artificial target sequences. However, two point mutations in the core Puf-binding motif resulted in a significant reduction in binding affinity, indicating that TgPuf1 also binds to conserved Puf-binding motif.
  
  
  CONCLUSIONS
  TgPuf1 appears to exhibit different expression levels in the tachyzoites and bradyzoites, suggesting that TgPuf1 may function in regulating the proliferation or/and differentiation that are important in providing parasites with the ability to respond rapidly to changes in environmental conditions. This study provides a starting point for elucidating the function of TgPuf1 during parasite development.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.forename: Jun
authors.initials: J
authors.lastname: Miao
authors.forename: Tingkai
authors.initials: T
authors.lastname: Liu
authors.forename: William J
authors.initials: WJ
authors.lastname: Sullivan
authors.affiliation: Department of Pathogen Biology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China. luc2@psu.edu.
authors.forename: Liwang
authors.initials: L
authors.lastname: Cui
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D005786:Gene Expression Regulation / Q000502:physiology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D021381:Protein Transport; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D014122:Toxoplasma / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D014774:Virulence
chemical_list: D015800:Protozoan Proteins; D016601:RNA-Binding Proteins