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Analysis of the processing body subunits Winnebago and Trailer Hitch in Drosophila melanogaster

  • Author(s): Jiang, Philip Yuan-Zheng
  • et al.
Abstract

To discover new ribonucleoprotein (RNP) components, Trailer Hitch (Tral), a RNP factor involved in endoplasmic reticulum (ER) exit site function, was immunoprecipiated and Winnebago (Winn) was identified as a novel Tral- associated protein. Analysis of the Winn sequence revealed a RNA binding motif and conservation with the human cyclo- ligase, methenyltetrahydrofolate synthetase (MTHFS). These observations led us to propose that Winn utilizes ATP to catalyze ring closure on RNA 5' caps in a manner similar to that of MTHFS. Experiments included recombinant expression of Bovine Winn protein, purification, and assay for ATPase activity. Despite a multistep protein purification strategy, our preparations were contaminated with low levels of ATPase that interfered with the assays. In parallel with identifying Winn, we sought to identify mRNA targets of Tral. From microarray analysis of mRNAs that co-immunoprecipitated with Tral, orc5 mRNA was identified. Orc5 protein is a component of the Origin Recognition Complex involved in DNA replication and membrane trafficking. Orc5 function in membrane trafficking suggested that it might be a functionally relevant target for the ER defects observed in tral mutants. We examined orc5 mutants for the ability to genetically interact with tral but these experiments did not reveal an interaction; therefore, orc5 mRNA is not a target of the Tral complex. We expanded our genetic screen using the Drosophila deletion collection to identify regions on Chromosome 3 that could dominantly enhance eggshell defects seen in tral mutants. Sixty genomic regions were tested for genetic interaction with tral; however, none of the regions tested interacted

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