Identification and characterization of novel components of a localized mRNP in Drosophila

• Applicant: Dr. Fulvia Bono
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry ; Developmental Biology ; Structural Biology ; Cell Biology
• Term: from 2015 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 270067186

• Final Report Year: 2022

Final Report Abstract

The Bono lab applied an experimental approach that complemented the work of A. Ephrussi. As part of their common aim to understand the function of mRNPs during early Drosophila oogenesis, the Bono group solved the crystal structure of the Exuperantia (Exu) protein. Exu is a core factor for the localization of bicoid mRNA to the anterior pole of the oocyte. The structure revealed the dimeric state of this protein and identified the respective contribution of its 3’-5’ EXO like domain and its SAM domain for its tight binding to bicoid mRNA. This work resolved a long-standing issue regarding the function of this essential factor in Drosophila development. The Bono lab also aimed at establishing a double-mRNP native purification protocol involving MS2-tag based purification of a localized mRNA, followed by immunoprecipitation of associated RBPs. In this TAP tagging approach, both the RNA and a known trans-acting factor were tagged in parallel. Tagged oskar could be purified in vitro, albeit with low efficiency and specificity. Therefore, in vivo native mRNP purification from isolated egg chambers will require further optimization. The Bono lab further performed immunoprecipitation (IP) coupled to MS of several tagged RBPs that are known to regulate localization of one or more maternal mRNA at different developmental stages. Using the GFP-TRAP system, complexes from isolated egg chambers could be purified in presence of RNase to preserve direct protein-protein interactions. Using mass spectrometry (MS) and subsequent statistical analysis, proteins significantly enriched with each bait were identified to construct an interactome of proteins involved in mRNA regulation during Drosophila oogenesis. Functional analysis using bioinformatics annotation tools revealed that these RBPs are involved in a broad range of events in addition to their role in mRNA localization and translation during development. Co-IPs in human cells were used to validate the candidates identified by MS. Several novel interactions of potential RBP regulators, including some previously uncharacterized genes could be verified. This work has been published and well received. It forms the basis for future structural and functional studies that will be continued outside the FOR2333 network.

Publications

• (2016) The bicoid mRNA localization factor Exuperantia is an RNA-binding pseudonuclease. Nat Struct Mol Biol 23:705-715
  Lazzaretti D., Veith K., Kramer K., Basquin C., Urlaub H., Irion U. and Bono F.
  
• (2017) mRNA localization in metazoans: a structural perspective. RNA Biol., 14:1473-1484
  Lazzaretti D. and Bono F.
  
• (2017) Target identification and mechanism of action of picolinamide and benzamide chemotypes with antifungal properties. Cell Chem Biol, 25(3):279-290
  Pries V., Nöcker C., Johnen P., Khan D., Tripathi A., Hong Z., Keller A., Fitz M., Perruccio F., Filipuzzi I., Thavam S., Aust T., Riedl R., Ziegler S., Bono F., Schaaf G., Bankaitis V., Waldmann H., Hoepfner D.
  
• (2020) An Interaction Network of RNA-Binding Proteins Involved in Drosophila Oogenesis. Mol Cell Proteomics, Sep;19(9):1485-1502
  Bansal P., Madlung J., Schaaf K., Macek B., Bono F.