GYF domain mediated protein: protein interactions

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Proteins containing GYF domains act in the context of mRNA surveillance in the cytoplasm (Smy2, PERQ2) or in spliceosome assembly in the nucleus (CD2BP2). The GYF domain thereby confers binding to proline-rich sequence (PRS) and this interaction has been defined at the atomic level for Smy2 and CD2BP2-type subfamilies. Structural knowledge serves as a basis to further incorporate peptide-mimetic and non-proteinogenic amino acid analogs into peptide scaffolds that specifically address GYF in comparison to other PRS recognition domains. In addition, we want to identify inhibitors that capture the second binding site of the CD2BP2-GYF domain that mediates interactions with the snRNP protein U5-15K. Proteomic profiling of immobilized GYF domain inhibitors will be achieved by combining mass spectrometry and stable isotope labeling in cell culture (SILAC). Furthermore, we will characterize co-precipitated RNA by RT-PCR of selected candidate mRNAs and deep sequencing. Functional assays comprise splicing of reporter mRNAs and the measurement of mRNA stability and modification of their 3’-untranslated regions. Finally, we will use CD2BP2 knockout mice to directly compare chemical and genetic interference with the function of this paradigmatic protein.