Identification and functional analysis of RNA-binding sites in proteins involved in nuclear mRNP assembly in S. cerevisiae

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The Hermes of gene expression is the messenger RNA (mRNA), which carries the information of a protein-coding gene to the ribosome. During nuclear mRNP biogenesis many proteins bind to the mRNA, processing and packaging it into a messenger ribonucleoprotein particle (mRNP). These RNA-binding proteins (RBPs) of the mRNP not only function at the time of their binding, but often also affect later steps of gene expression. Consequently, the process of mRNP assembly in the nucleus is essential and at the same time dauntingly complex.In the project proposed here, we combine mass-spectrometric and biochemical approaches to unravel the process of nuclear mRNP assembly and the functions of the RBPs involved. During the last three years we identified RNA-binding sites, developed new techniques for the identification of protein-RNA cross-links and functionally characterized RNA-binding mutants of the first nuclear RBP, Npl3. We will continue to systematically identify the RNA-binding sites in these RBPs in S. cerevisiae by “classical” photo-cross-linking and additionally by chemical cross-linking combined with high-resolution mass spectrometry. Using mutants in the RNA-binding sites thus identified, we will assess each RBP's function in mRNP assembly, nuclear mRNA export and mRNA stability. We expect that the results will elucidate the process of nuclear mRNP assembly and the influence of mRNP composition on the life cycle of mRNA in the cell.