Applications of mass spectrometry-based proteomics: the developmental proteome of D. melanogaster and the RNA-fold interactome of conserved RNA structures in yeast
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Abstract
Mass spectrometry-based proteomics is a widely used technology that enables identification and quantification of proteins as well as their post-translational modifications. In this thesis, quantitative proteomics has been applied to the study of 1) protein developmental dynamics in Drosophila melanogaster and 2) the protein interactors to a set of evolutionary conserved RNA structures in yeast.
In Chapter III we apply label-free quantitative proteomics to comprehensively investigate protein remodelling across development in Drosophila melanogaster, a widely used model organism in genetic and developmental studies. To this end, we provide two datasets: the whole life cycle proteome consisting of 7952 proteins and the highly temporal-resolved embryogenesis proteome that comprises 5458 proteins. These large proteomic datasets allowed us to identify maternally provided proteins important in maternal-to-zygotic transition and early development, as well as stage- and gender-specific proteins. We also quantify isoform-specific expression of 34 different genes during development, validate expression of 268 small proteins and evidence the pseudogene Cyp9f3Ψ as a protein-coding gene. Integration with available transcriptomic data revealed moderate mRNA-protein correlation, with a protein delay of 4-5 hours. The combination of proteomic data with tissue-specific data uncovers proteins with tissue-specific developmental regulation, as exemplified by two yet uncharacterized proteins with an impaired muscle phenotype upon knockdown. The two large-scale proteomic datasets can be explored in detail in our interactive web interface and serve as a powerful resource for future studies.
Chapter IV focuses on the identification of RNA-binding proteins (RBP) associated with evolutionary conserved RNA structures in yeast. Using a SILAC-based quantitative RNA pull-down approach, we map 162 proteins to individual RNA structures within messenger RNA. The majority of them are associated with RNA-binding features, whereas approximately one third are previously unrelated to RNA-binding and lack canonical RNA-binding domains. Intriguingly, we report a significant number of proteins binding to RNA folds in the coding regions of mRNAs, despite current knowledge about RNA-binding proteins regulation on the 5’- or 3’-UTR. Available PAR-CLIP datasets show high overlap with our reported protein-RNA interactions and RNA immunoprecipitation experiments confirmed our findings for selected mRNA-protein pairs. Using a reporter system and pulsed SILAC, we explored a role in translational control of a subset of mRNA-RBP pairs and find Nsr1 and YDR514C as possible translational regulators. This study presents a scalable immunoprecipitation-mass spectrometry (IP-MS) approach to map protein binders to individual RNA folds that connects structural RNA features to functionality.
Overall, during my PhD I applied quantitative MS-based proteomics to a broad range of biological questions, with special focus on the study of protein developmental dynamics and RNA-fold interactomics.