Combination of transcriptome-wide and structural approaches to study SHE binding to RNA localization elements in budding yeast

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In Saccharomyces cerevisiae, ASH1 mRNA and further transcripts are actively transported to the growing bud to support asymmetric cell division. A central player in this paradigm example of RNA localization is the RNA-binding protein (RBP) She2p. As part of the SHE machinery, She2p associates with its cargo mRNAs in the nucleus and promotes their subcellular localization in the cytoplasm. During this process, She2p engages into subcomplexes with different adapter proteins, namely Loc1p in the nucleus and She3p in the cytoplasm, which stabilize its binding to the RNA localization elements (LEs). In the first funding period, we could unravel how She2p and She3p jointly bind to an ASH1 LE RNA. Combining crystallography and chemical probing, we could directly visualize how the RNA adopts a distinct fold upon association of the adapter protein She3p. Based on the detailed structural information, we computationally derived a unifying descriptor, which allowed to predict in silico the location of SHE binding sites in known target mRNAs. Notably, we could validate in vitro She2p/She3p binding to newly predicted LEs, underlining that we indeed captured major molecular features of SHE recognition.In the second funding period, we propose to take our analysis to a transcriptome-wide scale to investigate how LEs are specifically recognized by the SHE machinery in the nucleus and the cytoplasm. Combining transcriptome-wide in vitro and in vivo RBP binding maps with structural biology and bioinformatics, we will address (i) which LEs are recognized by the SHE machinery in the cytoplasm, (ii) how SHE binding changes during nucleo-cytoplasmic remodeling of the transport complexes, and (iii) which RNA features are critical to define LEs. Our combined approach ranges from global binding patterns to high-resolution binding confirmations and will enable us to disentangle the combinatorial contribution of multiple proteins to SHE binding and mRNA localization this important model system.