Colaboration with Dr. Janosch Hennig and Dr. Christoph Müller
PROJECT INFORMATION :
Recent interactome studies have accumulated a wealth of information about the formation of mRNP complexes, including mRNA binding protein (mRBP) specificity, identification of targeted mRNAs, and the discovery of novel mRBPs. However, two important issues have been so far neglected. First, comparison of the mRNA interactome at two different developmental stages, and second, a following up with coordinated structural genomics investigations. Our proposal will cover both. Information retrieved by the latter will be used as a feedback-loop to accelerate functional studies and mutational analysis in vivo. In short, preliminary data of mRNA interactome studies of Drosophila larvae at two different developmental stages identified all mRBPs. Interestingly, some of the mRBPs were active during the early developmental stage (2 hours) but not during a later stage (5 hours) and vice versa. Prior to further investigations, the number of mRBP candidates will be reduced to those, which indeed show this temporally dynamic mRNA binding, are featuring novel/non-classical RNA binding domains, and/or feature low-complexity regions, which affect their mRNA binding activity. To enable structural interaction studies, these filtered candidates will be subjected to CLIP studies to identify the cognate RNA sequence, which will be validated in vitro and in vivo. Near-high-throughput nuclear magnetic resonance spectroscopy (NMR) screening will be employed to identify protein residues, which bind directly to mRNA. This information will already enable rational mutational analysis in vivo to monitor the effect mutations have on the development of the fly. Furthermore, the screening will reveal which candidates are suitable for high-resolution structure determination by NMR or X-ray crystallography. These structures will provide a wealth of information, which can be translated to orthologous systems and further improves the rational design of mutations for in vivo studies. The mRBP-RNA complex structures will improve our understanding of the protein-RNA recognition code, especially considering that novel/non-classical RNA binding proteins are part of the repertoire. Moreover, structures and interaction studies of intrinsically disordered regions and proteins, also part of the candidate list, will reveal if and how they contribute to RNA binding. We hope that we will be able to discover what triggers temporally dynamic mRNA binding activity to get insight into how mRNP complexes regulate gene expression and development.
Focus of the group:
RNA localisation and localised translation in development
KEY TECHNOLOGIES :
Protein and RNA biochemistry
Nucleic acid detection
Jeske, M., Müller, C.W*, and Ephrussi, A.* (2017). The LOTUS domain is a conserved DEAD-box RNA helicase regulator essential for the recruitment of Vasa to the germ plasm and nuage. Genes and Development 31: 1-13. doi: 10.1101/gad.297051.117
Sysoev, V. O., Fischer, B, Frese, C.K., Gupta, I., Krijgsveld, J., Hentze, M.W., Castello, A., Ephrussi, A. (2016). Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila. Nature Communications 7:12128. doi: 10.1038/ncomms12128.
Jeske, M., Bordi, M., Glatt, S., Müller, S., Rybin, V., Müller, C.W.*, and Ephrussi, A.* (2015). The Crystal Structure of the Drosophila Germline Inducer Oskar Identifies Two Domains with Distinct Vasa Helicase- and RNA-Binding Activities. Cell Reports 12:1-12.
Halstead, J.M., Lionnet, T., Wilbertz, J.H., Wippich, F., Ephrussi, A.*, Singer, R.H.* and Chao, J.A.* (2015). An RNA biosensor for imaging the first round of translation from single cells to living organisms. Science 347, 1367-1371.
Ghosh, S., Marchand, V., Gaspar, I. and Ephrussi A. (2012). Control of RNP motility and localization by a splicing-dependent structure in oskar mRNA. Nat Struct Mol Biol. 19, 441-449.
* joint corresponding author
INFO EXTRA :