Abstract

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 packing in the nucleus is essential and at the same time dauntingly complex.

Here, we combine biochemical and mass-spectrometric approaches to unravel the process of nuclear mRNP packaging and the functions of the RBPs involved. We systematically identify the RNA-binding sites in these RBPs in S. cerevisiae by photo- and chemical cross-linking combined with high-resolution mass spectrometry. We further develop methods that enable the mass spectrometry based identification of proteins including their protein domains and amino acids that are chemically crosslinked to RNA. Chemical crosslinking, e.g. with diepoxybutan or nitrogen mustard, has the advantage over UV-induced crosslinking, where mainly uridine (and cytosine) are linked to proteins with their specific RNA-interacting regions, that other nucleotides, namely guanosine and adenosine, are linked as well. Chemical crosslinking therefore adds another layer of information about protein regions that are in direct contact to RNA. We are currently applying chemical protein-RNA and -DNA crosslinking not only to isolated protein-nucleic acid complexes but also to entire cellular systems. Using mutants in the RNA-binding sites thus identified, we assess each RBP's function in mRNP packaging, nuclear mRNA export and mRNA stability. We expect that the results will elucidate the process of nuclear mRNP packaging and the influence of mRNP composition on the life cycle of mRNA in the cell.

Project-related publications

Minocha, R, Popova, V, Kopytova, D, Misiak, D, Hüttelmaier, S, Georgieva, S and Sträßer, K. Mud2 functions in transcription by recruiting the Prp19 and TREX complexes to transcribed genes. Nucleic Acids Res. 2018;46:9749-9763 doi:10.1093/nar/gky640.

Stützer A, Welp LM, Raabe M, Sachsenberg T, Kappert C, Wulf A, Lau AM, David SS, Chernev A, Kramer K, Politis A, Kohlbacher O, Fischle W, Urlaub H (2020) Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry. Nat Commun., 11, 5250. doi: 10.1038/s41467-020-19047-7.

Johannsson S, Neumann P, Wulf A, Welp LM, Gerber HD, Krull M, Diederichsen U, Urlaub H, Ficner R (2018) Structural insights into the stimulation of S. pombe Dnmt2 catalytic efficiency by the tRNA nucleoside queuosine. Sci Rep., 8, 8880. doi: 10.1038/s41598-018-27118-5.

Veit J, Sachsenberg T, Chernev A, Aicheler F, Urlaub H, Kohlbacher O (2016) LFQProfiler and RNP(xl): Open-Source Tools for Label-Free Quantification and Protein-RNA Cross-Linking Integrated into Proteome Discoverer. J Proteome Res., 15, 3441-8. doi: 10.1021/acs.jproteome.6b00407.