Biochemical dissection of RNA-protein complexes in the host response to infection ; Biochemische Dissektion von RNA-Protein-Komplexen in der Wirtsantwort auf Infektionen

RNA is not only central to cellular gene expression - as a messenger of genetic information, the main component of ribosomes and in the form of transfer RNAs translating the genetic code. It also fulfills a wide range of functions in the form of non-coding RNAs and is used by RNA viruses to store and replicate their genetic cargo. With the COVID-19 pandemic, researchers have embarked on an unprecedented endeavor to unravel how SARS-CoV-2 executes its replication cycle based on its complex RNA genome. To date, the true repertoire of RNA functions cannot be inferred from sequence and structural information alone. The interactions with proteins can provide important information on individual RNA functions. To identify which proteins directly interact with RNA, state-of-the-art methods apply UV crosslinking. UV irradiation induces formation of covalent bonds and the resulting complexes can be isolated using tagged DNA antisense probes and affinity purification. While these approaches have been used to reveal important RNA-based mechanisms, they come with important limitations: • only highly abundant RNA targets have been successfully analyzed • investigating separate regions as part of a transcript is not possible and • high cost and labor limit their application As a consequence, knowledge on the interactions of RNAs with moderate or low expression levels is missing. Further, proteins have to be individually mapped to identify corresponding RNA-binding sites and the number of targets investigated to date is still low. Recently, organic phase separation-based methods have emerged enabling the highly efficient isolation of the full repertoire UV-crosslinked RNA-protein complexes from a diversity of biological sources to globally identify RNA-binding proteins. Inspired by this potential, I set out to develop an approach combining organic phase separations with a sequence-specific RNA targeting strategy to isolate proteins interacting with a defined RNA sequence. Using this new method termed SHIFTR, I was able to ...