Functional characterisation of DHX proteins in the regulation of RNA metabolism and genome stability

von Rebecca Rossen Falk geb. Rebecca Falk
Dissertation
Datum der mündl. Prüfung:2023-03-01
Erschienen:2023-08-08
Betreuer:Prof. Dr. Markus Bohnsack
Gutachter:Prof. Dr. Markus Bohnsack
Gutachter:Dr. Ricarda Richter-Dennerlein
Zum Verlinken/Zitieren: http://dx.doi.org/10.53846/goediss-10037

 

 

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Englisch

Cells contain a multitude of different RNAs, which are highly versatile biomolecules that have crucial functions in almost all cellular processes. Furthermore, the inter-relationship between RNA and proteins is essential, as ribonucleoprotein complexes are responsible for the execution and control of fundamental cellular processes. RNA helicases are nucleoside triphosphate-dependent RNA-binding proteins capable of remodelling RNAs and ribonucleoprotein complexes and are, consequently, a key driving force in almost all aspects of RNA metabolism and are expressed not only in all three domains of life but also in some viruses. DHX proteins are a diverse group of Superfamily 2 RNA helicases with shared structural features that display a wide array of RNA remodelling activities in a multitude of cellular processes. Consequently, strict spatial, temporal, and catalytic regulation of DHX proteins is essential in cells, and the dysregulation of helicase activity is often associated with tumorigenesis and disease. However, the cellular function and mode of regulation of several DHX proteins remain elusive. The objective of this study was to broaden the understanding of the functional and regulatory repertoires of DHX proteins. To this end, a toolbox was established for exploring DHX protein functions in the cellular context. The toolbox included verified siRNAs, stably transfected cell lines for inducible expression of tagged DHX proteins, and tested antibodies. The usefulness of this toolbox was exemplified by the functional characterisation of DHX40. Analysis of DHX40 protein interactions by immunoprecipitation and mass spectrometry revealed that DHX40 interacts with several proteins implicated in regulating LINE-1 retrotransposition. Biochemical analysis using recombinantly expressed and purified proteins showed that DHX40 is an RNAdependent ATPase that is stimulated by the putative prolyl isomerase PPIL4, a novel protein cofactor identified in this study. DHX40 was confirmed to be a substrate of the deubiquitinase USP7, and mutagenesis analysis was used to investigate the interaction of DHX40 with the E3 ligase TRIM27. Comprehensive analysis of the RNA interactome by the crosslinking and analysis of cDNA (CRAC) approach revealed that DHX40 interacts with LINE-1 RNAs in HEK293, and complementary differential expression analysis showed that LINE-1 RNAs are upregulated upon depletion of DHX40. Finally, a cell-based LINE-1 retrotransposition assay showed that DHX40 and PPIL4 are suppressors of LINE-1 retrotransposition in HEK293 cells.
Keywords: RNA; Helicase; Retrotransposition; Genome stability; RNA regulation
 
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