Funktionelle Charakterisierung zellulärer RNA-bindender Proteine, die die Replikation von Flaviviren unterstützen
Zusammenfassung der Projektergebnisse
RNA viruses such as flaviviruses essentially require the function of cellular ‘host factors’ for the replication of the viral genome. Numerous of such host factors have already been identified. This was done, for example, by knock-out experiments in which depletion of these factors was found to inhibit viral replication. Accordingly, for the vast majority of these factors, their exact function is still unknown. In previous work for this project, we identified a host factor of the Flavivirus West Nile, the cellular RNA binding protein (RBP) AUF1, specifically the AUF1 isoform AUF1 p45. Our studies showed that AUF1 p45 has two RNA remodeling activities, an RNA chaperone and an RNA annealing activity, both of which are important for the realization of a step essential for viral replication - circularization of the viral RNA genome. This circularization is a prerequisite for the association of the viral replicase to the 5’ end of the viral RNA and for the subsequent initiation of RNA synthesis at the 3’ end of the genome. During the course of the project, we elucidated other important aspects of the activities of AUF1 in its function as a Flavivirus host factor: We could show that a post-translational modification, methylation of arginine residues in the C-terminus of the protein by the methyltransferase PRMT1, significantly increases the RNA chaperone activity of AUF1 p45. - We demonstrated that the RRM and the RGG/RG motifs in the AUF1 protein contribute to efficient RNA binding as well as RNA remodeling and observed that both motifs tightly cooperate during these activities. Interestingly, our data showed that specific arginine residues of the RGG/RG motif have defined roles in the RNA remodeling activity of the protein. - We could show that AUF1 p45 is a general host factor of Flavivirus replication: it supports not only WNV but also DENV and ZIKV replication. - We defined specific stem-loop structures within the 5’ and 3’-ends of the flaviviral genome, which are destabilized by AUF1 p45. We could further show that this destabilization assists genome cyclization and concurrently enables the viral replicase to initiate RNA synthesis. - We identified a metastable element in a 3’-terminal structure element of the viral genome to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45. Investigations with mosquito analogues of AUF1 p45 revealed the cell protein-mediated destabilization of the 3’SL to be less efficient in mosquito cells and supported a model in which the 3’SL acts as an RNA thermometer that adapts Flavivirus replication to different host cells. - We also investigated the role of the protein and its isoforms in mRNA repression mediated by micro RNAs and demonstrated distinct functions of the helix-destabilizing RNA chaperone and RNA annealing activities of AUF1 in this process. Finally, our data revealed the YGG motif of the AUF1 protein acting as another key element of its RNA chaperone activity. In sum, the project considerably extended our knowledge in several respects. On the one hand, additional insights were gained into the molecular mechanisms of how a cellular RNA binding protein (RBP) can modulate the activity of a viral replication process. On the other hand, important new information was obtained on how an RBP/RNA chaperone generally controls the structures and functions of RNA molecules, for example, in the posttranscriptional regulation of gene expression at the mRNA level.
Projektbezogene Publikationen (Auswahl)
- Arginine methylation enhances the RNA chaperone activity of the West Nile virus host factor AUF1 p45. RNA. 2016 Oct;22(10):1574-91
Friedrich S, Schmidt T, Schierhorn A, Lilie H, Szczepankiewicz G, Bergs S, Liebert UG, Golbik RP, Behrens SE
(Siehe online unter https://doi.org/10.1261/rna.055269.11) - Coordinated action of two double-stranded RNA binding motifs and an RGG motif enables Nuclear Factor 90 to flexibly target different RNA substrates. Biochemistry. 2016 Feb 16;55(6):948-59
Schmidt T, Knick P, Lilie H, Friedrich S, Golbik RP, Behrens SE
(Siehe online unter https://doi.org/10.1021/acs.biochem.5b01072) - NF90-NF45 is a selective RNA chaperone that rearranges viral and cellular riboswitches: biochemical analysis of a virus host factor activity. Nucleic Acids Res. 2017 Dec 1;45(21):12441-12454
Schmidt T, Friedrich S, Golbik RP, Behrens SE
(Siehe online unter https://doi.org/10.1093/nar/gkx931) - The properties of the RNA-binding protein NF90 are considerably modulated by complex formation with NF45. Biochem J. 2017 Jan 15;474(2):259-280
Schmidt T, Knick P, Lilie H, Friedrich S, Golbik RP, Behrens SE
(Siehe online unter https://doi.org/10.1042/BCJ20160790) - The Host Factor AUF1 p45 Supports Flavivirus Propagation by Triggering the RNA Switch Required for Viral Genome Cyclization. J Virol. 2018 Feb 26;92(6). pii: e01647-17
Friedrich S, Engelmann S, Schmidt T, Szczepankiewicz G, Bergs S, Liebert UG, Kümmerer BM, Golbik RP, Behrens SE
(Siehe online unter https://doi.org/10.1128/JVI.01647-17) - The RGG/RG motif of AUF1 isoform p45 is a key modulator of the protein’s RNA chaperone and RNA annealing activities. RNA Biol. 2019 Apr 5:1-12
Meyer A, Golbik RP, Sänger L, Schmidt T, Behrens SE, Friedrich S
(Siehe online unter https://doi.org/10.1080/15476286.2019.1602438) - An RNA thermometer activity of the West Nile virus genomic 3’-terminal stem-loop element modulates viral replication efficiency during host switching. Viruses 2020, 12, 104
Meyer A, Freier M, Schmidt T, Rostowski K, Zwoch J, Lilie H, Behrens SE, Friedrich S
(Siehe online unter https://doi.org/10.3390/v12010104) - Real-time fluorescence-based approaches to disentangle mechanisms of a protein’s RNA chaperone activity. Methods Mol Biol. 2020;2106:89-106
Schmidt T, Friedrich S, Golbik RP, Behrens SE
(Siehe online unter https://doi.org/10.1007/978-1-0716-0231-7_5) - RNA remodeling by RNA chaperones monitored by RNA structure probing. Methods Mol Biol. 2020;2106:179-192
Friedrich S, Schmidt T, Behrens SE
(Siehe online unter https://doi.org/10.1007/978-1-0716-0231-7_11) - Alternatively spliced isoforms of AUF1 regulate a miRNA-mRNA interaction differentially through their YGG motif. RNA Biol. 2021 Jun;18(6):843-853
Sänger L, Bender J, Rostowski K, Golbik R, Lilie H, Schmidt C, Behrens SE, Friedrich S
(Siehe online unter https://doi.org/10.1080/15476286.2020.1822637)
