Specifying the rules for target recognition and regulation by Roquin in vivo
Biochemistry
Final Report Abstract
Post-transcriptional gene regulation is crucial for the correct temporal and tight control of gene expression. In case of dysregulation, it is the underlying cause of various pathologies. At the mRNA level, this control is mediated by cis-regulatory elements encoded in the untranslated regions. These elements are recognized by RNA-binding proteins (RBPs) based on their sequence and/or structure. The binding of an RBP to a target mRNA e.g., controls its stability. The immunoregulatory Roquin proteins are of central importance in limiting inflammatory reactions by destabilizing mRNAs that encode proinflammatory proteins. Roquin proteins are characterized by their unique recognition of the shape of RNA stem-loops, the so-called CDEs (constitutive decay elements) and ADEs (alternative decay elements). CDEs are stem-loops with a trinucleotide-loop, ADEs carry a hexanucleotide-loop. It is unclear exactly which sequences mediate the binding of Roquin proteins and thus the destabilization of mRNAs. Furthermore, stem-loops with other loop sizes are also discussed in the literature as a binding motif for Roquin proteins. This points to several new, as yet undefined, cis-regulatory elements which are crucial for efficient mRNA repression by Roquin. Furthermore, the importance of the flanking sequence context in Roquin-mediated mRNA degradation is unclear. In addition to the unique ROQ domain that recognizes stem-loops in target mRNAs, Roquin proteins also encode a zinc finger (ZnF) domain with unknown RNA binding preference. Using RNA Bind-n-Seq (RBNS), we systematically characterized the RNA-binding preference of both the ROQ domain and the ZnF. This allowed us to uncover novel CDE and ADE motifs that efficiently mediate Roquin binding in vitro. Moreover, we were able to show that these motifs also control mRNA stability in vivo and thereby identified new bona fide Roquin target mRNAs. In addition, we investigated interactions between Roquin-mediated mRNA degradation and ARE (AU-rich elements)-mediated degradation, both important modulators of inflammatory responses. We showed that AU-rich CDEs in their unfolded form can interact with RBPs that recognize linear AU-rich sequences, suggesting a dual function of these CDEs in mRNA degradation. Interestingly, the ZnF also recognizes AU-rich sequences, which in interaction with stem-loops contribute to the binding of Roquin to target mRNAs. Overall, we were able to elucidate the RNA-binding preference of a central immune regulator in detail and to lay a foundation for the central function of RNA structures in mRNA metabolism.
Publications
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Structural basis for the recognition of transiently structured AU-rich elements by Roquin. Nucleic Acids Research.
Binas, Oliver; Tants, Jan-Niklas; Peter, Stephen A.; Janowski, Robert; Davydova, Elena; Braun, Johannes; Niessing, Dierk; Schwalbe, Harald; Weigand, Julia E. & Schlundt, Andreas
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mRNA-Strukturen steuern die posttranskriptionelle Genregulation. BIOspektrum, 27(4), 351-354.
Lichtenthaeler, Chiara; Oberstrass, Lasse & Weigand, Julia E.
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Heterogenous nuclear ribonucleoprotein D-like controls endothelial cell functions. Biological Chemistry, 405(4), 229-239.
Fischer, Sandra; Lichtenthaeler, Chiara; Stepanenko, Anastasiya; Heyl, Florian; Maticzka, Daniel; Kemmerer, Katrin; Klostermann, Melina; Backofen, Rolf; Zarnack, Kathi & Weigand, Julia E.
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Arid5a uses disordered extensions of its core ARID domain for distinct DNA- and RNA-recognition and gene regulation.
von Ehr, Julian; Oberstrass, Lasse; Yazgan, Ege; Schnaubelt, Lara Ina; Blümel, Nicole; McNicoll, Francois; Weigand, Julia E.; Zarnack, Kathi; Müller-McNicoll, Michaela; Korn, Sophie Marianne & Schlundt, Andreas
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Structure and RNA-binding of the helically extended Roquin CCCH-type zinc finger.
Tants, Jan-Niklas; Oberstrass, Lasse; Weigand, Julia E. & Schlundt, Andreas
