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Identification of Roquin-regulated mRNAs in T helper cells and molecular characterization of the Roquin-RNA interaction.
München, Ludwig-Maximilians-Universität, Fakultät für Biologie, Diss., 2015, 186 S.
The immune system combines potent humoral and cellular mechanisms fighting invading pathogens and infections. To prevent excessive inflammation and autoimmunity, immune responses are subject to extensive regulation on different levels of gene expression. Signaling via costimulatory receptors for example sets a threshold for T cell activation. Roquin-1 is an RNA-binding protein targeting the inducible costimulator (Icos) mRNA in T cells for post-transcriptional repression. The importance of Roquin-1 function for immune regulation became obvious in sanroque mice developing severe lupus-like autoimmune disease due to a single amino acid exchange in Roquin-1. A lack of Roquin-1 in T cells is compensated by its paralog Roquin-2, suggesting that Roquin-2 similarly represses mRNAs. In this thesis work, assays were established to identify additional mRNAs that are bound and regulated by Roquin proteins in T helper cells. Acute deletion of Roquinencoding genes in primary CD4+ T cells led to an upregulation of more than 50 mRNAs. One newly identified target was the mRNA of the costimulator Ox40, being specifically bound and post-transcriptionally regulated by Roquin proteins via its 3’UTR. To address the molecular requirements of Roquin-RNA interactions, I performed affinity-based selection experiments (SELEX) from a random RNA library and found a novel RNA sequence motif embedded in an RNA hexaloop to be preferentially bound by the Roquin-1 N-terminus. In collaboration with structural biologists we investigated the crystal structure of a protein-RNA complex that contains the RNA-binding ROQ domain and an RNA stem-loop structure from the Tnf 3’UTR, called constitutive decay element (CDE). The structure of this complex identified amino acids forming close RNA contacts and showed mainly non-sequence-specific protein- RNA interactions via the phosphate backbone of the RNA stem. In cell-based reporter assays using Roquin-1 mutants, I proved that the ability to bind RNA is required for Roquin-mediated post-transcriptional regulation of Icos and Ox40. Furthermore, I analyzed the Ox40 3’UTR and identified a CDE-like triloop and a SELEX-like hexaloop as the two functional cis-elements through which Roquin-1 regulates Ox40 expression. Taken together, my studies identified potential mRNA targets of Roquin in T helper cells and shed light on the molecular picture of Roquin-RNA interactions. Thereby, I could show that Roquin accepts a number of different cis-regulatory RNA elements, which are rather defined by structural than sequence requirements. My work contributes to the understanding of Roquin-mediated post-transcriptional gene
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Publikationstyp Sonstiges: Hochschulschrift
Typ der Hochschulschrift Dissertationsschrift
Quellenangaben Seiten: 186 S.
Fakultät Fakultät für Biologie
Institut(e) Abteilung für Molekulare Immunregulation (AMIR)