title: RNA Affinity Purification and Characterization of Roquin Proteins in CDE-mediated mRNA Decay
creator: Leppek, Kathrin
subject: 570
subject: 570 Life sciences
description: Tumor necrosis factor (TNF)-α is the most potent pro-inflammatory cytokine in mammals. The degradation of TNFα mRNA is critical for restricting TNFα synthesis and involves an AU-rich element (ARE) and a constitutive decay element (CDE) in the 3' untranslated region (UTR) of the mRNA. In the first part of my thesis, I optimized an RNA-based method to identify RNA-binding proteins (BPs) associated with TNFα mRNA. For this, I developed a modified streptavidin-binding RNA aptamer termed S1m. It has improved affinity for streptavidin and I found a four-fold repeat (4xS1m) to be most efficient. I then used TNFα ARE-4xS1m RNA to purify ARE-BPs from cellular extracts. By this, I found the majority of established ARE-BPs and confirmed Rbms1 and Roxan as novel ARE-BPs. The optimized 4xS1m aptamer therefore provides a powerful tool for the discovery of ribonucleoprotein (RNP) components.  In the second part of my thesis, I investigated the TNFα CDE in detail and found that the CDE is a 17 nucleotide long structured motif. Structural probing and mutagenesis provide evidence that it folds into a short RNA stem-loop in its active conformation. Using my 4xS1m protocol, I then identified CDE-associated proteins by mass spectrometry. Thereby, I found that the CCCH-type zinc and RING finger proteins Roquin (Rc3h1) and its paralog Roquin2 (Rc3h2) are stem-loop specific CDE-BPs. Next, I confirmed that the ROQ domain of Roquin specifically and directly binds to the CDE stem-loop. I could further show that Roquin is required for CDE-mediated mRNA decay and suppression of TNFα production in macrophages. TNFα expression was also increased by introduction of a morpholino that interferes with CDE-Roquin binding. My data provide evidence that Roquin proteins promote mRNA degradation by recruiting the Ccr4-Caf1-Not deadenylase complex. CDE motifs are highly conserved and are found in over 50 vertebrate mRNAs, many of which encode regulators of development and inflammation. In macrophages, I confirmed that CDE-containing mRNAs are the primary targets of Roquin on a transcriptome-wide scale. Thus, Roquin proteins act broadly as mediators of mRNA deadenylation by recognizing a conserved class of stem-loop RNA degradation motifs. In all, I unraveled a mechanism that adds an important component to the complex network that governs posttranscriptional control of gene expression.
date: 2014
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/17405/1/Kathrin_Leppek_PhDThesis.pdf
identifier: DOI:10.11588/heidok.00017405
identifier: urn:nbn:de:bsz:16-heidok-174056
identifier:   Leppek, Kathrin  (2014) RNA Affinity Purification and Characterization of Roquin Proteins in CDE-mediated mRNA Decay.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/17405/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng