TY  - GEN
KW  - Streptavidin
KW  -  S1
KW  -  S1m
KW  -  RNP
KW  -  mRNP
KW  -  mRNA
KW  -  RNA-binding protein
KW  -  AU-rich element
KW  -  ARE
KW  -  RNA structure
KW  -  mRNA deadenylation
KW  -  RNA-binding protein
KW  -  constitutive decay element
KW  -  CDE
KW  -  Rc3h1
KW  -  Rc3h2
KW  -  macrophage
KW  -  TNF
KW  -  tumor necrosis factor-?
Y1  - 2014///
TI  - RNA Affinity Purification and Characterization of Roquin Proteins in CDE-mediated mRNA Decay
ID  - heidok17405
A1  - Leppek, Kathrin
N2  - Tumor necrosis factor (TNF)-? is the most potent pro-inflammatory cytokine in mammals. The degradation of TNF? mRNA is critical for restricting TNF? 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 TNF? 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 TNF? 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 TNF? 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 TNF? production in macrophages. TNF? 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.
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/17405/
AV  - public
ER  -