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Control of mRNA degradation by AU-rich elements
The first step in general mRNA degradation is deadenylation, which is carried out by the Pan2-Pan3 and the Ccr4-Caf1-Not deadenylase complexes. After deadenylation, mRNAs are usually decapped by Dcp2 and further degraded either from the 5' end by the exoribonuclease Xrn1, or from the 3' end by the exosome. AU-rich elements (AREs) represent, besides micro-RNA binding sites, the second most abundant and possibly most potent class of regulatory elements that control the stability of specific mRNAs. Mechanisms of mRNA degradation AREs mediate tight control over the expression of potentially harmful proteins such as cytokines and proto-oncogenes (Schott & Stoecklin, 2010).
Our first focus in the lab is to determine mechanisms that control mRNA degradation. TTP is an RNA-binding protein that recognizes AREs and mediates rapid decay of the corresponding mRNA. Following up on our previous work on the regulation of TTP through phosphorylation (Stoecklin et al., 2004), we could show that TTP induces mRNA degradation by recruiting the Ccr4-Caf1-Not deadenylase complex (Sandler et al., 2011). We also developed an algorithm by which AREs can be assessed at a genome-wide level (Spasic et al., 2012).
Since specific RNA-protein interactions are central to the regulation and function of cellular RNAs, we improved a method to capture RNA-binding proteins using a streptavidin-binding aptamer. This method allowed us to isolate almost all of the currently known ARE-binding proteins (Leppek et al., 2014).
The CDE represents a novel class of stem-loop RNA degradation elements
Based on our earlier description of the Constitutive Decay Element (CDE) in the 3'UTR of TNF mRNA (Stoecklin et al., 2003), we determined that the CDE is a stem-loop RNA motif and identified Roquin as a specific CDE-binding protein. Roquin promotes mRNA degradation by recruiting the Ccr4-Caf1-Not deadenylase complex. The CDE represents a novel class of RNA degradation motifs with more than 50 highly conserved CDEs that could be identified in vertebrate mRNAs (Leppek et al., 2013). In collaboration with Teresa Carlomagno (EMBL Heidelberg), the structure of the CDE RNA stem-loop was solved by NMR (Codutti et al., 2015).
Pat1b drives the assembly of Processing (P)-bodies
P-bodies are cytoplasmic foci that contain mRNAs stalled in translation together with numerous components of the mRNA decay machinery, and are thus thought to represent sites where mRNAs are kept away from translation and/or degraded (Stoecklin & Kedersha, 2013, Kulkarni et al., 2010). Our work showed that human Pat1b, together with the RNA helicase Rck/DDX6, drives the assembly of P-bodies (Oezguer & Stoecklin, 2013). Furthermore, Pat1b connects the deadenylation with the decapping machinery (Oezguer et al., 2010), indicating an important function in the remodeling of mRNPs.
Control of mRNA translation
The rate at which an mRNA is translated is an efficient level at which gene expression can be regulated in the cytoplasm. It is known that protein and mRNA levels show a poor correlation in genome-wide comparisons, suggesting widespread regulation of protein synthesis and degradation. Under conditions of cellular stress, global translation is suppressed, and stalled mRNAs assemble in cytoplasmic stress granules. Our second focus in the lab is to uncover mechanisms that regulate translation during macrophage activation and under stress conditions.
Translation control of specific mRNAs in macrophages
Stimulation of macrophages causes a rapid change in gene expression, resulting in the production of immune regulatory proteins such as cytokines. By polysome profile and microarray analysis, we identified specific mRNAs whose translation is actively regulated during macrophage stimulation. Since many of the identified mRNAs encode feedback inhibitors of Nf_B signaling, translation control is important for downregulating inflammatory responses. We could also show that translationally regulated Ier3 contributes the survival of activated macrophages (Schott et al., 2014).
Control of protein synthesis under stress conditions
Many types of stress induce a global repression of translation by phosphorylation of the translation initiation factor eIF2. Thereby, cells limit further protein damage and reallocate their resources to repair and survival processes. We discovered that translation suppression in response to cold shock is independent of eIF2-phosphorylation, requires AMP-kinase activation, and is essential for cells to survive cold shock (Hofmann et al., 2012). In collaboration with the lab of Bernd Bukau (Heidelberg University), Hsp104 was characterized as a chaperone required for the resolution of heat-induced stress granules and the re-initiation of translation (Cherkasov et al., 2013). Through a collaboration with Alessia Ruggieri and Ralf Bartenschlager (Heidelberg University), we also contributed to the discovery of oscillating stress granules upon virus infection (Ruggieri et al., 2012).
In addition to the above mentioned collaborations, we are working together with the lab of Frank Lyko (DKFZ Heidelberg) to understand how tRNA methylation influences protein synthesis (Tuorto et al., 2012; Tuorto et al., 2015). In a collaboration with Aurelio Teleman (DKFZ Heidelberg), we also contributed to the description of DENR-MCT1 as a factor important for translation re-initiation following upstream open reading frames (Schleich et al., 2014).