Structural basis for the sequence-specific RNA-recognition mechanism of human CUG-BP1 RRM3
- The CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-like factors (CELF) family or the Bruno-like family and is involved in the control of splicing, translation and mRNA degradation. Several target RNA sequences of CUG-BP1 have been predicted, such as the CUG triplet repeat, the GU-rich sequences and the AU-rich element of nuclear pre-mRNAs and/or cytoplasmic mRNA. CUG-BP1 has three RNA-recognition motifs (RRMs), among which the third RRM (RRM3) can bind to the target RNAs on its own. In this study, we solved the solution structure of the CUG-BP1 RRM3 by hetero-nuclear NMR spectroscopy. The CUG-BP1 RRM3 exhibited a noncanonical RRM fold, with the four-stranded b-sheet surface tightly associated with the N-terminal extension. Furthermore, we determined the solution structure of the CUG-BP1 RRM3 in the complex with (UG)3 RNA, and discovered that the UGU trinucleotide is specifically recognized through extensive stacking interactions and hydrogen bonds within the pocket formed by the b-sheet surface and the N-terminal extension. This study revealed the unique mechanism that enables the CUG-BP1 RRM3 to discriminate the short RNA segment from other sequences, thus providing the molecular basis for the comprehension of the role of the RRM3s in the CELF/Bruno-like family.
SBE13, a newly identified inhibitor of inactive polo-like kinase 1
- Poster presentation at 5th German Conference on Cheminformatics: 23. CIC-Workshop Goslar, Germany. 8-10 November 2009 Protein kinases are important targets for drug development. The almost identical protein folding of kinases and the common co-substrate ATP leads to the problem of inhibitor selectivity. Type II inhibitors, targeting the inactive conformation of kinases, occupy a hydrophobic pocket with less conserved surrounding amino acids . Human polo-like kinase 1 (Plk1) represents a promising target for approaches to identify new therapeutic agents. Plk1 belongs to a family of highly conserved serine/threonine kinases, and is a key player in mitosis, where it modulates the spindle checkpoint at metaphase/anaphase transition. Plk1 is over-expressed in all today analyzed human tumors of different origin and serves as a negative prognostic marker in cancer patients. The newly identified inhibitor, SBE13, a vanillin derivative, targets Plk1 in its inactive conformation . This leads to selectivity within the Plk family and towards Aurora A. This selectivity can be explained by docking studies of SBE13 into the binding pocket of homology models of Plk1, Plk2 and Plk3 in their inactive conformation. SBE13 showed anti-proliferative effects in cancer cell lines of different origins with EC50 values between 5 microM and 39 microM and induced apoptosis. Increasing concentrations of SBE13 result in increasing amounts of cells in G2/M phase 13 hours after double thymidin block of HeLa cells. The kinase activity of Plk1 was inhibited with an IC50 of 200 pM. Taken together, we could show that carefully designed structure-based virtual screening is well-suited to identify selective type II kinase inhibitors targeting Plk1 as potential anti-cancer therapeutics.
Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain
- Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg2+ for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gswloop) of the guanine-sensing riboswitch and their Mg2+-induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gswloop or the increase in temperature for both Gsw and Gswloop involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg2+. We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.
Metal-ion binding and metal-ion induced folding of the adenine-sensing riboswitch aptamer domain
- Divalent cations are important in the folding and stabilization of complex RNA structures. The adenine-sensing riboswitch controls the expression of mRNAs for proteins involved in purine metabolism by directly sensing intracellular adenine levels. Adenine binds with high affinity and specificity to the ligand binding or aptamer domain of the adenine-sensing riboswitch. The X-ray structure of this domain in complex with adenine revealed an intricate RNA-fold consisting of a three-helix junction stabilized by long-range base-pairing interactions and identified five binding sites for hexahydrated Mg2+-ions. Furthermore, a role for Mg2+-ions in the ligand-induced folding of this RNA was suggested. Here, we describe the interaction of divalent cations with the RNA–adenine complex in solution as studied by high-resolution NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping and intermolecular nuclear Overhauser effects (NOEs) indicate the presence of at least three binding sites for divalent cations. Two of them are similar to those in the X-ray structure. The third site, which is important for the folding of this RNA, has not been observed previously. The ligand-free state of the RNA is conformationally heterogeneous and contains base-pairing patterns detrimental to ligand binding in the absence of Mg2+, but becomes partially pre-organized for ligand binding in the presence of Mg2+. Compared to the highly similar guanine-sensing riboswitch, the folding pathway for the adenine-sensing riboswitch aptamer domain is more complex and the influence of Mg2+ is more pronounced.
Post-transcriptional regulation of 5-lipoxygenase mRNA expression via alternative splicing and nonsense-mediated mRNA decay
Bernd Lothar Sorg
- 5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes (LT), a group of inflammatory lipid mediators derived from arachidonic acid. Here, we investigated the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD). In the present study, we report the identification of 2 truncated transcripts and 4 novel 5-LO splice variants containing premature termination codons (PTC). The characterization of one of the splice variants, 5-LOΔ3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LOΔ3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression.
Interplay between N-WASP and CK2 optimizes clathrin-mediated endocytosis of EGFR
- Clathrin-mediated endocytosis (CME) involves spatially and temporally restricted molecular dynamics.
Although protein kinases and the actin cytoskeleton contribute to the process, whether and how
functions of kinases and actin are integrated remains unknown. Here, we demonstrate that neural
Wiskott-Aldrich syndrome protein (N-WASP) and protein kinase CK2 form a complex and localize on
clathrin-coated vesicles (CCVs). N-WASP binds to and is phosphorylated by CK2, thereby reducing the
kinase activity of CK2. By contrast, N-WASP-promoted actin polymerization is decreased upon both
phosphorylation and binding of CK2. Knockdown of N-WASP and CK2, alone or in combination, results
in impaired endocytosis of epidermal growth factor (EGF) and increased cell-surface levels of EGF
receptor (EGFR). In order to rescue the phenotype of N-WASP-CK2 knockdown cells, both N-WASP and
CK2 activities and abilities to assemble in a complex are required. In summary, this study shows that the
N-WASP-CK2 complex integrates in a single circuit different activities contributing to CME of EGFR and
that the interplay between the two proteins optimizes this process.
Habilitationsordnung der Mathematisch-Naturwissenschaftlichen Fachbereiche der Johann Wolfgang Goethe-Universität Frankfurt am Main vom 4. Februar 1992 : genehmigt durch Beschluss des Präsidiums der Johann Wolfgang Goethe-Universität Frankfurt am Main am 19. November 2013
Ordnung der Fachbereiche Medizin, Biowissenschaften, Biochemie, Chemie und Pharmazie sowie Psychologie und Sportwissenschaften der Johann Wolfgang Goethe[-Universität] für den Masterstudiengang Interdisciplinary Neuroscience mit dem Abschluss "Master of Science" (M. Sc.) vom 30. März 2009 in der Fassung vom 22. April 2009, zuletzt geändert am 16. April 2012 : genehmigt vom Präsidium der Johann Wolfgang Goethe-Universität Frankfurt a. M. am 27. April 2010 ; hier: Änderungen vom 08.11.2012 ; genehmigt vom Präsidium der Johann Wolfgang Goethe-Universität Frankfurt a. M. am 18. Dezember 2012
Ordnung der Fachbereiche Medizin, Biowissenschaften, Biochemie, Chemie und Pharmazie sowie Psychologie und Sportwissenschaften der Johann Wolfgang Goethe-Universität für den Masterstudiengang Interdisciplinary Neuroscience mit dem Abschluss "Master of Science" (M.Sc.) vom 30. März 2009 in der Fassung vom 22. April 2009 : genehmigt durch das Präsidium am 11. Oktober 2011 ; hier: Änderung der Wahlpflichtmodule vom 16. April 2012 ; genehmigt durch das Präsidium am 11. September 2012
Gemeinsame Ordnung der Fachbereiche Biochemie, Chemie und Pharmazie, Biowissenschaften, Gesellschaftswissenschaften sowie Geowissenschaften, Geographie der Johann Wolfgang Goethe-Universität Frankfurt am Main für den Masterstudiengang Umweltwissenschaften mit dem Abschlussziel Master of Science (M.Sc.) vom 21.11.2011 : genehmigt durch das Präsidium der Johann Wolfgang Goethe-Universität Frankfurt am Main am 20.03.2012