10:15am - 10:30am
Dicer-Like Proteins with Sequence Cleavage Preference
Institute of Cell Biology, University of Bern, Switzerland
During the sexual development, Paramecium eliminates thousands of germline specific sequences from the somatic genome (Internal eliminated sequences, IESs) with the help of two classes of small RNAs. The so-called scan and iesRNAs are produced by three Dicer-like enzymes and contain characteristic signature motifs. scanRNAs are produced by Dcl2 and Dcl3, they are 25nt long and contain a 5’ UNG signature. Indirect evidences suggest that scanRNAs are produced from non-coding transcripts from the germline genome. iesRNAs are produced by Dcl5 and contain 5’ UAG and a 3’ CNAUN motif. Recently, it was shown that iesRNAs are produced from excised IESs, which form concatemers and circularize upon excision in order to be transcribed. Although iesRNAs are produced from concatemers, most of the iesRNAs map to the ends of IESs and not to IES-IES junctions.
In our study, we show that the signature motifs from both small RNA classes result from specific sequence preferences of the Dicer-like proteins while processing their dsRNA template. We could show that Dcl5 has a preference for UAG and a general preference for IES ends. This specific cleavage preference of Dcl5 ensures that most iesRNAs are produced from the ends of IESs supporting the precise excision. Moreover, we could also show that Dcl3 has a sequence preference for UNG and Dcl2 has a strict size preference of 25 nucleotides.
10:30am - 10:45am
Investigation of MET Oncogene Addiction and the MET-DDR Crosstalk: A Phosphoproteomic Approach
1Department of Radiation Oncology, Inselspital, Bern University Hospital and University of Bern, Switzerland; 2Institute of Molecular Systems Biology, ETH Zurich, Switzerland; 3Department for BioMedical Research, University of Bern, Switzerland; 4Faculty of Science, University of Zurich, Switzerland
The MET receptor tyrosine kinase has acquired widespread attention in the field of cancer research. Indeed, its role in promoting oncogesis, invasiveness and metastatic events has been well established. Moreover, MET inhibition (METi) was found to confer radiosensitivity to MET-addicted cell lines, potentially through the re-wiring of the DNA damage response (DDR) machinery. The concept of oncogene addiction postulates that, despite the diverse array of genetic lesions characterizing cancer cells, some tumors rely on individual oncogenes to sustain survival. While the importance of this concept was proven in basic research as well as in therapeutic settings, the molecular pathways underlying this phenomenon remain poorly understood. Therefore, the major aim of this project is to investigate oncogene addiction further, particularly through the identification of crucial players involved in oncogene-mediated apoptosis and in the crosstalk with the DDR. To this purpose, MET was used as a model and further investigated by targeted proteomics based on Selected Reaction Monitoring (SRM) in order to monitor key phosphorylation changes in MET-positive cellular models upon METi, IR and the combinational regime. Remarkably, METi and IR appear to regulate key phosphorylations synergistically in MET-addicted cellular models. Moreover, METi alone seems to control DDR-related phosphorylation events in MET-addicted cell lines, as well as a plethora of other cellular processes. Finally, subsequent analysis of cellular models addicted to other oncogenes upon exposure to their respective inhibitors seems to suggest that oncogene addiction is governed by shared dominant pathways that, once disrupted, lead to radiosensitivity. In this respect, the identification of a composite phosphosignature for oncogene addiction would allow more accurate patient stratification and personalized clinical settings, both for single agents as well as for combination therapies.
10:45am - 11:00am
Site-Directed Mutagenesis of Pestiviral RNase and Its Effect on Innate Immune Evasion
1Institute of Virology and Immunology, Vetsuisse Faculty, University of Bern, Switzerland; 2Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Switzerland.; 3Graduate School for Cellular and Biomedical Sciences, University of Bern, Switzerland.
Bovine virus diarrhea virus (BVDV), a pestivirus in the family Flaviviridae, causes persistent infection (PI) in cattle by entering the fetus early in gestation prior to development of adaptive immunity. The inhibition of interferon (IFN) type I synthesis plays an important role in escaping innate immunity and is a prerequisite for the birth of PI calves. Thus, pestiviruses express two IFN antagonists, Npro and Erns. In infected cells, the non-structural protein Npro inhibits IFN induction by inducing the degradation of the transcription factor IRF-3. By contrast, the structural envelope glycoprotein Erns gets also secreted from infected cells in a soluble form and, by virtue of its endoribonuclease activity, it is able to degrade its own viral RNA in order to prevent the activation of an IFN response also in non-infected cells.
Here, we show that the glycosaminoglycan (GAG) binding site located at the C-terminus of Erns is important for its IFN antagonism. We previously showed that Erns is endocytosed via clathrin-mediated endocytosis and that this cellular uptake is required to prevent IFN induction. Substitution of all four positive charged lysine residues in the GAG binding site of Erns to neutral alanines resulted in a complete loss of its activity against IFN induction. In addition, this mutant could also not be detected in cells by immunofluorescence staining. This effect was only moderate when mutating only one or two lysines instead of all four. As all of the mutants were still able to degrade dsRNA in an in vitro RNase assay, we conclude that in addition to its RNase activity, the correct localization of Erns inside the cells is crucial for its IFN antagonistic effect.
With this data, we predict that in a PI animal, Erns is secreted from infected cells followed by endocytosis also into non-infected cells in order to prevent aberrant induction of an innate immune response by Npro and Erns in infected and non-infected cells, respectively.
11:00am - 11:15am
Mitochondrial Protonpower: Protonmotive Force and ATP Synthesis
1Department of Chemistry and Biochemistry, University of Bern, Switzerland; 2Department of Biochemistry and Biophysics, Stockholm University, Sweden; 3Research Group Mitochondrial Structure and Dynamics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
In the inner membrane of mitochondria, the respiratory enzymes pump protons out of the organelle to maintain the protonmotive force essential for the synthesis of adenosine triphosphate by the ATP synthase. Since the ATP synthase forms rows of dimers along the edges of cristae and protons move faster along the membrane surface than to the bulk, Davies et al. proposed that the arrangement of the respiratory complexes and ATP synthase allow the generation of a lateral proton gradient at the membrane surface. This suggestion is particularly tempting, since the bulk-to-bulk proton gradient between the cytoplasm and the matrix is usually very low (<0.5 pH unit).
In the present project, the group of Martin Ott probed the pH at the level of the crista edges of yeast mitochondria, using a pH-sensitive GFP, with extensive controls. The localization of the pH probe was also confirmed by the group of Stefan Jakobs using high-resolution microscopy techniques. Their results confirm that there is no bulk-to-bulk proton gradient in the cristae. This, however, would not contradict the proposition of Davies et al., because the hypothetical lateral proton gradient is very thin and hence out of reach for the pH probes used so far.
Nevertheless the proposition is still challenged by an alternative hypothesis: the membrane potential in mitochondria is very high (~170 mV) and could compensate for the low proton gradient. To test this, we have reconstituted the yeast ATP synthase into liposomes and tried to trigger ATP synthesis by applying different proton gradient and membrane potential values, in presence or absence of a respiratory enzyme to pump protons. Our results so far show that the membrane potential fails to compensate for low proton gradient values and that substantial ATP synthesis is only achieved when proton pumping takes place, likely because pumped protons would rapidly form a lateral proton gradient.