Project acronym FRONTIERS OF RNAI
Project The role of RNA silencing in immunity and development in eukaryotes
Researcher (PI) Olivier Voinnet
Host Institution (HI) EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH
Country Switzerland
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary RNA silencing is a pan-eukaryotic gene regulation process that involves RNA molecules 19-30nt in length. These molecules are produced by RNAse-III proteins in the Dicer family and engage into sequence-specific regulation of complementary DNA or RNA upon their incorporation into effector complexes. RNA silencing serves essential roles in biology but the molecular bases of its mechanisms are still poorly understood. One major aspect of the proposed project is to decipher genetically the composition of RNA silencing effector complexes and to understand how those complexes orchestrate the regulation of fundamental processes involved in cell differentiation, notably the process of dosage compensation during chromosome X inactivation in mammals. The second aspect is part of our ongoing efforts to understand the implication of small RNAs in plant and animal innate immunity, their impact on pathogen’s fitness and evolution, and how pathogens counteract small RNA-directed immune pathways.
Summary
RNA silencing is a pan-eukaryotic gene regulation process that involves RNA molecules 19-30nt in length. These molecules are produced by RNAse-III proteins in the Dicer family and engage into sequence-specific regulation of complementary DNA or RNA upon their incorporation into effector complexes. RNA silencing serves essential roles in biology but the molecular bases of its mechanisms are still poorly understood. One major aspect of the proposed project is to decipher genetically the composition of RNA silencing effector complexes and to understand how those complexes orchestrate the regulation of fundamental processes involved in cell differentiation, notably the process of dosage compensation during chromosome X inactivation in mammals. The second aspect is part of our ongoing efforts to understand the implication of small RNAs in plant and animal innate immunity, their impact on pathogen’s fitness and evolution, and how pathogens counteract small RNA-directed immune pathways.
Max ERC Funding
900 000 €
Duration
Start date: 2008-08-01, End date: 2012-07-31
Project acronym MRNA QUALITY
Project Quality control of gene expression: mechanisms for recognition and elimination of nonsense mRNA
Researcher (PI) Oliver Muehlemann
Host Institution (HI) UNIVERSITAET BERN
Country Switzerland
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary Analogous to quality control checks along the assembly line in industrial manufacturing processes, cells possess multiple quality control systems that ensure accurate expression of the genetic information throughout the intricate chain of biochemical reactions. “Nonsense-mediated mRNA decay” (NMD) represents a quality control mechanism that recognizes and degrades mRNAs of which the protein coding sequence is truncated by the presence of a premature termination codon (PTC). By eliminating these defective mRNAs with crippled protein-coding capacity, NMD substantially reduces the synthesis of potentially deleterious truncated proteins. Given that 30 % of all known disease-causing mutations in humans lead to the production of a nonsense mRNA, NMD serves as an important modulator of the clinical manifestations of genetic diseases, and manipulating NMD therefore represents a promising strategy for future therapies of many genetic disorders. However, the underlying molecular mechanisms of NMD are currently not well understood. One goal of our research is to understand at the molecular level how PTCs are recognized and distinguished from correct termination codons and how this recognition of nonsense mRNAs subsequently triggers their rapid degradation. In addition to triggering NMD, we have recently discovered that PTCs in certain immunoglobulin genes can also lead to the transcriptional silencing of the corresponding gene. We now search for the biological relevance of this novel quality control mechanism termed “nonsense-mediated transcriptional gene silencing” (NMTGS) and want to identify the involved molecules and their interactions. Using mainly mammalian cell cultures, we study the effect on the expression of engineered NMD and NMTGS reporter genes upon various treatments of the cells. State-of-the-art biochemical and molecular biology techniques are employed with the goal to further our understanding of these processes and their regulation at the molecular level.
Summary
Analogous to quality control checks along the assembly line in industrial manufacturing processes, cells possess multiple quality control systems that ensure accurate expression of the genetic information throughout the intricate chain of biochemical reactions. “Nonsense-mediated mRNA decay” (NMD) represents a quality control mechanism that recognizes and degrades mRNAs of which the protein coding sequence is truncated by the presence of a premature termination codon (PTC). By eliminating these defective mRNAs with crippled protein-coding capacity, NMD substantially reduces the synthesis of potentially deleterious truncated proteins. Given that 30 % of all known disease-causing mutations in humans lead to the production of a nonsense mRNA, NMD serves as an important modulator of the clinical manifestations of genetic diseases, and manipulating NMD therefore represents a promising strategy for future therapies of many genetic disorders. However, the underlying molecular mechanisms of NMD are currently not well understood. One goal of our research is to understand at the molecular level how PTCs are recognized and distinguished from correct termination codons and how this recognition of nonsense mRNAs subsequently triggers their rapid degradation. In addition to triggering NMD, we have recently discovered that PTCs in certain immunoglobulin genes can also lead to the transcriptional silencing of the corresponding gene. We now search for the biological relevance of this novel quality control mechanism termed “nonsense-mediated transcriptional gene silencing” (NMTGS) and want to identify the involved molecules and their interactions. Using mainly mammalian cell cultures, we study the effect on the expression of engineered NMD and NMTGS reporter genes upon various treatments of the cells. State-of-the-art biochemical and molecular biology techniques are employed with the goal to further our understanding of these processes and their regulation at the molecular level.
Max ERC Funding
1 300 000 €
Duration
Start date: 2008-06-01, End date: 2013-05-31
Project acronym PLANT-MEMB-TRAFF
Project Plant endomembrane trafficking in physiology and development
Researcher (PI) Niko Geldner
Host Institution (HI) UNIVERSITE DE LAUSANNE
Country Switzerland
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary Understanding the structure and function of endomembrane compartments is central to a mechanistic understanding of eukaryotic cell behavior. Multi-cellular organisms show an increased complexity and specialization in their endomembrane trafficking pathways. Higher plants have independently developed multi-cellularity and show a differently structured, but highly complex endomembrane system that regulates numerous, fundamental processes, such as cell wall composition, plant nutrition or immune responses. However, the specificities of plant endomembrane trafficking are only insufficiently addressed by homology-based approaches, which are inherently biased and limited to modules and pathways that are conserved between animals/yeast and plants. I propose to address this by undertaking forward genetic approaches for regulators of endocytic trafficking in Arabidopis with newly developed tools. In addition, I will establish the root endodermis as a model to address the mechanism of epithelial polarity establishment in plants. Epithelia are a fundamental feature of multi-cellular organisms and have independently evolved in plants and animals. The root endodermis is a tissue of central importance for plant nutrition. It is accessible to analysis and displays all the defining features of an epithelium. Studying the endodermis will allow me to investigate how independent or conserved the mechanisms of epithelial polarity are. Apart from the immediate interest for a number of plant developmental and adaptive responses, I contend that both parts of my proposal are also of general, fundamental interest. Current comparisons between yeast and animals do not give us any reliable and coherent idea about what is truly fundamental or derived in eukaryotic membrane organization. Unbiased research on plant membrane trafficking will provide insight into an additional, divergent type of eukaryotic cell and allow a better appreciation of the evolution of eukaryotic membrane organization.
Summary
Understanding the structure and function of endomembrane compartments is central to a mechanistic understanding of eukaryotic cell behavior. Multi-cellular organisms show an increased complexity and specialization in their endomembrane trafficking pathways. Higher plants have independently developed multi-cellularity and show a differently structured, but highly complex endomembrane system that regulates numerous, fundamental processes, such as cell wall composition, plant nutrition or immune responses. However, the specificities of plant endomembrane trafficking are only insufficiently addressed by homology-based approaches, which are inherently biased and limited to modules and pathways that are conserved between animals/yeast and plants. I propose to address this by undertaking forward genetic approaches for regulators of endocytic trafficking in Arabidopis with newly developed tools. In addition, I will establish the root endodermis as a model to address the mechanism of epithelial polarity establishment in plants. Epithelia are a fundamental feature of multi-cellular organisms and have independently evolved in plants and animals. The root endodermis is a tissue of central importance for plant nutrition. It is accessible to analysis and displays all the defining features of an epithelium. Studying the endodermis will allow me to investigate how independent or conserved the mechanisms of epithelial polarity are. Apart from the immediate interest for a number of plant developmental and adaptive responses, I contend that both parts of my proposal are also of general, fundamental interest. Current comparisons between yeast and animals do not give us any reliable and coherent idea about what is truly fundamental or derived in eukaryotic membrane organization. Unbiased research on plant membrane trafficking will provide insight into an additional, divergent type of eukaryotic cell and allow a better appreciation of the evolution of eukaryotic membrane organization.
Max ERC Funding
1 199 889 €
Duration
Start date: 2008-04-01, End date: 2013-03-31
Project acronym SINGLEMOLFOLDING
Project Towards Protein Folding in the Cell with Single Molecule Spectroscopy
Researcher (PI) Benjamin Schuler
Host Institution (HI) University of Zurich
Country Switzerland
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary Many aspects of the physical principles governing protein folding in vitro have been elucidated in the past decades. At the same time, a large number of cellular components involved in protein folding in vivo have been identified. But our mechanistic understanding of how these cellular components affect the energy landscape of the folding process has remained very limited, largely due to a lack of suitable methods. An opportunity to bridge this gap is single molecule fluorescence spectroscopy in combination with Förster resonance energy transfer (FRET), a new technique that allows the investigation of distance distributions and dynamics of individual protein molecules even in complex and heterogeneous environments. In a multi-disciplinary project employing methods ranging from molecular biology and protein biochemistry to optics, microfluidic mixing, and development of instrumentation and software, we plan to use single molecule spectroscopy for investigating the question how proteins find their native three-dimensional structure in a cell. Our initial focus will be on molecular chaperones, which assist protein folding in vivo, but the approach is applicable to a wide range of related phenomena and will be extended to other cellular components. This project is thus expected to nucleate a comprehensive biophysical analysis of intracellular protein folding and dynamics, eventually within live cells. A detailed investigation of these processes will be crucial for understanding the fine balance between protein folding and misfolding in the cell, and the large number of diseases associated with protein misfolding and aggregation.
Summary
Many aspects of the physical principles governing protein folding in vitro have been elucidated in the past decades. At the same time, a large number of cellular components involved in protein folding in vivo have been identified. But our mechanistic understanding of how these cellular components affect the energy landscape of the folding process has remained very limited, largely due to a lack of suitable methods. An opportunity to bridge this gap is single molecule fluorescence spectroscopy in combination with Förster resonance energy transfer (FRET), a new technique that allows the investigation of distance distributions and dynamics of individual protein molecules even in complex and heterogeneous environments. In a multi-disciplinary project employing methods ranging from molecular biology and protein biochemistry to optics, microfluidic mixing, and development of instrumentation and software, we plan to use single molecule spectroscopy for investigating the question how proteins find their native three-dimensional structure in a cell. Our initial focus will be on molecular chaperones, which assist protein folding in vivo, but the approach is applicable to a wide range of related phenomena and will be extended to other cellular components. This project is thus expected to nucleate a comprehensive biophysical analysis of intracellular protein folding and dynamics, eventually within live cells. A detailed investigation of these processes will be crucial for understanding the fine balance between protein folding and misfolding in the cell, and the large number of diseases associated with protein misfolding and aggregation.
Max ERC Funding
1 314 000 €
Duration
Start date: 2008-09-01, End date: 2014-08-31
Project acronym SUPERCOMPETITORS
Project GENETIC AND GENOMIC STUDY OF CELL COMPETITION IN DROSOPHILA
Researcher (PI) Eduardo Moreno
Host Institution (HI) UNIVERSITAET BERN
Country Switzerland
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary During initial stages a cell accumulating tumor-promoting mutations is usually surrounded by normal cells. Current cancer models do not incorporate this transition from a single cell to a field of cells, although it is probably critical, since the behavior of an individual tumor cell within the cell community has to be dictated by the hard-wired genetic program that controls its aberrant cell biology and modulated by the plastic interactions with neighboring normal cells. Study of model organisms, such as yeast, C. elegans, or Drosophila, has historically pioneered crucial contributions to processes with important implications in neoplasia. Recent work in Drosophila has proposed a role for cell-competition and super-competition in early stages of cancer formation. Cell competition is a type of cell-cell interaction in which more competitive cells replace less competitive cells (Morata and Ripoll, 1975; Moreno et al. 2002). During the last year, my laboratory has performed the first microarrays to find genes involved in cell competition, as well as developed an in vitro system for cell competition. The genes identified in the microarrays are not downstream dMyc but are rather induced at the boundaries where cell competition takes place and seem to be upstream apoptosis induction. The new genes will be studied in vivo in Drosophila and in vitro with RNAi. We will also perform other microarray settings to subdivide the genes in different categories. Thanks to the complete genome sequences of both Drosophila and humans, those genes could be used to find human homologues that could serve as novel markers and targets for the detection and/or treatment of cancer at earlier stages. The possible use of cell competition as a tool for cell replacement will also be pursued. We expect to patent at least two or three of the novel uncharacterized genes with human homologs.
Summary
During initial stages a cell accumulating tumor-promoting mutations is usually surrounded by normal cells. Current cancer models do not incorporate this transition from a single cell to a field of cells, although it is probably critical, since the behavior of an individual tumor cell within the cell community has to be dictated by the hard-wired genetic program that controls its aberrant cell biology and modulated by the plastic interactions with neighboring normal cells. Study of model organisms, such as yeast, C. elegans, or Drosophila, has historically pioneered crucial contributions to processes with important implications in neoplasia. Recent work in Drosophila has proposed a role for cell-competition and super-competition in early stages of cancer formation. Cell competition is a type of cell-cell interaction in which more competitive cells replace less competitive cells (Morata and Ripoll, 1975; Moreno et al. 2002). During the last year, my laboratory has performed the first microarrays to find genes involved in cell competition, as well as developed an in vitro system for cell competition. The genes identified in the microarrays are not downstream dMyc but are rather induced at the boundaries where cell competition takes place and seem to be upstream apoptosis induction. The new genes will be studied in vivo in Drosophila and in vitro with RNAi. We will also perform other microarray settings to subdivide the genes in different categories. Thanks to the complete genome sequences of both Drosophila and humans, those genes could be used to find human homologues that could serve as novel markers and targets for the detection and/or treatment of cancer at earlier stages. The possible use of cell competition as a tool for cell replacement will also be pursued. We expect to patent at least two or three of the novel uncharacterized genes with human homologs.
Max ERC Funding
970 100 €
Duration
Start date: 2008-09-01, End date: 2013-08-31
