Project

Discipline

High-throughput sequencing; microarrays; microRNA; C. elegans; germ cell; cross-linker; NMR spectroscopy; surface plasmon resonance; translation regulation; bioinformatics; Protein-RNA interactions; RNA binding proteins; apoptosis; structural biology; oligonucleotide synthesis

Lead
Lay summary
Gene expression in eukaryotes is regulated at multiple levels. Whereas the mechanisms that regulate the early and late steps in this cascade - transcription and post-translational modifications - are well characterized, much less is known about the intervening steps. The regulatory processes controlling the fate of mRNAs in the cytosol are still poorly understood. It has become recently apparent that mRNA metabolism is heavily and dynamically regulated by RNA-binding proteins (RBPs) and the microRNAs (miRNAs). In this grant application, we propose to use a multi-disciplinary approach to construct a comprehensive view of the involvement of RBPs and miRNAs in the regulation of a physiologically relevant process: germ cell apoptosis (programmed cell death) in the nematode C. elegans. We chose this system for the following reasons. First, previous studies have shown that RBPs and miRNAs can regulate apoptosis in C. elegans. Second, C. elegans is a model organism that is easily amenable to genetic and reverse genetic studies. Third, translational control is particularly prevalent in the C. elegans germ line, controlling nearly every single fate decision and differentiation step. In order to construct a comprehensive view of this translational regulatory network, we have assembled a team of five research groups that covers a broad range of technical expertises. Our team includes a developmental geneticist (M. Hengartner) who will screen for novel RBPs and miRNAs involved in germ line apoptosis and perform in vivo functional characterization of novel targets. We will apply genomics tools to identify the in vivo targets of known and novel RBPs/miRNAs that regulate apoptosis (A. Gerber/University of Surrey) and through computational analyses will define common structural and functional features among the specific mRNAs targeted by the RBPs/miRNAs (M. Zavolan). Studies in vivo will be complemented by quantitative in vitro approaches to define the chemical determinants behind functional RBP/miRNA-mRNA interactions (J. Hall), and by the structural elucidation of several RNA-protein complexes (F. Allain). During the first three-years period on the current grant, we focused our effort on the biochemical, structural and functional characterization of two C. elegans RBP GLD-1 and GLA-3.   We also identified new apoptosis regulators and we developed new chemical methods to study RBP-RNA and miRNA-RNA interactions.  Building on the momentum created in this period, we aim in this second three-year period to finalize some of the long-term projects already initiated and to go beyond by establishing the regulatory network of RBP and miRNAs controlling the apoptotic outcome of a cell. Since RBP/miRNAs play important roles in a plethora of physiological processes, this multidisciplinary approach could become a 'landmark' attempt that may guide similar studies on other important biological processes. Finally, since many of the core components of the apoptosis network are evolutionarily conserved, the characterization of regulatory pathways in C. elegans may be relevant for further investigations of human diseases.

Direct link to Lay Summary

Last update: 21.02.2013

Number	Title	Start	Funding scheme

Gene expression in eukaryotes is regulated at multiple levels. Whereas the mechanisms that regulate the early and late steps in this cascade - transcription and post-translational modifications - have been studied in great detail, much less is known about the intervening steps. In particular, the regulatory processes controlling the fate of mRNAs in the cytosol (mRNA stability, subcellular localization, translation) are still poorly understood. In the last few years, it has become apparent that mRNA metabolism is heavily and dynamically regulated by RNA-binding proteins (RBPs) and microRNAs (miRNAs). Hundreds of RBPs and miRNAs are present in metazoan organisms, rivaling in number other classes of regulatory molecules such as transcription factors and kinases. Moreover, both RBPs and miRNAs appear to have on average between dozens and hundreds of targets. Consequently, it is estimated that at least two thirds of all human genes are regulated at the mRNA level by RBPs and miRNAs (Bartel, 2009). miRNAs preferentially regulate transcription factors and the RBPs frequently regulate their own mRNAs, suggesting that RBPs and miRNAs might be used to modulate coordinately almost every aspect of a cell's life. Aberrations in RBP or miRNA expression can readily lead to human diseases, further underscoring the importance of these regulatory molecules for proper development and homeostasis. As the significance of RBPs and miRNAs in the regulation of gene expression is increasingly recognized, the interest in characterizing their mode of action, the targets that they regulate, and how RBP and miRNA systems interact with each other is rising. In this Sinergia grant application, we propose to use a multi-disciplinary approach to construct a comprehensive view of the involvement of RBPs and miRNAs in the regulation of a physiologically relevant process: germ cell apoptosis in the invertebrate nematode C. elegans. We chose this system for the following three reasons. First, previous studies have shown that RBPs and miRNAs can regulate apoptosis in a variety of species, including C. elegans (reviewed in (Gartner et al., 2008; Jovanovic and Hengartner, 2006)). Second, C. elegans is a model organism that is easily amenable to genetic and reverse genetic studies. Third, translational control is particularly prevalent in the C. elegans germ line, controlling nearly every single fate decision and differentiation step. In order to construct a comprehensive view of the translational regulatory network that controls germ line apoptosis, we have assembled a team of five research groups that covers a broad range of technical expertise from chemistry to physiology and computational modeling. Our team includes a developmental geneticist (M. Hengartner) who will screen for novel RBPs and miRNAs involved in germ line apoptosis and who will perform in vivo functional characterization of novel targets. We will apply genomics tools to identify the in vivo targets of known and novel RBPs/miRNAs that regulate apoptosis (A. Gerber) and through computational analyses we will define common structural and functional features among the specific mRNAs targeted by the RBPs/miRNAs (M. Zavolan). Studies in vivo will be complemented by quantitative in vitro approaches to define the chemical determinants behind functional RBP/miRNA-mRNA interactions. To date, genetics, bioinformatics, biology and biochemistry are the disciplines which have contributed most in this field. Chemistry has played a relatively minor role, yet, understanding the physiochemical nature of the interactions between mRNAs and RBPs/miRNAs which is one cornerstone of this Sinergia proposal, is essentially a chemical problem. We will bring chemistry to bear during this collaboration in three synergistic ways: investigation of protein-RNA interactions at the atomic level (NMR structure: F. Allain), generation of high quality libraries to enable the discovery of determinants of RBP specificity and the prediction of RBP targets transcriptome-wide with bioinformatics methods, and development of chemical tools which will allow us to isolate miRNA-mRNA and protein-RNA interactions in the cell (J. Hall). During the two-year period on the current grant, we focused our effort on the biochemical, structural and functional characterization of two C. elegans RBP GLD-1 and GLA-3. We identified of a new apoptosis regulator, C41G7.3 and we developed new chemical methods to study RBP-RNA and miRNA-RNA interactions. Building on the momentum created in this period, we aim in this second three-year period to finalize some of the long-term projects already initiated and to go beyond by establishing the regulatory network of RBP and miRNAs controlling the apoptotic outcome of a cell. Since RBP/miRNAs play important roles in a plethora of physiological processes, this multidisciplinary approach could become a 'landmark' attempt that may guide similar studies on other important biological processes. Finally, since many of the core components of the apoptosis network are evolutionarily conserved, the characterization of regulatory pathways in C. elegans may be relevant for further investigations of human diseases.