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.