MicroRNAs (miRNAs) play a crucial role in regulating many biological processes by binding to the 3 ́-untranslated region of target mRNAs, thereby inducing translational repression or mRNA degradation. Although the principle mechanisms by which miRNAs mediate their biological effects are well known, detailed analysis of miRNA-dependent gene regulation revealed that the change in target gene expression is different under stress conditions. Our own preliminary data demonstrate that pharmacological inhibition or genetic deletion of miR-92a profoundly affects gene expression under ischemic stress in vivo, but has little effect under conditions of tissue homeostasis. Additionally, we showed that miRNA loading to the active and mRNA-associated high molecular weight RNA-induced silencing complex (HMW-RISC) was augmented after ischemic stress. Moreover, a striking cell type specificity of miRNA target regulation was observed. In the next funding period, we will determine the mechanisms underlying the cell type-specific and stress-dependent change in miRNA target regulation. In the first aim, we will determine how stress influences the association of miRNAs with the RISC complex. For that purpose, we will use cell culture models and elucidate the effects of hypoxia to mimic low oxygen conditions as occurring after infarction or in tumors. Subsequently, we will determine molecular mass profiles of Argonaute (Ago) protein complexes and assess their loading with miRNAs and mRNAs. Proteomic analysis of Ago immunoprecipitations will provide first insights into the changes of the complexes under stress conditions. This may include a change in protein composition and/or posttranslational modifications. Promising results will be validated and functionally investigated. In the second aim, we will determine cell type-specific effects of miRNAs using different cells lines and primary cells. MiRNA-target interactions will be studied by Ago2 UV crosslinking and immunoprecipitation. Resulting changes in target expression will be detected by RNA sequencing. We will identify the mechanisms underlying differential cell type-specific targeting, which may involve i) expression ranges at which mRNAs are most susceptible to miRNA regulation, ii) alternative 3 ́-UTR usage, iii) RNA binding proteins, which control miRNA binding sites, or iv) the interplay with competing endogenous RNAs. Finally, this project will also provide the cell and in vivo models to determine the function of novel light-regulated antimiRs developed in project A6.