Final Report Summary - SNORD104 MICRORNA (SNORD104 gene-encoded microRNA and its role in immune homeostasis) Over the last decades, microRNAs evolved as essential posttranscriptional regulators of gene expression affecting almost all biological processes including immune function. In addition, dysregulated microRNA expression can cause or contribute to a broad variety of pathological conditions and diseases. Almost three thousand microRNAs have been identified in man and a variable set of several hundred microRNAs is expressed in a single cell dependent on the current state of function, differentiation and activation. Due to redundant and pleiotropic effects of individual microRNAs, the complexity of the microRNA regulatory network remains largely elusive.In a large small RNA profiling study we identified small RNA fragments encoded by the small nucleolar RNA 104 (Snord104). Using murine and human in vitro cell culture and lentiviral based vector systems we evaluated and proved microRNA function of murine and human Snord104-encoded 3p and 5p small RNAs. Of note, the human Snord104-3p constitutes the hitherto longest microRNA with 28 nucleotides in length. In consecutive experiments we identified the ribosomal protein S3 (RPS3), an essential NF-kappaB binding partner as putative target of Snord104-encoded microRNAs. Employing in vitro cell culture and lentiviral gene transduction we validated RPS3 as target of murine and human Snord104-5p and 3p. Snord104 microRNA represents the second microRNA only to target a specific transcript with both, its 5p and 3p arm. In agreement with the number of predicted Snord104-5p and -3p binding sites within the RPS3 transcript, the inhibitory effect of 3p on the RPS3 expression is 10-100 times stronger than the effect of the 5p. Finally, we confirmed the indirect regulatory effect of Snord104 microRNAs on NF-kappaB activity in vitro.In parallel to the in vitro experiments addressing microRNA function and regulation of RPS3 and NF-kappaB activity by Snord104 microRNAs, we reconstituted Snord104 deficient mice and backcrossed them to C57/BL6 background for phenotypic analysis. Snord104 knockout mice on a pure C57/BL6 background exhibit a phenotype that is fully compatible with the non-redundant NF-kappaB regulatory function of Snord104 microRNAs observed in vitro. Snord104 deficient B cells exhibit an enhanced proliferative and blasting response to immune receptor stimulation compared to wild type B cells. In addition, Snord104 knockout mice have a higher frequency of germinal center B cells and memory CD4 T cells. Furthermore, Snord104-deficient lymphocytes express higher levels of CD95 (Fas) than lymphocytes from C57/BL6 wild type mice. Interestingly the phenotype of Snord104 knockout mice becomes more prominent during progressive aging of the mice supporting the hypothesis of an enhanced sensitivity of Snord104 deficient cells for immune receptor stimulation. In all in vitro experiments and analysis of cells ex vivo, Snord104 heterozygous mice on a pure C57/BL6 background exhibit an intermediate phenotype indicating haploinsufficiency. The fact, that the phenotype of Snord104 knockout mice resembles the phenotype of RPS3 transgenic mice further supports the essential NF-kappaB regulatory effect of Snord104 microRNAs by targeting RPS3.In summary, we identified and validated Snord104 microRNAs as new and non-redundant factors of the NF-kappaB regulatory network. Our work contribute to a better understanding of the complex and still partially elusive NF-kappaB regulatory network that is essential for immune homeostasis. To our knowledge, Snord104 knockout mice are the first and only animals exhibiting a constitutively reduced immune receptor activation threshold. Therefore, Snord104 knockout mice constitute a unique and valuable animal model to study the effect of immune receptor signaling strength on innate and adaptive immune responses and to evaluate the effect of Snord104 regulated NF-kappaB activity on pathogen clearance and the predisposition to and severity of autoimmune diseases.
