Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Periodic Report Summary 1 - SNORD104 MICRORNA (SNORD104 gene-encoded microRNA and its role in immune homeostasis)

Our research focuses on the immunoregulatory function and molecular mode of action of murine and human small nucleolar RNA 104 (Snord104).
In a large small RNA profiling study Snord104-derived small RNA fragments have been identified to exhibit a cleavage and sequence distribution pattern compatible with microRNA processing and function in mice and man. In addition, preliminary findings of a phenotypic and functional analysis of Snord104 deficient mice and cells indicate an essential immunoregulatory function of Snord104: Mice lacking Snord104 develop a splenomegaly and lymphadenopathy in association with an enhanced frequency of germinal center B cells and memory T cells within secondary lymphoid organs; In agreement with this phenotype, Snord104 deficient lymphocytes exhibit enhanced proliferation and blasting properties in response to immune receptor stimulation in vitro.
Therefore, the goals of the research effort are to evaluate and prove microRNA function of murine and human Snord104, to identify and validate putative targets of Snord104 derived small RNA fragments, and to investigate the role of Snord104 in innate and adaptive immune responses as well as in autoimmune diseases.
Using Snord104 small RNA 3p and 5p fragment specific fluorescence-based artificial target constructs, Snord104 expression vectors and Snord104 small RNA fragment specific antagomirs, microRNA function of both Snord104-3p and -5p was evaluated and confirmed in vitro. Interestingly, with a length of 28 nucleotides, the human Snord104-3p represents the largest human small RNA sequence hitherto shown to execute microRNA function.
Transcriptome analysis of in vitro activated primary B cells from Snord104 competent and deficient mice identified the Ribosomal Protein S3 (RPS3) as putative target of Snord104. Interestingly, multiple putative Snord104-3p specific binding sites as well as a single Snord104-5p specific binding site were predicted by miRanda, a miRNA target prediction and interaction software. RPS3 as Snord104 target was experimentally validated and confirmed by using fluorescence-based RPS3 reporter constructs and by assessing endogenous RPS3 mRNA levels by RT-qPCR in cell lines transfected with Snord104-3p and 5p specific miRNA mimics. In addition, Snord104 prompter reporter constructs have been cloned to investigate the regulation of Snord104 expression and processing.
In summary, within the first reporting period we finally proved miRNA function of Snord104 derived small RNA fragments and identified and validated RPS3 as a Snord104 regulated target. Together with the phenotype of Snord104 deficient mice and cells, these findings indicate that Snord104 may executes its immunoregulatory function by tuning NF-kB activity through miRNA mediated regulation of RPS3.
The effect of Snord104-3p on NF-kB activity is currently evaluated in vitro. Furthermore, within the next support period the effect of Snord104 on adaptive immune responses and autoimmune diseases will be explored. To this end, Snord104 knockout and control animals will be immunized with various T cell-dependent and T cell-independent antigens and the levels of neutralizing antibodies will be determined by ELISA. In addition Snord104 knockout mice backcrossed to different genetic backgrounds will either be followed for the spontaneous development of autoimmunity or employed in models of induced autoimmunity to study the effect of Snord104 on the susceptibility to develop autoimmune diseases and to evaluate its effect on the course of the disease.
Overall, the evaluation of the immunoregulatory function of Snord104 will provide significant new insights into the regulation of NF-kB dependent immune receptor signaling, its impact on physiological and aberrant immune responses, and may points towards new strategies to modulate immune responses in health and disease.

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