The small RNA profile is a signature of RNA silencing processes. The availability of these profiles is the first important step to dissect gene regulation mediated by endogenously expressed double-stranded RNA (dsRNA). The most intriguing sources of dsRNA in mammalian systems are microRNA (miRNA) genes that encode short inverted repeat sequences that give rise to hairpin RNA precursors. The hairpin RNAs are processed to approximately 22-nucleotide (nt) miRNAs that target messages of protein-coding genes for cleavage or repression of translation. The emerging targets indicate the involvement of miRNAs and RNA silencing in numerous cellular and developmental processes, many of which may also be linked to genetic diseases. The control of transposable elements in germ line cells, and the regulation of epigenetic changes, for example during development of the immune system or in proliferate diseases, may involve small RNAs that target chromatin structures.
The specific aims of this grant are: 1. Recording of small RNA profiles and development of protocols that reduce sample size and processing time. 2. Development and application of biochemical and cell biological methods for the identification of target genes of RNA silencing in human and mouse. 2.1 Development of fractionation and immunoprecipitation techniques to isolate and clone the target RNA segments recognized by small RNAs, from cell lines and tissue. 2.2 Development of reporter-based systems to functionally validate miRNA/target RNA interactions.
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