Isolierung und Charakterisierung von gewebs- und entwicklungs-spezifischen mikroRNAs von Drosophila, Maus und Mensch

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants, animals and viruses. These small RNAs provide specificity to protein effector complexes mediating posttranscriptional silencing. The Caenorhabditis elegans lin-4 gene was the first miRNA characterized and for a long time was deemed to be unique to nematodes. Later, the let-7 miRNA was discovered in C. elegans, and it was found to be evolutionary conserved in the genomes of Drosophila melanogaster and human. This work describes the cloning of small RNAs from Drosophila, mouse and human. Among the cloned RNAs was let-7 and many novel invertebrate and mammalian miRNAs.Several miRNAs were found to be tissue-specific and developmentally regulated. Besides miRNAs, repeat-associated small interfering RNAs were cloned from Drosophila; this class of small regulatory RNAs derived from transposons and other repeat regions from fly. During the last few years miRNAs have been further characterized by many laboratories. Their mode of expression and biosynthesis pathway have been studied in depth, and proteins involved in the miRNA-guided silencing pathway have been identified. The current understanding of miRNA and related RNA silencing phenomena is presented. In addition, this work describes the optimization and validation of the cloning procedure used for the discovery of miRNAs. The improvements of the protocol are discussed in the context of other small RNA profiling methods