In the last years miRNAs have increasingly been recognised as potent posttranscriptional regulators of gene expression. Possibly, miRNAs exert their action on virtually any biological process by simultaneous regulation of numerous genes. The importance of miRNA-based regulation in health and disease has inspired research to investigate diverse aspects of miRNA origin, biogenesis, and function. Despite the recent rapid accumulation of experimental data, and the emergence of functional models, the complexity of miRNA-based regulation is still far from being well understood. In particular, we lack comprehensive knowledge as to which cellular processes are regulated by which miRNAs, and, furthermore, how temporal and spatial interactions of miRNAs to their targets occur. Results from large-scale functional analyses have immense potential to address these questions. In this review, we discuss the latest progress in application of high-content and high-throughput functional analysis for the systematic elucidation of the biological roles of miRNAs. 1. Introduction miRNAs (microRNAs) are 17-nt to 24-nt long noncoding RNAs that regulate gene expression in metazoans. miRNAs act by partially or completely complementary binding to their target mRNAs, resulting in translational repression and/or mRNA degradation [1, 2]. miRNAs are predicted to affect the expression of nearly 60% of protein-coding mammalian genes [3, 4] and, thereby, to control many, if not all, biological processes. Fundamental changes at the cellular and organismal level, including development [5], aging [6], the stress response [7], cell proliferation [8, 9], and apoptosis [10, 11], were shown to be regulated by miRNAs. Furthermore, miRNAs have been implicated in various diseases, such as diabetes [12–14], cancer [15, 16], hepatitis C [17], neurodevelopmental (reviewed in [18]), and mental [19] disorders. Rapidly growing knowledge of miRNAs as potent regulators in health and disease makes miRNAs attractive as targets for therapeutic intervention [20, 21] as well as for diagnostic markers [22, 23]. Numerous previous publications have addressed miRNA biogenesis and action (for detailed reviews see [24, 25]). Briefly, miRNAs are transcribed as long primary transcripts (pri-miRNAs), most of which are polyadenylated and capped. Pri-miRNAs are initially cleaved in nucleus by a multiprotein complex, called Microprocessor, yielding ~70-nt long stem-loop structured precursor miRNAs (pre-miRNAs). The key components of the Microprocessor complex are the RNase III enzyme Drosha and the double-stranded
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