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Unveiling the Molecular Basis of RNA Interference with Single Molecule Fluorescence

Final Report Summary - RNAI (Unveiling the Molecular Basis of RNA Interference with Single Molecule Fluorescence)

In the central dogma of molecular biology, RNA was traditionally viewed as a passive information carrier. Recent groundbreaking discoveries have changed this paradigm and a group of non-coding RNAs are now recognized as regulators of gene expression. MicroRNA (miRNA) is a short, single-stranded RNA of ~22 nucleotides. This non-coding RNA works as a regulatory component in eukaryotes, controlling embryonic development, apoptosis, tumorigenesis, anti-viral defense and other cellular functions. This regulation process (RNA interference or RNAi) occurs when a microRNA-loaded RISC (RNA-induced silencing complex) binds to a target mRNA. A microRNA acts as a guide of the RISC since it is base-pairing between a microRNA and an mRNA that directs the target recognition.
When small RNAs are exogenously introduced into cells, microRNA machineries are hijacked and the exogenous small RNAs trigger artificial RNAi. The action mechanism of RNAi is sequence-specific and thus endogenous genes can be manipulated with custom-designed small RNAs (small interference RNAs, siRNAs). This has addressed a possibility of using siRNAs for biomedical applications. With the human genome available in hand and RNA synthesis easily accessible, this has instigated small RNA-based therapeutics. However, several technical problems emerged. One of them was ‘off-target effects’ in which siRNAs target other mRNAs non-specifically and induce side effects. The immense potential of RNAi in therapeutics will be restored when deep understanding on the molecular origin of off-target effects is achieved and it leads to designing artifact-free assays. In order to strategically enhance fidelity in the basepairing between a small RNA and an mRNA, it is demanded to understand the RISC-mediated target recognition process. Effective delivery of designed siRNA strands to desired targets will be achieved by understanding how a small RNA is processed by RISC-associated proteins and incorporated into RISC and how small RNA-loaded RISC suppresses mRNA expression.
Using single molecule fluorescence, the Joo group discovered novel molecular mechanisms of microRNA. They made impact on understanding how microRNA is generated (Fareh et al, Nature Communication) and is regulated (Kim et al, EMBO Journal) and how microRNA finds target sequence (Chandradoss et al, Cell; Schirle et al, eLife; Klum et al, EMBO Journal). Furthermore they expanded this study to bacterial systems of how microDNA is generated (Swarts et al, Molecular Cell) and how CRISPR RNA find target sequence (Blosser et al, Molecular Cell). The fruitful outcome of this research will help realizing RNAi-based gene therapeutics in the near future.