RISC is a 5′ phosphomonoester-producing RNA endonuclease
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Discussion
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Implication
Biochemical analysis uncovers that human RISC cleaves target RNA via hydrolysis producing 5'-phosphate ends, indicating product release or conformational shifts limit reaction rates.
Key Points
- To elucidate the catalytic mechanism, reaction products, and structural requirements of target RNA cleavage mediated by human RNA-induced silencing complex (RISC).
- Purified minimal RISC from human cells using affinity purification.
- Assayed cleavage activity against target RNAs bearing site-specific 2'-deoxy and 2'-O-methyl chemical modifications as well as length and sequence variations.
- RISC cleaved target RNA through hydrolysis, yielding 3'-hydroxyl and 5'-phosphate termini.
- Substitution of the cleavage-site 2'-hydroxyl with 2'-deoxy did not alter cleavage rates, whereas 2'-O-methyl substitution substantially reduced cleavage activity.
- Target RNAs as short as 15 nucleotides were cleaved efficiently, and mismatches opposite the 5' or 3' ends of the siRNA had minimal impact on cutting efficiency.
Cite This Study
Martı̂nez et al. (2004) studied this question.