Double stranded RNA induces potent and specific gene silencing in a broad range of eukaryotic organisms. This mode of gene silencing, called RNA interference (RNAi), acts at the transcriptional level through formation of heterochromatin, and at the post-transcriptional leve,l through mRNA degradation and translational suppression. In all cases, RNAi begins with the processing of endogenous or introduced precursor RNA into micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) 21-25 nucleotides in length by the enzyme Dicer. The central objective of this project is to determine how RNA is recognized and cleaved by Dicer during induction of the RNAi pathway.
We aim to understand the structural and biochemical basis for Dicer activity, including its ability to recognize double stranded RNA targets, produce approximately 22- nucleotide duplex RNA products and deliver these products to downstream silencing pathways. Although miRNAs and siRNAs are the indispensable mediators of all RNAi gene silencing processes, the biochemical basis for their generation remains poorly understood. In order for Dicer to produce functional products from long duplex RNA precursors, the enzyme must coordinate two double stranded cleavages over a distance of 60 A. The structural mechanism underlying this coordination is entirely unknown. Well-diffracting crystals are in hand for an intact Dicer enzyme and will be the focus of initial structure determination efforts using X-ray crystallography. In parallel, a series of in vitro and in vivo experiments will be conducted to elucidate mechanisms of RNA recognition and catalysis by different kinds of Dicers. The proposed studies will reveal the molecular """"""""ruler"""""""" used to determine siRNA length and may enable the design of pre-siRNAs targeted to specific gene silencing pathways.
|Wilson, Ross C; Tambe, Akshay; Kidwell, Mary Anne et al. (2015) Dicer-TRBP complex formation ensures accurate mammalian microRNA biogenesis. Mol Cell 57:397-407|
|Sternberg, Samuel H; Redding, Sy; Jinek, Martin et al. (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507:62-7|
|Kidwell, Mary Anne; Chan, Jessica M; Doudna, Jennifer A (2014) Evolutionarily conserved roles of the dicer helicase domain in regulating RNA interference processing. J Biol Chem 289:28352-62|
|Staals, Raymond H J; Agari, Yoshihiro; Maki-Yonekura, Saori et al. (2013) Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus. Mol Cell 52:135-145|
|Lee, Ho Young; Zhou, Kaihong; Smith, Alison Marie et al. (2013) Differential roles of human Dicer-binding proteins TRBP and PACT in small RNA processing. Nucleic Acids Res 41:6568-76|
|Pattanayak, Vikram; Lin, Steven; Guilinger, John P et al. (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31:839-43|
|Koh, Hye Ran; Kidwell, Mary Anne; Ragunathan, Kaushik et al. (2013) ATP-independent diffusion of double-stranded RNA binding proteins. Proc Natl Acad Sci U S A 110:151-6|
|Taylor, David W; Ma, Enbo; Shigematsu, Hideki et al. (2013) Substrate-specific structural rearrangements of human Dicer. Nat Struct Mol Biol 20:662-70|
|Ma, Enbo; Zhou, Kaihong; Kidwell, Mary Anne et al. (2012) Coordinated activities of human dicer domains in regulatory RNA processing. J Mol Biol 422:466-76|
|Noland, Cameron L; Ma, Enbo; Doudna, Jennifer A (2011) siRNA repositioning for guide strand selection by human Dicer complexes. Mol Cell 43:110-21|
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