The Doudna Lab

Exploring molecular mechanisms of RNA-mediated gene regulation

Eukaryotic RNA Interference

Double-stranded RNA induces potent and specific gene silencing in a broad range of eukaryotic organisms through a pathway known as RNA interference (RNAi). 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. We previously determined the crystal structure of an intact Dicer enzyme, revealing how Dicer functions as a molecular ruler to measure and cleave duplex RNAs of a specific length. Current work focuses on the mechanism of a complex of proteins known as the RISC loading complex (RLC) which load miRNA into the endonuclease Argonaute. The RLC contains the enzyme Dicer as well as TRBP, an RNA-binding protein hypothesized to interact with miRNA and Dicer during RISC loading. We seek to determine the molecular underpinnings of these interactions, along with the role of TRBP in RISC loading.

Eukaryotic Dicer. The structure of Giardia intestinalis Dicer shows how the enzyme works as a molecular ruler to generate siRNAs of the desired size. Read more...

A model of human Dicer. A model of the human Dicer enzyme based on known structures and EM data. Read more...

While it is known how human Dicer precisely measures and cleaves RNA, the role of its helicase domain remains a mystery. Dicer’s helicase belongs to a family of dynamic proteins known to remodel RNA and RNA/protein complexes by unwinding or moving along RNA duplexes. Interestingly, there are hints that this domain is primarily required for the siRNA, but not the miRNA, pathway. Understanding how Dicer’s helicase domain functions could give key insights into Dicer's role in RNA interference and perhaps its involvement in other biological pathways. We are therefore also pursuing functional studies of the Dicer helicase domain in both siRNA/miRNA processing and RISC loading. (This work is supported by the NIH.)