Enzyme’s structure helped elucidate RNAi’s mechanism

Ten years ago, researchers obtained the first crystal structure of Dicer, a key enzyme in a biological process called RNA interference (RNAi). In RNAi, a process also known as gene silencing, a short RNA binds to mRNA, preventing mRNA’s sequence from being transcribed into a protein.

The determination of Dicer’s structure by Jennifer A. Doudna of the University of California, Berkeley, and coworkers helped confirm how the enzyme processes RNA and thus advanced RNAi technology (Science 2006, DOI: 10.1126/science.1121638). More recently, Doudna and coworkers also helped discover CRISPR-Cas9, a technology with broader gene-editing applications.

RNAi was discovered about 20 years ago by Andrew Z. Fire and Craig C. Mello when they were researchers at the Carnegie Institution of Washington. Fire and Mello won a 2006 Nobel Prize for this work because it revealed RNAi to be a previously unknown mechanism for gene regulation and opened up the potential for a new class of antisense oligonucleotide therapeutics.

Dicer kick-starts the RNAi process by cleaving either a type of hairpin RNA, called precursor miRNA, or double-stranded RNA. The cleavage products—microRNA (miRNA) and small interfering RNA (siRNA), respectively—are approximately 22-nucleotide double-stranded RNA fragments. A protein named Argonaute 2 then takes up these fragments, releases one strand (the passenger) of each fragment, and uses the other strand (the guide) to bind target mRNA. Finally, Argonaute 2 cleaves the target mRNA, silencing the gene it encodes.

The Doudna group’s Dicer structure showed that a formation called the “handle,” located between the enzyme’s PAZ and RNase III domains, is about the same length as 25 RNA nucleotides. This helped confirm that Dicer acts as a molecular ruler, cleaving its RNA substrates into approximately handle-sized pieces during the first step of RNAi. The study also helped confirm that two magnesium ions in each of the enzyme’s two RNase III domains play essential roles in its mechanism of action.

Since its discovery, RNAi has been used as a research tool to study biological processes such as cancer and cell differentiation. It is also being developed commercially today as a means to block the production of disease-related proteins and potentially as a means to control insect pests in agriculture. But the Dicer structure per se didn’t help lead to RNAi therapeutics, which “bypass Dicer by delivering RNA that is already cut like the Dicer product,” explains Judy Lieberman of Boston Children’s Hospital, whose research interests include RNAi.

A number of these Dicer-bypassing therapeutics—siRNAs—are now in the clinical trials pipeline for various diseases. But RNAi agents have had cell-access problems. “The real obstacle is getting them across the cell membrane” and released inside target cells, Lieberman says. Chemically conjugating siRNAs with a sugar recognized by a liver cell receptor or encapsulating siRNAs into lipid nanoparticles have largely solved these problems for the liver, but not other tissues in the body, she says.

So no RNAi agents have yet been approved for clinical use. Nevertheless, Lieberman says, “I remain cautiously optimistic that they will be a new class of very powerful drugs. We will know more in the coming year.”