Another Piece of the Puzzle

RNA interference (RNAI) has generated much excitement in the life sciences community as a way to do genetic studies and as a potential therapeutic agent. In this process, short pieces of double-stranded RNA are incorporated into a protein complex known as the RNA-induced silencing complex (RISC), which catalyzes the cleavage of messenger RNA and thus suppresses gene expression.

Biologists have not known what part of RISC is responsible for the cleavage, dubbing the unidentified nuclease "Slicer." Now, scientists at Cold Spring Harbor Laboratory in Long Island, N.Y., have identified the protein Argonaute 2 as Slicer.

Argonaute proteins were known to be conserved and key components [of RISC], but there was no obvious reason to tag Argonaute as the nuclease," says biologist Gregory J. Hannon.

The team, led by Hannon and structural biologist Leemor Joshua-Tor, used a combination of X-ray crystallography and biochemical and genetic tests to confirm the role of Argonaute in RISC. They solved the X-ray crystal structure of the Argonaute protein obtained from the archaebacterium Pyrococcus furiosus [Science, published online July 29, http://www.sciencemag.org/cgi/content/abstract/1102514v1].

Characteristics of the structure helped pinpoint Argonaute as the portion responsible for mRNA cleavage. The protein has a crescent-shaped base with a domain at the center of the crescent that resembles the enzyme RNAse H.

This domain came as a surprise, Joshua-Tor says. "RNAse H enzymes specialize in cleaving RNA that is 'guided' by a DNA strand in a double-stranded RNA-DNA hybrid," she says. "Argonautes appear to specialize in single-stranded RNA cleavage--in particular, mRNA--guided by an RNA strand" in a double-stranded RNA substrate known as short interfering RNA (siRNA), which is incorporated into RISC.

The RNA strand probably binds in a distinct groove that is lined with positive charges suitable for interacting with the negatively charged RNA backbone. In a model, the researchers incorporated double-stranded RNA into this groove. The model suggests a cleavage site that matches what is observed with mRNAs in RNAi.

"Not only does the structure reveal Argonaute as the RISC nuclease, but it provides a concrete model for how cleavage is positioned in the target mRNA," Joshua-Tor says.

Four Argonaute proteins are found in mammals: Ago1 to Ago4. Using biochemical and genetic tests, the Cold Spring Harbor team finds that only Ago2 can catalyze mRNA cleavage [Science, published online July 29, http://www.sciencemag.org/cgi/content/abstract/1102513v1]. When selected amino acids in Ago2 were changed to match the corresponding amino acids in Ago1 or Ago3, the protein lost its ability to cleave mRNA.

There's plenty left to figure out about how RNAi works. "We still need to understand precisely how the siRNA and mRNA substrates fit into Argonaute, how Argonaute controls protein synthesis rather than mRNA cleavage, and how RNAi can regulate chromatin," Hannon says.