In Sophocles' Oedipus the King, the citizens of Thebes are cursed by a plague that will end only when the murderer of Laius, their former ruler, is identified and banished. The culprit is eventually revealed to be none other than the current king (and Laius' son), Oedipus. Although there are many things to be learned from this drama, one conclusion relates directly to the process of discovery: The object of an intensive search sometimes turns out to be hiding in plain view. So it is with the study of RNA interference (RNAi), as illustrated by two papers in this issue by Liu et al. on page 1437 (1) and by Song et al. on page 1434 (2). These authors now identify the long-sought catalytic subunit that executes RNAi, and show that it has been staring us in the face for years.
In most eukaryotes, RNAi is one of several mechanisms that silence the expression of specific genes in response to double-stranded RNA (dsRNA) (3). Within cells, the dsRNA silencing triggers are cleaved into 21- to 23-nucleotide short interfering RNA (siRNA) fragments. These fragments then associate with a large protein assembly called the RNA-induced silencing complex (RISC) (see the figure). An siRNA within RISC recognizes specific messenger RNAs (mRNAs) through base pairing, and in this way guides RISC to the appropriate targets. The complex harbors a catalytic activity that specifically cleaves the bound mRNA without affecting the guide siRNA. RISC was first discovered 4 years ago (4), and the race to identify its resident proteins--especially the catalytic subunit "Slicer"--has been raging ever since.
The first protein subunit discovered in RISC was Argonaute2 (5), one of a family of Argonaute proteins. Members of the Argonaute family are defined by the presence of PAZ and PIWI domains (6). The PAZ domain was recently shown to be important for binding to RNA (probably siRNA) (7), but beyond that the biochemical functions of Argonaute proteins within RISC remained elusive. The Hannon and Joshua-Tor laboratories have now used a battery of biophysical, biochemical, and genetic approaches to examine Argonaute structure and function (1, 2). In doing so, they have lifted our understanding of RNAi to a new level. Most notably, they provide compelling evidence that human Argonaute2 (and, by extension, other Argonaute proteins in different species) is the elusive Slicer subunit.
Song et al. (2) have solved the crystal structure of a complete Argonaute ortholog from the archaebacterium Pyrococcus furiosis. The PAZ domain sits atop a crescent composed of three other domains (including PIWI) and matches the structures of other well-characterized PAZ domains. Most tellingly, the tertiary structure of the PIWI domain clearly resembles that of ribonuclease H (RNase H) enzymes, which cleave the RNA strand of RNA/DNA hybrid duplexes. The structural similarity includes three carboxylate residues that are thought to bind and position a catalytically important divalent metal ion. The proposed catalytic site lies at the edge of a positively charged groove that extends into the PAZ domain, providing a plausible binding site for the siRNA/mRNA substrate duplex. Because RISC and RNase H are both metal-dependent enzymes that cleave one specific strand of a nucleic acid duplex and leave chemically similar termini in the products, the structural similarity immediately suggests that the PIWI domain may harbor RISC's target mRNA cleavage activity.
Of course, functional conclusions require functional data, and to this end Liu et al. (1) generated mammalian cells that express epitope-tagged versions of four human Argonaute proteins. Although siRNAs and miRNAs (microRNAs; endogenous RNAs that silence gene expression via translational control) bind to all four tagged proteins, only Argonaute2 was associated with cleavage of target mRNAs. This suggests that the other Argonautes might operate in different forms of RNA silencing. The cleavage activity remained even when the immunoprecipitates were washed under harsh conditions, indicating that it associates tightly with Argonaute2 and may reside within Argonaute2 itself. Similar conclusions have also been reported recently by Meister et al. (8). Liu et al. further demonstrate that Argonaute2 is essential for mouse embryonic development. Mice deficient in Argonaute2 display multiple abnormalities including defects in neural tube closure and heart development. Furthermore, cells cultured from Argonaute2-deficient mice fail to mount an RNAi response upon siRNA transfection, consistent with a role for Argonaute2 in RISC.
The crystal structure of Argonaute suggests that these proteins are nucleases, and functional data indicate that mRNA target cleavage activity is associated specifically with Argonaute2. Thus, Liu et al. proceeded to map some of the determinants of cleavage activity in Argonaute2. Several point mutations within the PIWI domain specifically blocked cleavage of target mRNAs without affecting Argonaute2 protein expression or siRNA binding. Notable among these are two of the three carboxylate residues proposed to bind to a catalytic metal ion. As the authors themselves note, these results do not formally prove that those residues constitute part of an active site; the mutations could block activity by disrupting an interaction with a separate enzymatic subunit. Nonetheless, when the mutagenesis results are combined with other functional data as well as with the structural similarity of RNase H to the PIWI domain, it makes for a compelling argument that human Argonaute2 is, in fact, the long-sought Slicer subunit of RISC. Thus, the answer to one of the RNAi field's most important questions appears to be in hand.
Naturally, many questions remain. What parts (if any) do Argonaute proteins play in earlier stages of the RNAi pathway, such as dsRNA cleavage and RISC assembly? Given that specific Argonautes are essential for distinct silencing pathways--including those that affect protein synthesis (9) and heterochromatin assembly (10)--how is functional specificity established for the different Argonaute family members? Do all Argonautes require or contain nuclease activity as part of their normal duties? A new entry into the nuclease lexicon is a reminder that a search for the familiar can lead to surprising discoveries.
