Argonaute is a key player in the formation of the RNA-induced silencing complex (RISC), a major component of RNA interference (RNAi). RNAi is a sequence-specific post-transcriptional gene silencing (PTGS) mechanism involved in a plethora of biological functions. RNAi is present in plants, fungi, insects, bacteria, and mammals. It has been shown to protect the host from viruses, and to suppress the activity of transposons, which are segments of DNA that can move from one location to another and sometimes cause abnormal gene products (9).
RNAi can be induced by double-stranded RNA (dsRNA) from exogenous sources or dsRNA transcribed from endogenous noncoding RNA. Endogenous RNAs are cleaved into microRNAs (miRNAs) in the nucleus by the enzyme Drosha. Exogenous RNAs are cleaved in the cytoplasm by the enzyme Dicer into short fragments, about 20 bases long, with charachteristic two-nucleotide 3' overhangs, called small interfering RNAs (siRNAs) (1, 2). Click here for a schematic overview of RNAi.
The siRNAs, whether produced by the cleaving action of Dicer or Drosha, are bound to the RNA-induced silencing complex (RISC) by their two-nucleotide 3' overhangs. Argonatue proteins are key components of the RISC. The Pyrococcus furiosus Argonaute protein (PfAgo) has been shown to bind the two-nucleotide 3' overhangs of siRNAs at the PfAgo Piwi Argonaute Zwille (PAZ) domain (1).
PfAgo, as well as mammalian Argonaute2 (3), have been shown to posess RNase H-like mRNA cleaving activity for which miRNAs and siRNAs are the substrates. (Click here to view a typical RNAse H protein from Archaeoglobus fulgidus.) Argonaute proteins associated with such cleaving activity, referred to as "Slicer" activity, enable the RISC to cleave mRNA as directed by siRNA or miRNA (1). However, the enzyme involved in Slicer activity has yet to be identified. In light of recent data, it seems highly probable that Argonautes involved in formation of the RISC are the catalytic engines of RNAi, what we believe to be "Slicer."
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The full-length structure of Argonaute from the archaebacterium P. furiosus is 770 amino acids long and has six characteristic domains < N-terminal domain (residues 1-151), the Piwi Argonaute Zwille (PAZ) domain (residues 152-275), the interdomain connector (residues 276-361), the middle domain (residues 362-544), and the PIWI domain are represented schematically [Here] (1).
It has been shown that the PAZ domain of PfAgo contains well-conserved amino acids that bind two-nucleotide 3' siRNA overhangs, and that siRNA can bind mRNA and thus direct the PIWI domain to a specific mRNA sequence for subsequent mRNA cleavage [Here]. Since Argonaute proteins are characterized by their PAZ and PIWI domains (4), we will now focus on their respective features.
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It is important to note that even though the PAZ domain of PfAgo is structurally similar to other PAZ domains, the primary structure of PfAgo is not well conserved (4). There are, however, several conserved amino acids, four of which are involved in binding of the two-nucleotide 3' overhang: Tyr-190, Tyr-212, Tyr-216, and His-217 < These aromatic residues interact specifically with the two-nucleotide 3' overhangs of an siRNA by hydrogen bonding (1). The last nucleotide of an siRNA interacts with residues Ile-261, Leu-263, and Trp-213 < Arg-220 < is in contact with the second to last nucleotide of the siRNA (1). All of these molecular contacts indicate that the PAZ domain of PfAgo binds the overhanging 3' ends of siRNA.
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The center of the PIWI domain has significant RNase H homology implying that PIWI is an RNase H domain (1). This is significant becuase RNase H endoribonucleases are responsible for hydrolyzing the phosphodiester bonds of of RNA, although typically hybridized to DNA. [Here]. Moreover, the PIWI domain has three highly conserved catalytic carboxylates: Asp-558, Asp-628, and Glu-635 < DDE motif, are present at the active center of similar RNase H enzymes.
In a seperate experiment, the corresponding aspartate residues from human Ago2 were changed to alanines by site-directed mutagenisis (3). The result of this experiment was that even though the RISC complex formed and was able to bind siRNAs (by the unaffected PAZ domain), it lost its mRNA cleavage activity. This data corroborates the previous notion that the conserved DDE motif of the PfAgo PIWI domain is involved in cleavage of mRNA in RNAi.
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RNAi is likely to have the greatest impact as a therapeutic tool in two key clinical areas, cancer and infectious disease, but it also has the potential as a therapy for other disorders including some dominant genetic diseases (6). In a recent study, researchers used RNAi in mice to knock-down the gene Th that encodes the dopamine synthesis enzyme, tyrosine hydroxylase. This resulted in behavioral changes, and demonstrated how RNAi induced by RNA in viral vectors could be readily accomplished for specific uses, potentially including human gene therapy (7) [Here].
In several other recent studies, siRNAs have been shown to protect mice from viral infection, tumor growth, hepatitis, sepsis, and ocular neovascularization, a proliferation of abnormal blood vessels beneath the retina (8). Two companies have recently come forth with RNAi treatments for Macular degeneration. The treatments inhibit the vascular endothelial growth factor (VEGF) that is involved in neovascularization (Alnylam, Sirna). The National Eye Institute estimates that over 1.6 million 50+ year-old adults in the United States suffer from advanced age-related macular degeneration, a condition that causes severe deterioration of vision and may ultimately cause blindness (10).
Although there are technical challenges associated with the therapeutic application of siRNAs, such as synthesis, delivery, and specificity (8), using siRNA as an alternative therapeutic approach has several benefits over viral vectors in gene therapy applications [Here]. The siRNA approach for gene silencing holds great therapeutic promise, as siRNAs, like miRNAs, are naturally used by cells to regulate gene expression and are therefore nontoxic and highly effective (8).
(1) Song, Ji-Joon, Stephanie K. Smith, Gregory J. Hannon, Leemor Joshua-Tor. 2004. Crystal Structure of Argonaute and Its Implications for RISC Slicer Activity. Science 305: 1434-1437.
(2) Meister, Gunter, and Thomas Tuschi. 2004. Mechanisms of Gene Silencing by Double Stranded RNA. Nature 431: 343-349.
(3) Liu, Jidong, Michael A. Carmell, Fabiola V. Rivas, Carolyn G. Marsden, J. Michael Thompson, Ji-Joon Song, Scott M. Hammond, Leemor Joshua-Tor, Gregory J. Hannon. 2004. Argonaute2 is the Catalytic Engine of Mammalian RNAi. Science 305: 1437-1441.
(4) L. Cerutti, N. Mian, A. Bateman, T. 2000. Domains in gene silencing and cell differentiation proteins: the novel PAZ domain and redefinition of the Piwi domain. Trends in Biochemical Sciences 10: 481-482.
(5) J. J. Song et al. 2003. The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes. Nature Struct. Biol. 10, 1026-1032.
(6) Natasha J Caplen et al. 2003. RNAi as a gene therapy approach . Expert Opinion Biol. Therapy. 3(4): 575 -586.
(7) Hommel JD, Sears RM, Georgescu D, Simmons DL, DiLeone RJ. 2003. Local gene knockdown in the brain using viral-mediated RNA interference. Nat Med. 9(12):1539-44.
(8) Yair Dorsett and Thomas Tuschl. 2004. siRNAs: Applications in Functional Genomics and Potentials as Therapeutics. Nat Rev. 3: 318-329.
(9) Archana Thakur. 2003. RNA interference Revolution. Electronic Journal Biotechnology 6(1).
(10) National Eye Institute, US National Institutes of Health. http://www.nei.nih.gov/health/