BIOLOGICAL OVERVIEW

Arabidopsis Argonaute1 (AGO1) is the founder of the Argonaute gene superfamily that is conserved from fission yeasts to humans (Carmell, 2002). AGO1, and several members of this superfamily are necessary for stem cell renewal or RNA interference. However, little has been reported about their roles in animal development or about the molecular activities of any of the members. A Drosophila homolog of Arabidopsis AGO1 has been isolated in an attempt to search genetically for regulators of Wingless (Wg) signal transduction. Drosophila Argonaute 1 (AGO1) is broadly expressed in the embryo and imaginal discs. AGO1 over-expression at wing margins suggests that it behaves as a positive regulator in the genetic background employed. Unexpectedly, loss-of-function mutations of AGO1, do not give typical segment polarity phenotypes of the wg class; instead, AGO1 maternal and zygotic mutant embryos show developmental defects, with malformation of the nervous system being the most prominent. The mutant exhibits a decrease in the numbers of several types of neurons and glia examined. The Drosophila AGO1 protein is distributed in the cytoplasm and co-sediments with poly(U)- or poly(A)-conjugated beads (Kataoka, 2001). Mutations in Drosophila AGO1 suppress Double stranded RNA interference (RNAi) in embryos. This defect corresponds to a reduced ability to degrade mRNA in response to dsRNA in vitro. Furthermore, AGO1 is not required for short interfering RNA (siRNA) production in vitro nor can the introduction of siRNA bypass AGO1 mutants in vivo. These data suggest that AGO1 functions downstream of siRNA production (Williams, 2002).

To identify new components of the Wg signal transduction pathway, a genetic screen was performed for dominant modifiers of the wing-margin phenotype caused by the over-expression of Shotgun, the Drosophila E-cadherin. Focus was placed on a P-element insertion line, l(2)k08121, in which the transposon was inserted into gene CG6671. This gene is homologous to Arabidopsis AGO1 (Bohmert, 1998); therefore it was designated AGO1. The lethality of l(2)k08121 is due to a loss of AGO1 function, as shown by the fact that remobilization of the P-element recovers the lethality and that expression of a cDNA clone (LD09501) under a heat-shock promoter made l(2)k08121 homozygotes and l(2)k08121/Df develop to adulthood. l(2)k08121 is a strong allele, as was shown by a great reduction in the level of mRNA and was used in subsequent studies (Kataoka, 2001).

Considering that loss-of-function mutations of AGO1 do not give typical segment polarity phenotypes of the wg class, it seems unlikely that AGO1 is an essential regulator of the Wg signaling in normal development. Nevertheless, AGO1 over-expression is able to release the block generated by sequestration of Arm. Therefore, it would be interesting to investigate in other animal species if the expression of AGO1 homologs under strong promoters can cope with situations in which ß-catenin is depleted (Kataoka, 2001).

The Argonaute gene family was first defined by the Arabidopsis Argonaute gene (AGO1) and encodes related proteins of unknown molecular function in plants, animals, and fungi. Members of the Argonaute gene family, including rde-1 in Caenorhabditis elegans, qde-2 in Neurospora, AGO1 in Arabidopsis, and piwi in Drosophila, are genetically defined as being required for PTGS. The Drosophila Argonaute2 (AGO2) protein was biochemically identified as a component of the RISC complex (see RNAi and PTGS - functions and processes). Thus far, AGO2 is the only identified RISC component. The conservation of genes involved in PTGS extends beyond members of the Argonaute family. The Dicer enzyme, whose activity was identified in Drosophila, has homologs in plants, C. elegans, and mammals. Also, RNA-dependent RNA polymerases have been implicated in PTGS in plants, Neurospora, and C. elegans. Thus, homologous proteins may have similar functions in PTGS throughout a diverse range of eukaryotic organisms (Williams, 2002 and references therein).

The Drosophila genome encodes five Argonaute-like proteins. Consistent with their embryonic expression patterns, piwi and aubergine are defined by mutations that affect aspects of germ-line development. piwi is required to maintain germ-line stem cells perhaps by regulating a somatically derived stem cell promoting signal. piwi has also been shown to affect transgene-mediated cosuppression in Drosophila. aub is required for embryo patterning, by regulating oskar and gurken translation, and for pole cell formation. aub is also required for Su(Ste)-mediated suppression of Stellate in the testis. Interestingly, this regulation seems to involve an RNAi-like mechanism. The related AGO3 gene has an embryonic expression pattern very similar to piwi and aub, suggesting that it may have a role in gonad and/or germ-line development as well (Williams, 2002 and references therein).

With the availability of loss-of-function mutations in AGO1, its role as a possible component of the RNAi machinery in Drosophila embryos was examined. Mutations in AGO1 result in late embryonic/early larval lethality and have defects in the central and peripheral nervous system (Kataoka, 2001). Precise excision of the l(2)k08121 P element reverted lethality, as did a heat shock-driven AGO1 cDNA. l(2)k08121, an insertion near the transcription start site of two of the AGO1 isoforms, has been shown to be a strong allele (Kataoka, 2001). This out-crossed insertion line is referred to as AGO1^k08121 (Williams, 2002).

Tests were performed for RNAi in vivo by assaying the ability of dsRNA corresponding to the eve gene to produce an eve phenotype when injected into wild-type and AGO1 mutant embryos. AGO1^k08121 was maintained as a heterozygous stock balanced over a CyO-armGFP chromosome. One-quarter of the embryos produced in this stock are AGO1^k08121 homozygous mutants, as identified by the absence of zygotic GFP expression. The remaining three-quarters of the embryos, which express GFP and represent both heterozygous and CyO-armGFP homozygous embryos, are referred to as AGO1⁺. Wild-type embryos, wild-type embryos injected with control dsRNA corresponding to the white gene, and uninjected AGO1^k08121 mutant embryos all have eight ventral denticle belts. In comparison, eve mutant embryos or wild-type embryos injected with eve dsRNA have a visible reduction in the number of denticle belts. eve dsRNA was injected into AGO1^k0812 and AGO1⁺ embryos. AGO1^k08121 zygotic mutants were less responsive than AGO1⁺ embryos to eve dsRNA. Specifically, only 8% of AGO1^k08121 zygotic mutants had fewer than 7 denticle belts whereas 38% of their GFP-expressing AGO1⁺ siblings exhibited an eve phenotype. A similar reduction in RNAi-induced embryonic phenotypes (12% vs. 34%) was observed by using dsRNA corresponding to the ftz gene. AGO1^k16601 zygotic mutants, resulting from an independent P element insertion 8 nucleotides downstream of the l(2)k08121 insertion site, also exhibited a reduced response to eve dsRNA (7% vs. 28%). It should be noted that RNAi is not completely inhibited in AGO1^k08121 mutants, since a small percentage of eve embryos are observed after injection with eve dsRNA. However, the percentage of embryos exhibiting an RNAi-induced phenotype is clearly reduced in AGO1 mutants when compared with wild type (Williams, 2002).

A number of control experiments were performed to confirm that the decrease in RNAi-induced phenotypes was a specific effect of AGO1 reduction-of-function. Of the embryos homozygous for a l(2)k08121 revertant chromosome that were injected with eve dsRNA, 33% exhibited an eve phenotype. Similarly, AGO1^k08121 embryos containing the heat shock-driven AGO1 transgene that rescues lethality also had a near wild-type response to dsRNA (29%). Therefore, the P element insertion in AGO1 causes the reduced RNAi response exhibited in AGO1^k08121 embryos (Williams, 2002).

As a first step toward understanding the molecular function of AGO1, an in vitro extract was used to narrow down the step(s) in RNAi that are affected in AGO1 mutants. Previous in vitro analysis of RNAi in Drosophila relied on extracts, prepared from either early syncitial embryos or S2 cells, which were capable of processing dsRNA into siRNA and then degrading mRNA in a sequence-specific manner. Extracts were prepared from older 12-16-h cellular embryos, when the zygotic expression of the armGFP clearly distinguishes CyO-containing embryos from AGO1^k08121 embryos (Williams, 2002).

An assessment was made of the ability of extracts prepared from AGO1^k08121 and GFP-expressing embryos, laid by an AGO1^k08121/CyO-armGFP balanced stock, to degrade target mRNA in response to dsRNA. mRNA incubated in the extracts without dsRNA for 1 h were only modestly degraded. Extracts prepared from AGO1⁺ embryos and preincubated with eve dsRNA efficiently degrade eve mRNA. Similarly, white dsRNA can target white mRNA for degradation. This process is sequence-specific because eve dsRNA does not affect the stability of white mRNA nor does white dsRNA affect the stability of eve mRNA, suggesting that the embryo extracts faithfully reproduce RNAi in vitro. Extracts prepared from AGO1^k08121 embryos, however, did not degrade mRNA when preincubated with a homologous dsRNA. The ability to degrade mRNA in a sequence specific manner is restored in extracts prepared from the revertant line and in extracts prepared from the line where the AGO1^k08121 insertion is rescued by the expression of an AGO1 cDNA. The reduced RNAi observed in living AGO1^k08121 embryos correlates with the lack of dsRNA-triggered mRNA degradation in vitro (Williams, 2002).

To determine whether AGO1 is required for the initial Dicer-mediated cleavage of dsRNA into siRNA, synthetic eve siRNA was injected into embryos laid by w¹¹¹⁸ and AGO1^k08121/CyO-armGFP females and they were scored for the number of denticle belts. siRNA is capable of producing an eve phenotype in wild-type embryos, albeit at a lower penetrance than longer dsRNA (26% vs. 60% for w¹¹¹⁸ and 28% vs. 38% for GFP-expressing embryos). This lower penetrance may be the result of incomplete phosphorylation of the 5' ends of the siRNA, which is required for efficient degradation of mRNA or may reflect a lower potency of the siRNA. AGO1^k08121 mutants, however, produced only 1% eve embryos after injection with eve siRNA. Therefore, AGO1^k08121 mutants have a reduced response to synthetic siRNA, suggesting that AGO1 functions downstream of siRNA production (Williams, 2002).

The ability of extracts from AGO1^k08121 mutant embryos and their AGO1⁺;GFP-expressing siblings to process dsRNA into short ~21-nt fragments was assayed. Extracts prepared from both AGO1^k08121 zygotic mutants and AGO1⁺ embryos were able to cleave dsRNA corresponding to both the eve and white genes into ~21-nt fragments. The rate of siRNA production was similar in both AGO1^k08121 and AGO1⁺ extracts. Taken together, these experiments indicate that wild-type AGO1 activity is required after the Dicer-mediated production of siRNA (Williams, 2002).

Thus AGO1 mutant embryos still exhibit some RNAi activity. There are several possible explanations for this observation. (1) AGO1^k08121 may not be a null allele. The P element is inserted near the transcriptional start site for two of the isoforms and in the second intron of a third isoform, leaving the possibility that a functional mRNA could be produced; however, there is a strong reduction of all AGO1 transcripts in the l(2)k08121 allele (Kataoka, 2001). (2) Because AGO1 and AGO2 have similar expression patterns and both may function in RNAi, they may share functional redundancy. Partial redundancy has been demonstrated for two related Argonaute-like genes during C. elegans and Arabidopsis development. An interesting and untested idea is that AGO1 and AGO2 may have some redundancy during early embryogenesis but then later tissue-specific differences in their expression patterns would uncover the lethality associated with AGO1 mutants. This model would be consistent with the continued up-regulation of AGO1 mRNA, especially in the ventral nerve cord, near the end of embryogenesis. (3) The maternal contribution of AGO1 mRNA might provide some level of AGO1 activity, although it cannot support wild-type levels of RNAi (Williams, 2002).

There are several possible steps in which AGO1 may function. Recent studies of C. elegans rde-1 mutants have shown that in vitro extracts are capable of processing dsRNA into siRNA, but siRNA is greatly reduced in vivo. This work suggested that wild-type rde-1 activity is required to stabilize siRNA. Thus, AGO1/rde-1 may protect siRNA from a nuclease. AGO1 may help to incorporate siRNA into a functional RISC or, like AGO2, is itself a component of RISC. Such activity could include maintaining the required 5' phosphate group on siRNA, acting as a scaffold to assemble the multiprotein complex, unwinding siRNA/strand selection for base pairing to target mRNA, or acting as a component of the nuclease that cleaves mRNA. These possible functions for AGO1 are not exclusive. For example, siRNA could be stabilized by incorporation into RISC. However, the biochemical activity or in vivo partners of the AGO1 protein are not known (Williams, 2002).

AGO1 mutants are late embryo/early larval lethal and exhibit defects in the embryonic nervous system (Kataoka, 2001). This finding is not surprising because other components of the RNAi pathway have been shown to function during development. Dicer/dcr-1 and the rde-1 homologs alg-1 and alg-2 are required for the production of the small temporal RNA (stRNA) in C. elegans. stRNAs, encoded by the lin-4 and let-7 genes, are 21-22-nt single-stranded RNAs that function by base pairing to the 3' untranslated regions and inhibiting translation of genes that control developmental timing. stRNAs are initially made as ~70-nt primary transcripts that can fold into a hairpin structure. The double-stranded stem portion of the RNA is cleaved and processed into the functional 21-22-nt stRNA. Recent studies have uncovered a large and diverse population of endogenous microRNA that share many of the characteristics of lin-4/let-7. These observations raise the possibility that small RNA represents a common mode of gene regulation and their production/usage requires a mechanism similar to RNAi (Williams, 2002).

The neuronal defect in AGO1 mutants is particularly intriguing. Lai and Posakony (1998) have proposed that short regions of RNA:RNA duplex formation between the 3' untranslated regions (UTRs) of the proneural genes and members of the Enhancer of split complex [E(spl)] may represent a level of gene regulation during neurogenesis. Most recently, it was noted that several Drosophila microRNAs have regions of complementarity to negative regulatory elements in the 3' UTRs of multiple members of the Bearded and E(spl) complexes (Lai, 2002). Because members of these gene families regulate development of the nervous system, it would be very interesting if the developmental defects in AGO1 mutants are the results of a defect in microRNA processing or use (Williams, 2002).

GENE STRUCTURE

cDNA clone length - 3586

Bases in 5' UTR - 293 (CT42236)

Exons - 10 (3 common exons and 7 upstream exons including 3 alternative start sites)

Bases in 3' UTR - 440

PROTEIN STRUCTURE

Amino Acids - 950

Structural Domains

At least three alternatively spliced transcripts are made from Drosophila AGO1, and focus was placed on one of them, CT42236, which is equivalent to the EST clone LD09501. The predicted AGO1 protein consists of 950 amino acids, and its molecular weight was estimated as ~106 kDa. As in the case of all members of the AGO1 superfamily, amino acid sequences of AGO1 provided no definite information about its molecular activity. Phylogenetic trees and multiple alignments of amino acid sequences suggest that this superfamily consists of two distinct subfamilies and several orphans. One of these subfamilies was named the AGO1 subfamily, which includes Arabdopsis AGO1, Drosophila AGO1 and an S. pombe protein, SPCC736.11. The AGO2 protein of Drosophila was biochemically identified as a component of the RNA-induced silencing complex (RISC) and has thus been implicated in the degradation of target mRNA in response to dsRNA (Hammond, 2001). The other subfamily was designated as the PIWI subfamily, because the founder is a Drosophila protein of the piwi gene, which controls the division of germ-line stem cells. The orphans whose mutants were isolated, are C. elegans rde-1, which is required for RNA interference (Tabara, 1999), and Neurospora QDE-2 (Cogoni, 1997; Fagard, 2000), which is required for quelling, a phenomenon similar to co-suppression (Kataoka, 2001).

Every member of the superfamily shares a conserved box of 43 residues near the carboxy terminal, and proteins of the AGO1 subfamily share a longer stretch of 86 residues on average (the AGO1 box). What distinguishes the two subfamilies most is the presence or absence of a region that is positioned closer to the amino terminal, and this is termed the N region. The N region is highly conserved in the AGO1 subfamily, and to different degrees among orphans; however, it is not found in the PIWI subfamily. A given organism has multiple genes that belong to this superfamily; for example, more than 20 genes are encoded in the C. elegans genome. The Drosophila genome appears to have five family members, piwi, sting/aubergine, AGO1, CG7439 (Argonaute 2)] and a fifth predicted from genomic sequences (Kataoka, 2001).

Searches of the Drosophila genome and expressed sequence tag (EST) databases reveal five separate transcribed members of the Argonaute gene family. Argonaute-like genes are defined as encoding two conserved domains: a well conserved region of approximately 300 amino acids, called the PIWI box, near the C-terminal region of the ORF, and a more centrally located and less well conserved region of 110 amino acids called the PAZ domain. The molecular functions of Argonaute-like proteins are not known; however, two of the genes, piwi and aubergine (aub)/sting, are necessary for proper germ-line development. The other three members include AGO1, AGO2 and AGO3 (the latter has not yet been characterized) (Williams, 2002).

Based on sequence alignments, the five genes can be subdivided into two subcategories. Interestingly, the embryonic RNA expression patterns of the five Drosophila genes reflect this sequence-based grouping as well. AGO1 and AGO2 are maternally deposited and have fairly ubiquitous embryonic expression patterns. They are, however, more strongly expressed in the ventral and cephalic furrows. Later, AGO1 is also up-regulated in the developing nervous system. piwi, aub, and AGO3, which are similar in sequence, are also expressed maternally but unlike AGO1 and AGO2, their expression disappears by embryonic stages 10-12. Zygotic transcription is then restricted to the presumptive gonad, suggesting they have tissue-specific roles during embryo development and are less likely to a part of the general RNAi machinery that is assumed to be ubiquitous (Williams, 2002).

The discovery of RNA-mediated gene-silencing pathways, including RNA interference, highlights a fundamental role of short RNAs in eukaryotic gene regulation and antiviral defence. Members of the Dicer and Argonaute protein families are essential components of these RNA-silencing pathways. Notably, these two families possess an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain whose biochemical function is unknown. The nuclear magnetic resonance solution structure of the PAZ domain from Drosophila melanogaster Argonaute 1 (Ago1) is reported. The structure consists of a left-handed, six-stranded beta-barrel capped at one end by two alpha-helices and wrapped on one side by a distinctive appendage, which comprises a long beta-hairpin and a short alpha-helix. Using structural and biochemical analyses, it has been demonstrated that the PAZ domain binds a 5-nucleotide RNA with 1:1 stoichiometry. The RNA-binding surface has been mapped to the open face of the beta-barrel, which contains amino acids conserved within the PAZ domain family, and the 5'-to-3' orientation of single-stranded RNA bound within that site was defined. Furthermore, PAZ domains from different human Argonaute proteins also bind RNA, establishing a conserved function for this domain (Yan, 2003).

date revised: 10 June 2002

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