REVIEWS
`
` N O N - C O D I N G R N A
`
`Argonaute proteins: functional
`insights and emerging roles
`
`Gunter Meister
`
`Abstract | Small-RNA-guided gene regulation has emerged as one of the fundamental
`principles in cell function, and the major protein players in this process are members of the
`Argonaute protein family. Argonaute proteins are highly specialized binding modules that
`accommodate the small RNA component — such as microRNAs (miRNAs), short interfering
`RNAs (siRNAs) or PIWI-associated RNAs (piRNAs) — and coordinate downstream
`gene-silencing events by interacting with other protein factors. Recent work has made
`progress in our understanding of classical Argonaute-mediated gene-silencing principles,
`such as the effects on mRNA translation and decay, but has also implicated Argonaute
`proteins in several other cellular processes, such as transcriptional regulation and splicing.
`
`Argonaute proteins are key players in all small-RNA-
`guided gene-silencing processes that have been identi-
`fied and characterized so far. They are highly conserved,
`and family members are found in all eukaryotes, with the
`exception of Saccharomyces cerevisiae, which has lost
`the small RNA machinery1. Also, some bacteria and
`archaea contain Argonaute genes2: their specific functions,
`however, remain elusive.
`Argonaute proteins are the direct binding partners
`of small RNAs. The eukaryotic Argonaute family can be
`divided into AGO proteins (also referred to as the AGO
`clade), which are similar to Arabidopsis thaliana AGO1,
`and PIWI proteins (also referred to as the PIWI clade),
`which are homologous to Drosophila melanogaster
`Piwi. AGO proteins mainly interact with microRNAs
`(miRNAs) or short interfering RNAs (siRNAs) and
`are involved in cytoplasmic post-transcriptional
`gene-silencing processes3,4. PIWI proteins are mainly
`expressed in germline cells, where these proteins bind
`to PIWI-interacting RNAs (piRNAs) and function
`in the silencing of transposable genetic elements5. In
`Caenorhabditis elegans, in which 26 different Argonaute
`genes exist, a third clade (known as WAGO) has evolved
`that is equally distant from the AGO and PIWI clades.
`These proteins mainly serve as secondary Argonaute
`proteins and acquire their small RNA load through
`‘primary’ Argonaute proteins or through small RNA
`amplification processes6. Plant Argonaute proteins clus-
`ter to the AGO clade2. However, some of these proteins
`are exclusively expressed in the germ line and have plant-
`specific functions7. The biogenesis of small RNA classes
`is summarized in BOXES 1,2.
`
`In the past few years, novel insights into the func-
`tion of Argonaute proteins have been reported. Among
`them are additional mechanistic details that improve
`our understanding of known aspects, such as structural
`information on human Argonaute proteins, the mecha-
`nism of small RNA loading, Argonaute post-translational
`modifications and Argonaute protein function in germ
`cells. In addition, novel cellular Argonaute functions
`are emerging, including roles for Argonaute in tran-
`scription, alternative splicing and even DNA repair.
`This Review focuses on the recent literature of the
`diverse aspects of Argonaute protein function with an
`emphasis on AGO clade proteins. The first part dis-
`cusses insights into known functions, and the second
`part addresses novel pathways in which Argonaute
`proteins have been implicated.
`
`Structural insights
`Structural studies have revealed how Argonaute pro-
`teins act as highly specialized small-RNA-binding
`modules. They are characterized by amino-terminal
`(N), PAZ (PIWI–ARGONAUTE–ZWILLE), MID
`(middle) and PIWI domains. Initial structural studies
`on bacterial and archaeal Argonaute proteins revealed
`that Argonaute proteins consist of two lobes compris-
`ing the N–PAZ and the MID–PIWI domains, respec-
`tively. A hinge connects the two lobes, and structural
`rearrangements occur during RNA binding. The PAZ
`domain anchors the 3′ end of the small RNA by bend-
`ing it into a specific binding pocket8. PAZ domains of
`the PIWI subfamily members specifically accommo-
`date methylated piRNA 3′ ends9. The MID domain
`
`Biochemistry Center
`Regensburg, Laboratory
`for RNA Biology,
`University of Regensburg,
`Universitätsstrasse 31,
`93053 Regensburg,
`Germany.
`e-mail: gunter.meister@vkl.
`uni-regensburg.de
`doi:10.1038/nrg3462
`Published online 4 June 2013
`
`NATURE REVIEWS | GENETICS
`
`© 2013 Macmillan Publishers Limited. All rights reserved
`
` VOLUME 14 | JULY 2013 | 447
`
`Alnylam Exh. 1049
`
`

`

`R E V I E W S
`
`Box 1 | Biogenesis of miRNAs and siRNAs
`
`snoRNA
`
`pri-miRNA
`
`DGCR8
`
`Drosha
`
`pre-mRNA
`
`Mirtron
`
`Exon
`
`Exon
`
`Microprocessor complex
`
`Spliceosome
`
`Exportin 5
`
`Nucleus
`
`Cytoplasm
`
`NPC
`
`Double-
`stranded RNA
`
`Dicer
`
`Dicer
`
`miRNA*
`
`miRNA
`miRNA*
`
`siRNA
`
`Guide
`Passenger
`
`Argonaute
`
`Degradation
`
`Argonaute
`
`miRNP or
`miRISC
`
`Argonaute
`
`siRISC
`
`Most microRNAs (miRNAs) are transcribed as primary transcripts (pri-miRNAs) by RNA polymerase II (RNA Pol II; see the
`upper left panel of the figure). Such transcripts contain one or more hairpins, and the miRNAs lie in their double-stranded
`stem. Processing by the nuclear microprocessor complex, which contains the RNase III enzyme Drosha, releases the
`hairpin: this part is referred to as the precursor miRNA (pre-miRNA)70. Pre-miRNAs can also be generated from short
`introns, which are referred to as mirtrons154 (see the upper right panel of the figure). Mirtrons are Drosha-independent
`and are liberated during splicing by the spliceosome. Some classes of small nucleolar RNAs (snoRNAs) can also produce
`miRNAs155 (see the upper centre panel of the figure); how these are processed is unclear. After export to the cytoplasm by
`exportin 5, Dicer (another RNase III enzyme) cleaves off the loop of the hairpin and generates a double-stranded RNA.
`The mature miRNA strand is subsequently incorporated into the RNA-induced silencing complex (RISC, or miRISC for
`miRNA-containing RISC or miRNP for microribonucleoprotein), where it directly binds to a member of the AGO protein
`family. The other strand is referred to as miRNA* and is normally degraded. In some cases, miRNAs* can be functional as
`well70. It has also been shown that miRNAs can be processed independently of Dicer. AGO2, for example, can cleave miRNA
`precursors and produce mature miRNAs67,156–159.
`Small interfering RNAs (siRNAs) are very similar to miRNAs and are widely used as research tools for sequence-specific
`gene inactivation. Depending on the organism, siRNAs are either introduced as short double-stranded RNAs (dsRNAs)
`by transfection or long dsRNAs, which are processed to siRNAs by Dicer73 (see the lower right panel of the figure). The
`so-called guide strand is incorporated into RISC (or siRISC for siRNA-containing RISC), and the other strand (known as
`the passenger strand) is destabilized. In many organisms, siRNAs from endogenous transcripts have been identified. In
`Drosophila melanogaster gonadal and somatic tissues, as well as cultured S2 cells, endogenous siRNAs mainly correspond
`to a subset of retrotransposons or stem–loop-structured RNAs160–162. In mouse oocytes, endogenous siRNAs derive from
`different dsRNA sources, including inverted repeat structures, bidirectional transcription or antisense transcription163,164.
`Pseudogenes seem to be sources for endogenous siRNAs as well. It is currently unclear, however, whether such
`endogenous siRNAs exist in mammalian somatic cells as well. NPC, nuclear pore complex.
`
`448 | JULY 2013 | VOLUME 14
`
` www.nature.com/reviews/genetics
`
`© 2013 Macmillan Publishers Limited. All rights reserved
`
`

`

`Box 2 | piRNA biogenesis and function
`
`Mobile genetic elements such as transposons are a constant threat for the genome.
`PIWI-interacting RNAs (piRNAs) protect germline cells from transposons in organisms as
`diverse as flies, fish or mammals. piRNAs are 25 to 33 nt in length, depending on the PIWI
`clade protein that they bind to. piRNAs derive from distinct transposons that are referred
`to as piRNA clusters, but the piRNAs from each locus are characterized by a complex
`mixture of sequences spanning large portions of the transposon5. piRNA clusters are
`transcribed in the sense or antisense direction, and the long single-stranded RNA serves
`as the basis for piRNA production.
`The biogenesis of piRNAs is independent of Dicer165 and requires other nucleases. Two
`biogenesis pathways are important for piRNA production. First, a primary processing
`pathway generates primary piRNAs, and these are then amplified by an amplification
`cycle referred to as the ping-pong loop5. In the primary biogenesis pathway, the long
`transposon transcript is initially cleaved by the nuclease zucchini (Zuc; see the
`figure)166–168, which probably generates the 5′ ends of primary piRNAs. The other steps of
`primary piRNA maturation are not understood5.
`In the ping-pong cycle (see the lower right panel of the figure), mature sense primary
`piRNAs guide PIWI clade proteins to complementary sequences on antisense transcripts
`from the same piRNA cluster. PIWI proteins use their slicer activity to cleave the target
`antisense transcript to generate a new 5′ end. This 5′ end is bound by another PIWI
`protein. In subsequent steps, the 3′ end is trimmed to the length of the mature piRNA,
`leading to a mature antisense secondary piRNA, which can now target sense transcripts
`transcribed from the piRNA cluster. In Drosophila melanogaster, the two Piwi proteins
`Aubergine and Ago3 cooperate in secondary piRNA production to generate sense and
`antisense piRNAs. However, antisense piRNAs dominate, and a protein called Qin, which
`contains E3 ligase and Tudor domains, seems to modulate such a heterotypic ping-pong
`cycle169. In the mouse germ line, the PIWI proteins MILI and MIWI collaborate in piRNA
`generation. After trimming, piRNAs receive a methyl group at the 3′ end by the
`methyltransferase HEN1. Primary piRNAs carry such modifications as well5. In
`Caenorhabditis elegans, an additional piRNA production pathway exists. Short and
`capped RNA polymerase II transcripts are decapped, processed and directly loaded into
`PIWI proteins170.
`piRNAs guide PIWI proteins to complementary RNAs derived from transposable
`elements. Similarly to in RNA interference, PIWI proteins cleave the transposon RNA,
`leading to silencing. In flies, mutations in piwi, aub and Ago3 (which encode the Piwi
`proteins in (cid:38)(cid:16)(cid:124)(cid:79)(cid:71)(cid:78)(cid:67)(cid:80)(cid:81)(cid:73)(cid:67)(cid:85)(cid:86)(cid:71)(cid:84)) are required for transposon silencing in the germ line5.
`Similar observations were made when the mouse PIWI proteins MILI and MIWI were
`genetically inactivated171. Here, long interspersed nuclear elements (LINE) and long
`terminal repeat (LTR) retrotransposons accumulated172.
`
`piRNA cluster
`
`Sense or antisense
`transcript
`
`Zuc
`
`Primary
`processing
`
`Antisense transcript
`
`Sense piRNA
`
`Secondary
`processing
`(ping-pong cycle)
`
`Antisense piRNA
`
`Trimming
`
`Sense transcript
`
`Trimming
`
`Mature
`antisense piRNA
`
`Primary
`piRNAs
`
`PIWI protein
`
`Mature sense
`piRNA
`
`R E V I E W S
`
`anchors the 5′ end of the small RNA by providing a
`binding pocket in which the 5′ terminal base engages
`in stacking interactions with a conserved tyrosine. In
`addition, several hydrogen bonds coordinate correct
`5′ end binding8. The N domain is required for small
`RNA loading and assists in unwinding the small RNA
`duplex10.
`The PIWI domain is structurally similar to RNase H,
`and it has indeed been shown that Argonaute proteins
`can function as endonucleases and cleave target RNA that
`is fully complementary to the bound small RNA8. The
`PIWI domain contains a catalytic triad composed of
`DDX (where X is D or H). Recent structural work on
`an Argonaute protein from yeast revealed that a fourth
`residue is also essential turning the catalytic centre
`into a tetrad of DEDX11. Generally, only a subset of
`Argonaute proteins possesses cleavage activity. In mam-
`mals, for example, only AGO2 of the AGO subfamily is
`catalytically active and functions as an endonuclease12,13.
`Besides the PIWI domain, an unstructured loop in the
`N domain is also important for cleavage14.
`Recently, crystal structures of human AGO2 have
`been reported15,16. Overall, their structure is strikingly
`similar to known bacterial and archaeal Argonaute pro-
`teins, underlining the high conservation of the basic
`principles of small RNA pathways.
`
`Mechanisms of Argonaute loading
`The AGO subfamily and the PIWI subfamily use differ-
`ent mechanisms for small RNA loading, and they will
`be discussed separately with an emphasis on AGO clade
`proteins.
`
`Loading AGO clade members. Dicer processing gener-
`ates a short double-stranded RNA of about 20–24 nt
`in length. However, only one strand associates with
`the AGO protein and becomes the guide strand. How
`is the correct strand selected, and how is it loaded into
`AGO proteins? One important determinant for strand
`selection lies in the small RNA duplex itself: both in the
`miRNA pathway and the siRNA pathway, the strand
`with the less stably paired 5′ end is preferentially loaded
`into AGO proteins. These thermodynamic differ-
`ences between the small RNA ends are known as the
`asymmetry rule17,18.
`In various organisms, Dicer proteins directly inter-
`act with AGO proteins and strand selection, and load-
`ing is achieved within such complexes19–21. In addition,
`Dicer proteins partner with various different dsRNA-
`binding proteins, which have been most extensively
`studied in D. melanogaster. In flies, two Dicer proteins
`exist: Dcr1 processes precursor miRNAs (pre-miRNAs)
`and loads Ago1 for miRNA-guided gene silencing,
`whereas Dcr2 cleaves perfectly paired long dsRNA
`and loads siRNAs into Ago2. Dcr1 interacts with
`loquacious (Loqs), which is essential for the miRNA
`pathway22. Dcr2 requires R2d2, which is important for
`the siRNA pathway. R2d2 functions as a sensor for the
`thermodynamic asymmetry within an siRNA duplex
`and positions Dcr2 for correct strand selection23,24. In
`mammals, the dsRNA-binding domain proteins TRBP
`
`NATURE REVIEWS | GENETICS
`
` VOLUME 14 | JULY 2013 | 449
`
`© 2013 Macmillan Publishers Limited. All rights reserved
`
`

`

`R E V I E W S
`
`a
`
`dsRNA or
`pre-miRNA
`
`Asymmetry sensing
`
`Dissociation or reassociation
`
`Argonaute
`
`HSP90
`
`Dicer
`
`TRBP
`
`Dicer
`
`Small RNA duplex
`
`Small RNA transfer
`to Argonaute
`
`HSP90
`
`Argonaute
`
`TRBP
`
`Dicer
`
`b
`
`Open conformation
`
`HSP90
`
`Argonaute
`
`Open conformation
`
`Closed conformation
`
`HSP90
`
`Argonaute
`
`Argonaute
`
`Co-chaperone
`
`HSP90
`
`+ ATP
`
`Co-chaperone
`
`HSP90
`
`Figure 1 | A model for loading of small RNAs into AGO clade proteins. a | Dicer binds long double-stranded RNAs
`(dsRNAs) or precursor microRNAs (pre-miRNAs) and uses its two RNase III domains to cleave both strands (indicated
`by the two arrows), generating a ~21 nt dsRNA. This short dsRNA dissociates from and reassociates with Dicer at a
`different position. During this step, a dsRNA-binding protein (here, TRBP) senses the asymmetry and positions the
`RNA in an orientation, allowing correct strand selection and loading. In the next step, the dsRNA is transferred
`to a bound Argonaute protein that is kept in an open conformation by heat shock protein 90 (HSP90).
`Cleavage-competent AGO proteins cleave the passenger strand (indicated by an arrow), allowing for rapid strand
`displacement. Cleavage-incompetent AGO proteins displace the passenger strand through a much slower process
`without prior cleavage. See REF. 28 for further details. b | An HSP90 dimer binds unloaded Argonaute proteins and
`holds them in an open conformation. Co-chaperones support HSP90 in this process. After small RNA binding, the
`passenger strand is removed, HSP90 hydrolyses ATP, and the AGO protein loaded with the single-stranded RNA
`transitions into a closed conformation. HSP90 and potential co-chaperones leave the complex.
`
`(also known as TARBP2) and PACT (also known as
`PRKRA) interact with Dicer and have been implicated
`in miRNA biogenesis25–27.
`Novel insights into how loading events might func-
`tion in humans have come from recent biochemical
`studies28. First, it was confirmed that human TRBP is
`an asymmetry sensor and functions similarly to R2d2
`in D. melanogaster. Furthermore, Dicer contains two
`different RNA-binding sites: one that positions long
`dsRNA for cleavage, and one that rebinds the cleaved
`siRNA after cleavage with the help of TRBP. During
`this second step, asymmetry sensing occurs (FIG. 1a).
`Within a minimal RNA-induced silencing complex-loading
`complex (RISC-loading complex) containing Dicer,
`TRBP and an AGO protein, the double-stranded
`siRNA or miRNA is loaded onto AGO. The transfer
`of the double-stranded siRNA or miRNA to the Ago
`protein is mediated by the multi-protein chaperone
`complex Hsc70–Hsp90 (heat shock cognate protein
`70 kDa–heat shock protein 90) in D. melanogaster or
`HSP90 in plants and humans; this complex hydrolyses
`ATP to keep AGO proteins in an open state, allowing
`them to accommodate the RNA duplex29–31 (FIG. 1b).
`Heat shock proteins are often assisted by co-chaperones.
`Indeed, a recent mass spectrometry study found that
`the co-chaperone FKBP5 associated with mouse AGO2
`(REF.  32). Whether it is required for RISC loading
`remains to be shown.
`
`In the next step, the duplex is unwound, and the pas-
`senger strand of the siRNA or the miRNA* strand of the
`miRNA is removed. There is recent evidence that the N
`domain of AGO proteins supports this step10. ‘Wedging’
`of the N domain, in which there are conformational
`changes in the AGO protein that drive the N domain
`between the two duplex strands, leads to duplex open-
`ing and further unwinding. How unwinding occurs, and
`whether additional factors are needed, remains unclear.
`It has also been reported that AGO proteins themselves
`might possess strand dissociation activity to remove
`one of the strands33. Putative helicases, such as RNA
`helicase A (RHA), MOV10 or Armitage, have also been
`implicated in duplex unwinding in human and D. mela-
`nogaster cells19,34–36. Strand removal correlates with the
`overall thermodynamic stability of the duplex, possibly
`arguing against the action of additional helicases, which
`might act independently of sequence35,37. Further studies
`are required to solve this controversy.
`Perfectly paired siRNA or miRNA duplexes are
`cleaved by catalytically active AGO proteins, such as
`AGO2 (REFS 38–40); this leads to efficient loading of
`catalytically active AGO proteins. AGO proteins that
`lack catalytic activity are loaded as well but require
`more time to remove the passenger strand. In flies
`and humans, the passenger strand nicked by AGO2 is
`removed from the RISC-loading complex by the endo-
`nuclease C3PO (also known as translin). C3PO interacts
`
`Dicer
`An RNase III family
`endonuclease that processes
`double-stranded RNA and
`precursor microRNAs into
`small interfering RNAs and
`microRNAs, respectively.
`
`RNA-induced silencing
`complex-loading complex
`(RISC-loading complex). A
`protein complex containing
`Dicer, a double-stranded
`RNA-binding protein,
`Argonaute, heat shock protein
`90 (HSP90) and potentially
`other proteins that is required
`for loading small RNAs onto
`the Argonaute protein.
`
`miRNA* strand
`The microRNA (miRNA) strand
`within the miRNA precursor
`that is not selected as the
`mature miRNA. miRNA*
`strands are typically degraded
`and are therefore less
`abundant in cells.
`
`450 | JULY 2013 | VOLUME 14
`
` www.nature.com/reviews/genetics
`
`© 2013 Macmillan Publishers Limited. All rights reserved
`
`

`

`R E V I E W S
`
`with AGO2 and activates RISC, which is now ready to
`fully cleave complementary target RNAs41,42. It remains
`unknown whether C3PO also functions in conjunction
`with non-catalytic AGO proteins. A minimal RISC-
`loading complex requires Dicer, a dsRNA-binding pro-
`tein, the HSC70–HSP90 system and an AGO protein.
`However, it is likely that additional species-specific or
`even sequence-specific factors influence RISC loading.
`
`Loading PIWI clade members. Loading PIWI proteins
`with piRNAs is tightly linked to piRNA biogenesis,
`which is summarized in BOX 2. Although the piRNA-
`loading mechanisms are largely unknown, several
`recent reports shed some light on this process. In
`D. melanogaster, genetic studies found that loss of the
`putative helicase Armitage reduced the levels of PIWI,
`suggesting that is has a role in PIWI–RISC formation
`or stabilization43. Interestingly, the Armitage orthologue
`in mice, MOV10L1, is also required for piRNA produc-
`tion44,45. The detailed mechanisms, however, remain
`unclear. Recent genetic data in D. melanogaster revealed
`that all piRNA classes are lost when the gene encod-
`ing the co-chaperone shutdown (Shu) is mutated46; Shu
`interacts with Hsp90 (REFS 46,47). In mice, the co-chap-
`erone FKBP6 seems to be required for the delivery of
`piRNAs to the PIWI protein MIWI2 (REF. 48). Therefore,
`it is likely that piRNA loading uses similar chaperone-
`assisted mechanisms to miRNA and siRNA loading in
`somatic cells.
`
`Distinct AGO clade proteins
`Biochemical isolation of Argonaute proteins followed
`by deep sequencing of the associated small RNAs has
`revealed that different classes of small RNAs can asso-
`ciate with distinct Argonaute proteins. In this section,
`examples for such associations in various organisms are
`discussed.
`
`Drosophila melanogaster. Sorting small RNAs into
`various Ago clade proteins has been extensively stud-
`ied in D. melanogaster. In flies, precursor RNAs with a
`near-perfect complementarity in the dsRNA are pro-
`cessed by Dcr2 and are specifically loaded into Ago2.
`By contrast, miRNA precursors, which are usually not
`fully paired, are processed by Dcr1 and loaded into Ago1
`(REFS 49–52). However, there are also exceptions to these
`rules: miR-277, for example, is processed by Dcr1 but is
`loaded into Ago2. This process seems to involve rebind-
`ing to Dcr2, which loads miR-277 into Ago2 (REF. 50).
`In addition, endogenous siRNAs (endo-siRNAs), which
`derive from mismatched precursors, are found in
`Ago2. These findings highlight that there are additional
`mechanisms that influence loading of small RNAs in
`D. melanogaster 53,54.
`
`Arabidopsis thaliana. In A. thaliana, ten different AGO
`proteins exist, and they also show preferences for dis-
`tinct classes of small RNAs. In addition, four different
`Dicer variants, DCL1–4, are required for the produc-
`tion and loading of AGO proteins. For example, DCL1
`generates 18–21 nt-long miRNAs and loads them into
`
`AGO1 (REF. 55). DCL2–4 produces slightly longer small
`RNAs, which are loaded into other Argonaute proteins.
`The mechanistic details, however, remain elusive55.
`AGO4 contains a specific class of small RNAs termed
`heterochromatic siRNAs, which are involved in small
`RNA-directed DNA methylation (RdDM) processes.
`AGO6 is similar to AGO4 and also interacts with
`heterochromatic siRNAs. It is involved in RdDM in the
`growing points of shoots and roots56. AGO2 is mainly
`loaded with trans-acting siRNAs (ta-siRNAs) and repeat-
`associated siRNAs. In addition, AGO7 has been sug-
`gested to function in ta-siRNA generation57,58. AGO5
`contains various siRNAs that originate from intergenic
`regions59. The small RNA content of the remaining
`A. thaliana AGO proteins has not yet been analysed
`in detail, mainly owing to the highly specific expres-
`sion patterns in germ cells. Interestingly, AGO9 is
`expressed in somatic companion cells of female gam-
`etes and preferentially interacts with siRNAs derived
`from transposable elements. It is required for trans-
`posable element silencing in the gametes, most prob-
`ably owing to transport processes between the two cell
`types60. Finally, AGO10 seems to sequester miR-166
`and miR-165 specifically and thus regulates shoot apical
`meristem development61.
`
`Mammals. In mammals, only one Dicer exists. All four
`human AGO proteins bind miRNAs and deep-sequenc-
`ing analysis has shown that distinct miRNA subpopu-
`lations might preferentially bind to AGO proteins62.
`However, it has also been reported that miRNAs bind
`randomly to AGO clade proteins63,64. It is possible that
`the non-catalytic AGO proteins — AGO1, AGO3 and
`AGO4 — might be at least partially redundant. AGO1
`and AGO3 are expressed in many cell lines and tissues,
`whereas AGO4 expression seems to be less broad37.
`Recent genetic inactivation of mouse AGO4 revealed
`that it is required for spermatogenesis65 and that it
`regulates entry into meiosis. Furthermore, AGO3 has
`been implicated in Alu RNA-guided mRNA decay dur-
`ing stem cell proliferation66, thus suggesting that non-
`catalytic mammalian AGO proteins are not redundant.
`Genetic studies on AGO1 and AGO3 will help to eluci-
`date potential specific functions of these AGO proteins
`as well. AGO2, which is the only catalytic AGO protein in
`mammals, is essential for early embryonic development.
`Its cleavage activity, however, is required only soon after
`birth, where it processes the highly specialized miRNA
`miR-451 (REF. 67). In addition to its slicer function,
`AGO2 binds miRNAs that are partially complementary
`to target RNAs and engages in gene silencing in a similar
`manner to AGO1, AGO3 or AGO4.
`Many small RNA classes possess specific sequence
`biases at the 5′ end, and it has become clear that
`Argonaute proteins are able to sense the 5′-terminal
`nucleotide of the small RNA. A rigid loop in the MID
`domain of human AGO2 allows specific contacts to a
`5′-terminal uridine or adenine. Small RNAs with Gs or
`Cs at the 5′ end bind to human AGO2 with low affini-
`ties68. Similar small-RNA-binding strategies have been
`found for plant and D. melanogaster AGO proteins54,69.
`
`Endogenous short
`interfering RNAs
`(endo-siRNAs). Short
`interfering RNAs (siRNAs) that
`originate from endogenous
`double-stranded RNA sources,
`such as long hairpin structures
`or sense or antisense
`transcripts from specific
`genomic loci that form
`double-stranded RNAs.
`
`Trans-acting siRNAs
`(ta-siRNAs). Small RNAs
`derived from specific genes;
`the transcripts are cleaved
`by specialized miRNAs and
`the cleavage products
`can be converted into
`double-stranded RNAs by
`RNA-dependent RNA
`polymerases. A Dicer enzyme
`cleaves the double stranded
`RNA to ta-siRNAs. ta-siRNAs
`are involved in DNA
`methylation.
`
`Repeat-associated siRNAs
`Small RNAs that originate from
`repetitive regions such as
`transposable elements. These
`RNAs are involved in silencing
`transcripts that are produced
`from repetitive elements.
`
`Shoot apical meristem
`A plant tissue that is located
`at the tip of the shoot axis. It
`contains undifferentiated cells
`and gives rise to lateral organs
`as well as the stem. It can
`regenerate.
`
`NATURE REVIEWS | GENETICS
`
` VOLUME 14 | JULY 2013 | 451
`
`© 2013 Macmillan Publishers Limited. All rights reserved
`
`

`

`R E V I E W S
`
`Poly(A)-binding
`protein-interacting motif 2
`(PAM2). This motif is present in
`several proteins and interacts
`with the poly(A)-binding
`protein domain, which is found
`in several proteins, including
`PABPC1.
`
`LIM domain
`Named after the proteins
`LIN1L, ISL1 and MEC3, LIM
`domain proteins have been
`linked to the cytoskeleton, and
`they have also been implicated
`in regulating cell growth,
`migration, cell–cell adhesion
`and signalling.
`
`Argonaute-binding partners
`Small RNAs guide Argonaute proteins to target RNAs,
`and Argonautes serve as platforms for interacting pro-
`teins that facilitate the major downstream events of gene
`silencing. Briefly, miRNAs guide AGO clade members to
`mRNAs, often to binding sites located in the 3′ untrans-
`lated region (UTR; for detailed reviews on miRNA func-
`tion, see REFS 70–72). In cases in which there is perfect
`complementarity between the miRNA and the target,
`and if the AGO is catalytic, the mRNA target is cleaved73.
`This process is rare in mammals but is frequently found
`in plants. In mammals, miRNAs usually bind to partially
`complementary sites. The so-called ‘seed sequence’ of the
`miRNA, comprising nucleotides 2 to 7 or 8, is often fully
`paired and essential for the interaction. As a consequence
`of miRNA binding, the mRNA is translationally silenced
`or degraded71. Recently, the dynamics of miRNA-guided
`gene silencing was investigated in different model
`systems, and it was found that translational inhibi-
`tion is the dominant form of repression at early time
`points, whereas mRNA decay is the major contributor
`at later time points74–77.
`
`GW proteins. miRNA-guided gene repression is medi-
`ated by and assisted by many additional proteins. For
`target mRNA repression, AGO proteins directly inter-
`act with a member of the GW protein family19,78–81. GW
`proteins are characterized by an N domain containing
`multiple glycine–tryptophan (GW) repeats. This domain
`directly interacts with AGO proteins and is therefore
`referred to as the AGO-binding domain71. The recent
`structure of human AGO2 crystallized in the presence
`of free tryptophan amino acids, identified two bind-
`ing pockets for tryptophans on the surface of the PIWI
`domain, suggesting that GW proteins use only two of
`their numerous tryptophans for AGO binding16. The
`many GW repeats in the GW proteins suggest that more
`than one AGO molecule could simultaneously bind to
`a single GW protein. However, the stoichiometry is
`unclear, and further biochemical and biophysical studies
`are required.
`The carboxy-terminal half of each GW protein con-
`tains several domains and short binding motifs, including
`an RNA recognition motif (RRM) and a poly(A)-binding
`protein-interacting motif 2 (PAM2) motif. Both of these
`motifs interact with poly(A)-binding protein C (PABPC)
`on the poly(A) tail of the mRNA. It has been proposed
`that GW binding to PABPC inhibits its interaction with
`the cap-binding complex and prevents the mRNA from
`circularization, which is a prerequisite for efficient trans-
`lational initiation82,83. Alternatively, a more direct role of
`PABPC in miRNA-guided gene silencing has recently
`been proposed84. PABPC and the poly(A) tail stimulate
`the association of AGO–GW protein complexes to the
`mRNA, which explains why silencing efficiency corre-
`lates with the length of the poly(A) tail and thus with the
`number of PABPC molecules that are bound to it84.
`Interestingly, the C-terminal silencing domain of GW
`proteins also contains a short stretch that is enriched in
`GW repeats. This motif interacts with NOT1, which
`is a part of the deadenylase CCR4–NOT complex,
`
`and recruits it to the mRNA, leading to removal of the
`poly(A) tail85,86,87. A short poly(A) tail leads to decap-
`ping of the mRNA and subsequent decay by the abun-
`dant 5′ to 3′ exonuclease XRN1 (REF. 71). However, the
`interaction of miRNA-containing RISC with PABPC is
`also required for translational repression, supporting a
`model in which PABPC recruits miRISC to the mRNA
`without the immediate removal of the poly(A) tail by the
`deadenylation machinery88.
`
`Additional AGO interaction partners. Several proteom-
`ics studies have sought to identify cellular interaction
`partners of AGO proteins19,32,89–92. In addition to the pro-
`teins that are required for RISC loading (for example,
`HSP90), many proteins with RNA-binding activity are
`among the binding partners. Many of these proteins
`are indirectly associated with AGO proteins by binding
`to the same RNAs. Nevertheless, such proteins can influ-
`ence the efficiency of miRNA-guided gene silencing.
`For example, the RNA-binding protein Pumilio (Pum)
`binds to the 3′ UTR of the E2F3 mRNA and enhances
`the activity of several miRNAs that regulate E2F3
`(REF. 93). It is possible that RNA-binding proteins such
`as Pumilio help to establish a stable interaction of the
`miRNA with the mRNA89. AGO proteins do not directly
`touch the mRNA, and the base pairing via the short seed
`region of the miRNA might not be sufficient for stable
`binding of miRISC to the target mRNA. Other pro-
`teins found in biochemical studies, such as importin 8
`(IPO8) or UPF1, seem to support AGO complexes in
`target association90,94. In addition, it has been proposed
`that LIM domain proteins help to stabilize the silenced
`complex by simultaneously interacting with the 5′ cap-
`binding complex and AGO proteins 95, and the mam-
`malian hyperplastic disc homologue EDD (also known
`as UBR5) helps in the recruitment of downstream factors
`by binding to GW proteins96. A protein that is associated
`with the 40S ribosomal subunit — namely, receptor for
`activated C kinase 1 (RACK1) — interacts with AGO
`proteins and recruits miRISC to sites of translatio