miRISC is a multi-protein assembly that uses microRNAs (miRNAs) to identify mRNAs targeted for repression. Dozens of miRISC-associated proteins have been identified, and interactions between many ...factors have been examined in detail. However, the physical nature of the complex remains unknown. Here, we show that two core protein components of human miRISC, Argonaute2 (Ago2) and TNRC6B, condense into phase-separated droplets in vitro and in live cells. Phase separation is promoted by multivalent interactions between the glycine/tryptophan (GW)-rich domain of TNRC6B and three evenly spaced tryptophan-binding pockets in the Ago2 PIWI domain. miRISC droplets formed in vitro recruit deadenylation factors and sequester target RNAs from the bulk solution. The condensation of miRISC is accompanied by accelerated deadenylation of target RNAs bound to Ago2. The combined results may explain how miRISC silences mRNAs of varying size and structure and provide experimental evidence that protein-mediated phase separation can facilitate an RNA processing reaction.
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•Ago2 has three pockets that bind Trp residues in the miRISC scaffold protein TNRC6B•Ago2-TNRC6B interactions promote phase separation of miRISC•miRISC droplets sequester miRNA target RNAs and recruit deadenylases•miRISC condensation correlates with accelerated target deadenylation
Phase separation of miRISC proteins leads to target mRNA sequestration and facilitates accelerated deadenylation.
Since their serendipitous discovery in nematodes, microRNAs (miRNAs) have emerged as key regulators of biological processes in animals. These small RNAs form complex networks that regulate cell ...differentiation, development and homeostasis. Deregulation of miRNA function is associated with an increasing number of human diseases, particularly cancer. Recent discoveries have expanded our understanding of the control of miRNA function. Here, we review the mechanisms that modulate miRNA activity, stability and cellular localization through alternative processing and maturation, sequence editing, post-translational modifications of Argonaute proteins, viral factors, transport from the cytoplasm and regulation of miRNA-target interactions. We conclude by discussing intriguing, unresolved research questions.
Nearly every cell in the human body contains a set of programmable gene-silencing proteins named Argonaute. Argonaute proteins mediate gene regulation by small RNAs and thereby contribute to cellular ...homeostasis during diverse physiological process, such as stem cell maintenance, fertilization, and heart development. Over the last decade, remarkable progress has been made toward understanding Argonaute proteins, small RNAs, and their roles in eukaryotic biology. Here, we review current understanding of Argonaute proteins from a structural prospective and discuss unanswered questions surrounding this fascinating class of enzymes.
Argonaute proteins form the functional core of the RNA-induced silencing complexes that mediate RNA silencing in eukaryotes. The 2.3 angstrom resolution crystal structure of human Argonaute2 (Ago2) ...reveals a bilobed molecule with a central cleft for binding guide and target RNAs. Nucleotides 2 to 6 of a heterogeneous mixture of guide RNAs are positioned in an A-form conformation for base pairing with target messenger RNAs. Between nucleotides 6 and 7, there is a kink that may function in microRNA target recognition or release of sliced RNA products. Tandem tryptophan-binding pockets in the PIWI domain define a likely interaction surface for recruitment of glycine-tryptophan-182 (GW182) or other tryptophan-rich cofactors. These results will enable structure-based approaches for harnessing the untapped therapeutic potential of RNA silencing in humans.
Structural basis for microRNA targeting Schirle, Nicole T.; Sheu-Gruttadauria, Jessica; MacRae, Ian J.
Science,
10/2014, Letnik:
346, Številka:
6209
Journal Article
Recenzirano
Odprti dostop
MicroRNAs (miRNAs) control expression of thousands of genes in plants and animals. miRNAs function by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for ...repression. We determined crystal structures of human Argonaute-2 (Ago2) bound to a defined guide RNA with and without target RNAs representing miRNA recognition sites. These structures suggest a stepwise mechanism, in which Ago2 primarily exposes guide nucleotides (nt) 2 to 5 for initial target pairing. Pairing to nt 2 to 5 promotes conformational changes that expose nt 2 to 8 and 13 to 16 for further target recognition. Interactions with the guide-target minor groove allow Ago2 to interrogate target RNAs in a sequence-independent manner, whereas an adenosine binding-pocket opposite guide nt 1 further facilitates target recognition. Spurious slicing of miRNA targets is avoided through an inhibitory coordination of one catalytic magnesium ion. These results explain the conserved nucleotide-pairing patterns in animal miRNA target sites first observed over two decades ago.
Small RNAs (sRNAs), including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are essential gene regulators for plant and animal development. The loading of sRNA duplexes into the proper ...ARGONAUTE (AGO) protein is a key step to forming a functional silencing complex. In Arabidopsis thaliana, the specific loading of miR166/165 into AGO10 (AtAGO10) is critical for the maintenance of the shoot apical meristem, the source of all shoot organs, but the mechanism by which AtAGO10 distinguishes miR166/165 from other cellular miRNAs is not known. Here, we show purified AtAGO10 alone lacks loading selectivity towards miR166/165 duplexes. However, phosphate and HSP chaperone systems reshape the selectivity of AtAGO10 to its physiological substrates. A loop in the AtAGO10 central cleft is essential for recognizing specific mismatches opposite the guide strand 3' region in miR166/165 duplexes. Replacing this loop with the equivalent loop from Homo sapiens AGO2 (HsAGO2) changes AtAGO10 miRNA loading behavior such that 3' region mismatches are ignored and mismatches opposite the guide 5' end instead drive loading, as in HsAGO2. Thus, this study uncovers the molecular mechanism underlying the miR166/165 selectivity of AtAGO10, essential for plant development, and provides new insights into how miRNA duplex structures are recognized for sRNA sorting.
Argonaute proteins play a central role in mediating post-transcriptional gene regulation by microRNAs (miRNAs). Argonautes use the nucleotide sequences in miRNAs as guides for identifying target ...messenger RNAs for repression. Here, we used single-molecule FRET to directly visualize how human Argonaute-2 (Ago2) searches for and identifies target sites in RNAs complementary to its miRNA guide. Our results suggest that Ago2 initially scans for target sites with complementarity to nucleotides 2–4 of the miRNA. This initial transient interaction propagates into a stable association when target complementarity extends to nucleotides 2–8. This stepwise recognition process is coupled to lateral diffusion of Ago2 along the target RNA, which promotes the target search by enhancing the retention of Ago2 on the RNA. The combined results reveal the mechanisms that Argonaute likely uses to efficiently identify miRNA target sites within the vast and dynamic agglomeration of RNA molecules in the living cell.
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•Argonaute uses one-dimensional diffusion as a search mechanism•Argonaute initially probes for target sites using a small region (nt 2–4) of miRNA•The seed (nt 2–8) is the minimal motif required for stable binding to target sites•The lateral diffusion promotes cooperativity between neighboring target sites
Argonaute identifies miRNA targets by scanning potential target RNAs using one-dimensional diffusion while probing for sites complementary to a small segment of the miRNA and remains stably associated to sites complementary to the full miRNA seed.
RNA interference is a powerful mechanism of gene silencing that underlies many aspects of eukaryotic biology. On the molecular level, RNA interference is mediated by a family of ribonucleoprotein ...complexes called RNA-induced silencing complexes (RISCs), which can be programmed to target virtually any nucleic acid sequence for silencing. The ability of RISC to locate target RNAs has been co-opted by evolution many times to generate a broad spectrum of gene-silencing pathways. Here, we review the fundamental biochemical and biophysical properties of RISC that facilitate gene targeting and describe the various mechanisms of gene silencing known to exploit RISC activity.
MicroRNAs (miRNAs) broadly regulate gene expression through association with Argonaute (Ago), which also protects miRNAs from degradation. However, miRNA stability is known to vary and is regulated ...by poorly understood mechanisms. A major emerging process, termed target-directed miRNA degradation (TDMD), employs specialized target RNAs to selectively bind to miRNAs and induce their decay. Here, we report structures of human Ago2 (hAgo2) bound to miRNAs and TDMD-inducing targets. miRNA and target form a bipartite duplex with an unpaired flexible linker. hAgo2 cannot physically accommodate the RNA, causing the duplex to bend at the linker and display the miRNA 3′ end for enzymatic attack. Altering 3′ end display by changing linker flexibility, changing 3′ end complementarity, or mutationally inducing 3′ end release impacts TDMD efficiency, leading to production of distinct 3′-miRNA isoforms in cells. Our results uncover the mechanism driving TDMD and reveal 3′ end display as a key determinant regulating miRNA activity via 3′ remodeling and/or degradation.
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•Structural and mutational analyses reveal mechanism of target-directed miRNA degradation•TDMD targets trap Ago2 in a conformation with miRNA 3′ end displayed for enzymatic attack•miRNA-TDMD target pairing features dictate miRNA 3′ end remodeling and fate•miRNA 3′ end display is a mechanism that controls miRNA stability and activity
MicroRNAs (miRNAs) shape post-transcriptional gene expression by repressing messenger RNAs. Conversely, certain target RNAs induce miRNA decay through a process called target-directed miRNA degradation (TDMD). Sheu-Gruttadauria et al. show how these targets expose the miRNA 3′ end for enzymatic attack, enabling 3′ end remodeling and degradation.
Argonaute proteins use small RNAs to guide the silencing of complementary target RNAs in many eukaryotes. Although small RNA biogenesis pathways are well studied, mechanisms for removal of guide RNAs ...from Argonaute are poorly understood. Here we show that the Argonaute2 (Ago2) guide RNA complex is extremely stable, with a half-life on the order of days. However, highly complementary target RNAs destabilize the complex and significantly accelerate release of the guide RNA from Ago2. This “unloading” activity can be enhanced by mismatches between the target and the guide 5′ end and attenuated by mismatches to the guide 3′ end. The introduction of 3′ mismatches leads to more potent silencing of abundant mRNAs in mammalian cells. These findings help to explain why the 3′ ends of mammalian microRNAs (miRNAs) rarely match their targets, suggest a mechanism for sequence-specific small RNA turnover, and offer insights for controlling small RNAs in mammalian cells.
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•The Ago2-guide RNA complex is extremely long-lived in vitro•Interaction with target RNAs destabilizes the Ago2-guide RNA complex•3′ mismatches can enhance silencing of abundant mRNAs in mammalian cells•3′ mismatches can enhance silencing of abundant mRNAs in mammalian cells
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