Multilayer checkpoints for microRNA authenticity during RISC assembly

MicroRNAs (miRNAs) function through the RNA-induced silencing complex (RISC), which contains an Argonaute (Ago) protein at the core. RISC assembly follows a two-step pathway: miRNA/miRNA* duplex loading into Ago, and separation of the two strands within Ago. Here we show that the 5' phosphate of the miRNA strand is essential for duplex loading into Ago, whereas the preferred 5' nucleotide of the miRNA strand and the base-pairing status in the seed region and the middle of the 3' region function as additive anchors to Ago. Consequently, the miRNA authenticity is inspected at multiple steps during RISC assembly.     

Cleavage of the star strand facilitates assembly of some microRNAs into Ago2-containing silencing complexes in mammals

In animals, microRNAs (miRNAs) and small interfering RNAs (siRNAs) repress expression of protein coding genes by assembling distinct RNA-induced silencing complexes (RISCs). It has previously been shown that passenger-strand cleavage is the predominant mechanism when siRNA duplexes are loaded into Argonaute2 (Ago2)-containing RISC, while an unwinding bypass mechanism is favored for miRNA duplexes with mismatches. Here I present experimental data indicating that some mammalian miRNAs are assembled into Ago2-containing RISC by cleaving their corresponding miRNA star strands. This phenomenon may depend on the secondary structure near the scissile phosphate of the miRNA duplex. In addition, I show that ATP is not required for star-strand cleavage in this process. Taken together, the data here provide insight into the miRNA-loading mechanisms in mammals.     

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

Small RNAs regulate genetic networks through a ribonucleoprotein complex called the RNA-induced silencing complex (RISC), which, in mammals, contains at its center one of four Argonaute proteins (Ago1-Ago4) (reviewed in [1-4]). A key regulatory event in the RNA interference (RNAi) and microRNA (miRNA) pathways is Ago loading, wherein double-stranded small-RNA duplexes are incorporated into RISC (pre-RISC) and then become single-stranded (mature RISC), a process that is not well understood [5, 6]. The?Agos contain an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain [7, 8] whose primary function is to bind the 3' end of small RNAs [9-13]. We created multiple PAZ-domain-disrupted mutant Ago proteins and studied their biochemical properties and biological functionality in cells.?We found that the PAZ domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer-substrate duplex RNAs,?PAZ-disrupted Agos bound duplex small interfering RNAs,?but were unable to unwind or eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved PAZ domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA- and RNAi-based therapeutics.     

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential

It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function.     

The nucleotide sequence features of the mature microRNA seem to be responsible for the affinity to human Ago2 AND Ago3 proteins

Mature miRNA of 20-24 nt in length are the endogenous sncRNA. They programs RISC to regulate functioning of mRNA with complimentary sites for these miRNAs. In case of Ago3 protein present in human RISC miRNAs direct inhibition of translation, whereas in case of Ago2 is in RISC, than mRNA cleavage in the middle of miRNA/mRNA heteroduplex is also possible. Using ACTIVITY system, that we developed earlier, we analyzed published data on miRNA affinity to human Ago2 and Ago3 proteins. We found increase in miRNA affinity to both Ago2 and Ago3 with the increase of the YRHB tetranucleotide abundance near 3'-end of these miRNAs (r = 0.613, alpha < 0.025). We also found that miRNA tendency to bind Ago2 in favor of Ago3 increases with the RHHK tetranucleotide abundance near miRNA center (r = 0.501, alpha < 0.05). Using these two findings we proposed two formulas to predict miRNA affinity to Ago2 and Ago3 proteins based on the YRHB and RHHK abundances within this arbitrary miRNA. Thereby we made reliable predictions of miRNA affinity to these proteins in RISC for both canonical (alpha < 0.00025) and non-canonical (alpha < 0.05) miRNAs in comparison with independent experimental data.     

Systematic identification of mRNAs recruited to argonaute 2 by specific microRNAs and corresponding changes in transcript abundance

microRNAs (miRNAs) are small non-coding RNAs that regulate mRNA stability and translation through the action of the RNAi-induced silencing complex (RISC). Our current understanding of miRNA function is inferred largely from studies of the effects of miRNAs on steady-state mRNA levels and from seed match conservation and context in putative targets. Here we have taken a more direct approach to these issues by comprehensively assessing the miRNAs and mRNAs that are physically associated with Argonaute 2 (Ago2), which is a core RISC component. We transfected HEK293T cells with epitope-tagged Ago2, immunopurified Ago2 together with any associated miRNAs and mRNAs, and quantitatively determined the levels of these RNAs by microarray analyses. We found that Ago2 immunopurified samples contained a representative repertoire of the cell's miRNAs and a select subset of the cell's total mRNAs. Transfection of the miRNAs miR-1 and miR-124 caused significant changes in the association of scores of mRNAs with Ago2. The mRNAs whose association with Ago2 increased upon miRNA expression were much more likely to contain specific miRNA seed matches and to have their overall mRNA levels decrease in response to the miRNA transfection than expected by chance. Hundreds of mRNAs were recruited to Ago2 by each miRNA via seed sequences in 3'-untranslated regions and coding sequences and a few mRNAs appear to be targeted via seed sequences in 5'-untranslated regions. Microarray analysis of Ago2 immunopurified samples provides a simple, direct method for experimentally identifying the targets of miRNAs and for elucidating roles of miRNAs in cellular regulation.     

Dicer is dispensable for asymmetric RISC loading in mammals

In flies, asymmetric loading of small RNA duplexes into Argonaute2-containing RNA-induced silencing complex (Ago2-RISC) requires Dicer-2/R2D2 heterodimer, which acts as a protein sensor for the thermodynamic stabilities of the ends of small RNA duplexes. However, the mechanism of small RNA asymmetry sensing in mammalian RISC assembly remains obscure. Here, we quantitatively examined RISC assembly and target silencing activity in the presence or absence of Dicer in mammals. Our data show that, unlike the well-characterized fly Ago2-RISC assembly pathway, mammalian Dicer is dispensable for asymmetric RISC loading in vivo and in vitro.     

NMDAR‐dependent Argonaute 2 phosphorylation regulates miRNA activity and dendritic spine plasticity

MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins to form the RNA‐induced silencing complex (RISC), underpinning a powerful mechanism for fine‐tuning protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)‐dependent synaptic plasticity by modulating the translation of proteins involved in dendritic spine morphogenesis or synaptic transmission. However, it is unknown how NMDAR stimulation stimulates RISC activity to rapidly repress translation of synaptic proteins. We show that NMDAR stimulation transiently increases Akt‐dependent phosphorylation of Ago2 at S387, which causes an increase in binding to GW182 and a rapid increase in translational repression of LIMK1 via miR‐134. Furthermore, NMDAR‐dependent down‐regulation of endogenous LIMK1 translation in dendrites and dendritic spine shrinkage requires phospho‐regulation of Ago2 at S387. AMPAR trafficking and hippocampal LTD do not involve S387 phosphorylation, defining this mechanism as a specific pathway for structural plasticity. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA‐mediated translational repression to control dendritic spine morphology.     

Sorting of Drosophila small silencing RNAs

In Drosophila, small interfering RNAs (siRNAs), which direct RNA interference through the Argonaute protein Ago2, are produced by a biogenesis pathway distinct from microRNAs (miRNAs), which regulate endogenous mRNA expression as guides for Ago1. Here, we report that siRNAs and miRNAs are sorted into Ago1 and Ago2 by pathways independent from the processes that produce these two classes of small RNAs. Such small-RNA sorting reflects the structure of the double-stranded assembly intermediates--the miRNA/miRNA( *) and siRNA duplexes--from which Argonaute proteins are loaded. We find that the Dcr-2/R2D2 heterodimer acts as a gatekeeper for the assembly of Ago2 complexes, promoting the incorporation of siRNAs and disfavoring miRNAs as loading substrates for Drosophila Ago2. A separate mechanism acts in parallel to favor miRNA/miRNA( *) duplexes and exclude siRNAs from assembly into Ago1 complexes. Thus, in flies small-RNA duplexes are actively sorted into Argonaute-containing complexes according to their intrinsic structures.     

Crystallin-αB Regulates Skeletal Muscle Homeostasis via Modulation of Argonaute2 Activity

The core functional machinery of the RNAi pathway is the RNA-induced silencing complex (RISC), wherein Argonaute2 (Ago2) is essential for siRNA-directed endonuclease activity and RNAi/microRNA-mediated gene silencing. Crystallin-αB (CryAB) is a small heat shock protein involved in preventing protein aggregation. We demonstrate that CryAB interacts with the N and C termini of Ago2, not the catalytic site defined by the convergence of the PAZ, MID, and PIWI domains. We further demonstrate significantly reduced Ago2 activity in the absence of CryAB, highlighting a novel role of CryAB in the mammalian RNAi/microRNA pathway. In skeletal muscle of CryAB null mice, we observe a shift in the hypertrophy-atrophy signaling axis toward atrophy under basal conditions. Moreover, loss of CryAB altered the capability of satellite cells to regenerate skeletal muscle. These studies establish that CryAB is necessary for normal Ago2/RISC activity and cellular homeostasis in skeletal muscle.     

Novel functional small RNAs are selectively loaded onto mammalian Ago1

Argonaute (Ago) proteins function in RNA silencing as components of the RNA-induced silencing complex (RISC). In lower organisms, the small interfering RNA and miRNA pathways diverge due in part to sorting mechanisms that direct distinct small RNA (sRNA) duplexes onto specific Ago-RISCs. However, such sorting mechanisms appear to be lost in mammals. miRNAs appear not to distinguish among Ago1–4. To determine the effect of viral infection on the sorting system, we compared the content of deep-sequenced RNA extracted from immunoprecipitation experiments with the Ago1 and Ago2 proteins using Epstein–Barr virus (EBV)-infected cells. Consistent with previous observations, sequence tags derived from miRNA loci in EBV and humans globally associate in approximately equivalent amounts with Ago1 and Ago2. Interestingly, additional sRNAs, which have not been registered as miRNAs, were associated with Ago1. Among them, some unique sequence tags derived from tandem loci in the human genome associate exclusively with Ago1 but not, or rarely, with Ago2. This is supported by the observation that the expression of the unique sRNAs in the cells is highly dependent on Ago1 proteins. When we knocked down Ago1, the expression of the Ago1-specific sRNAs decreased dramatically. Most importantly, the Ago1-specific sRNAs bound to mRNAs and regulated target genes and were dramatically upregulated, depending on the EBV life cycle. Therefore, even in mammals, the sorting mechanism in the Ago1–4 family is functional. Moreover, the existence of Ago1-specific sRNAs implies vital roles in some aspects of mammalian biology.     

Conversion of pre-RISC to holo-RISC by Ago2 during assembly of RNAi complexes

In the Drosophila RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) direct Argonaute2 (Ago2), an endonuclease, within the RNA-induced silencing complex (RISC) to cleave complementary mRNA targets. In vitro studies have shown that, for each siRNA duplex, RISC retains only one strand, the guide, and releases the other, the passenger, to form a holo-RISC complex. Here, we have isolated a new Ago2 mutant allele and provide, for the first time, in vivo evidence that endogenous Ago2 slicer activity is important to mount an RNAi response in Drosophila. We demonstrate in vivo that efficient removal of the passenger strand from RISC requires the cleavage activity of Ago2. We have also identified a new intermediate complex in the RISC assembly pathway, pre-RISC, in which Ago2 is stably bound to double-stranded siRNA.     

Specific features of the mature microrna nucleotide sequences can influence the affinity for the human Ago2 and Ago3 proteins

Mature microRNAs (miRNAs) with a length of 20–24 nucleotides are endogenous, small, non-coding RNAs. They program the RNA-induced silencing complex (RISC) to inhibit translation of the mRNAs carrying the sites complementary to these miRNAs. When the RISC contains Ago3, the mRNA translation is inhibited; however, in the case of Ago2, the mRNA can be also cleaved in the center of mRNA/miRNA heteroduplex. Using the earlier developed system ACTIVITY, we have analyzed the published data on the affinity of mature human miRNA sequences for the Ago2 and Ago3 proteins. It has been found that the higher the abundance of YRHB tetranucleotides near the miRNA 3′-end, the higher the miRNA affinity for both proteins (r = 0.613, α < 0.025) and that the miRNA binding to Ago2 increases relative to that Ago3 with the abundance of RHHK tetranucleotides near the miRNA center (r=0.501, α < 0.05). These two patterns allowed us to propose equations for predicting the affinity of mature miRNAs for the Ago2 and Ago3 proteins and to reliably predict the affinity of canonical (α < 0.00025) and noncanonical (α < 0.05) miRNAs for each protein using independent data.     

Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation

The mRNA-cleavage step of RNA interference is mediated by an endonuclease, Argonaute2 (Ago2), within the RNA-induced silencing complex (RISC). Ago2 uses one strand of the small interfering (si) RNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone at a specific site measured from the guide strand's 5' end. Here, we show that both strands of siRNA get loaded onto Ago2 protein in Drosophila S2 cell extracts. The anti-guide strand behaves as a RISC substrate and is cleaved by Ago2. This cleavage event is important for the removal of the anti-guide strand from Ago2 protein and activation of RISC.     

Argonaute蛋白与小RNA的作用机制

RNA干扰(RNA interference, RNAi)是在植物、动物、线虫、真菌以及昆虫等生物体中普遍存在的通过双链RNA(double strand RNA, dsRNA)诱导的抑制同源基因表达的一种保守的调控机制.小分子RNA通过特异性地识别结合RNA诱导的沉默复合体（RNA-induced silencing complex, RISC）对目标mRNA的表达在转录和翻译水平进行抑制.作为RISC的重要组成成分,Argonaute蛋白（Ago）发挥了至关重要的作用.为了进一步阐明Ago蛋白在RNA干扰中对小分子RNA的作用机制,本文介绍了Ago蛋白的结构、分类及其在RNA干扰机制中的作用,并着重阐述了目前已知的植物Ago蛋白对小分子RNA的几种作用机制,以及目前研究发现的Ago蛋白的功能作用,从而更进一步证实Ago蛋白对小分子RNA的作用是一个复杂的过程.     

Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character—as is found in small interfering RNAs (siRNAs)—directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.     

Expanded RNA-binding activities of mammalian Argonaute 2

Mammalian Argonaute 2 (Ago2) protein associates with microRNAs (miRNAs) or small interfering RNAs (siRNAs) forming RNA-induced silencing complexes (RISCs/miRNPs). In the present work, we characterize the RNA-binding and nucleolytic activity of recombinant mouse Ago2. Our studies show that recombinant mouse Ago2 binds efficiently to miRNAs forming active RISC. Surprisingly, we find that recombinant mouse Ago2 forms active RISC using pre-miRNAs or long unstructured single stranded RNAs as guides. Furthermore, we demonstrate that, in vivo, endogenous human Ago2 binds directly to pre-miRNAs independently of Dicer, and that Ago2:pre-miRNA complexes are found both in the cytoplasm and in the nucleus of human cells.