An mRNA m7G cap binding-like motif within human Ago2 represses translation

microRNAs (miRNAs) bind to Argonaute (Ago) proteins and inhibit translation or promote degradation of mRNA targets. Human let-7 miRNA inhibits translation initiation of mRNA targets in an m(7)G cap-dependent manner and also appears to block protein production, but the molecular mechanism(s) involved is unknown and the role of Ago proteins in translational regulation remains elusive. Here we identify a motif (MC) within the Mid domain of Ago proteins, which bears significant similarity to the m(7)G cap-binding domain of eIF4E, an essential translation initiation factor. We identify conserved aromatic residues within the MC motif of human Ago2 that are required for binding to the m(7)G cap and for translational repression but do not affect the assembly of Ago2 with miRNA or its catalytic activity. We propose that Ago2 represses the initiation of mRNA translation by binding to the m(7)G cap of mRNA targets, thus likely precluding the recruitment of eIF4E.     

Identification of eight members of the Argonaute family in the human genome

A number of genes have been identified as members of the Argonaute family in various nonhuman organisms and these genes are considered to play important roles in the development and maintenance of germ-line stem cells. In this study, we identified the human Argonaute family, consisting of eight members. Proteins to be produced from these family members retain a common architecture with the PAZ motif in the middle and Piwi motif in the C-terminal region. Based on the sequence comparison, eight members of the Argonaute family were classified into two subfamilies: the PIWI subfamily (PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2) and the eIF2C/AGO subfamily (EIF2C1/hAGO1, EIF2C2/hAGO2, EIF2C3/hAGO3, and EIF2C4/hAGO4). PCR analysis using human multitissue cDNA panels indicated that all four members of the PIWI subfamily are expressed mainly in the testis, whereas all four members of the eIF2C/AGO subfamily are expressed in a variety of adult tissues. Immunoprecipitation and affinity binding experiments using human HEK293 cells cotransfected with cDNAs for FLAG-tagged DICER, a member of the ribonuclease III family, and the His-tagged members of the Argonaute family suggested that the proteins from members of both subfamilies are associated with DICER. We postulate that at least some members of the human Argonaute family may be involved in the development and maintenance of stem cells through the RNA-mediated gene-quelling mechanisms associated with DICER.     

Argonaute MID domain takes centre stage

Two recent papers, one in EMBO reports and one in Nature give us the first eukaryotic structures of Argonaute MID domains; providing a structural basis for the 5′-nucleotide recognition of the guide strand and a possible explanation for the allosteric regulation of RNA binding.EMBO Rep (2010) advance online publication. doi: 10.1038/embor.2010.81Argonaute (AGO) proteins are the central component of small RNA-mediated gene silencing in eukaryotes. Functional AGO complexes are loaded with single-stranded small RNAs, which guide AGO to a messenger RNA (mRNA) target through base pairing. Although the structure of a full-length eukaryotic AGO has yet to be described, insights into the mechanism of guide RNA binding and target recognition have been revealed by the structures of distantly related AGO homologues from archaea and eubacteria (Song et al, 2004; Wang et al, 2008, 2009). These studies show that AGO proteins are composed of amino-terminal, PAZ (PIWI/Argonaute/Zwille), MID (middle) and PIWI (P-element-induced whimpy testes) domains. The phosphorylated 5′-end of the guide strand RNA is localized in the MID–PIWI domain interface with the 3′-end anchored to the PAZ domain. On binding to mRNA the catalytic RNase H-like active site located in the PIWI domain is in position to cleave the targeted mRNA.Two recent papers, one in EMBO reports (Boland et al, 2010) and one in Nature (Frank et al, 2010), give us the first eukaryotic structures of AGO MID domains. The human AGO MID domain structure provides a structural basis for the 5′-nucleotide recognition of the guide strand observed in eukaryotic AGOs, and the structure of the MID domain of QDE-2 from Neurospora crassa published in this journal offers a possible explanation for the allosteric regulation of RNA binding discovered earlier this year by Rachel Green''s group (Djuranovic et al, 2010)The two structures have a similar topology resembling a Rossmann-like fold with four β-strands forming a central β-sheet flanked by α-helices. Superposition of the two structures, which are 24% identical in sequence, shows that they are also similar in three dimensions, with a root mean square deviation (r.m.s.d.) of 2.1 Å. The archaeal and eubacterial AGO MID domains solved previously share less than 20% sequence identity and have a greater than 2.5 Å r.m.s.d. for backbone atoms from both QDE-2 and human AGO2, despite having a similar overall fold.Crystals of the QDE-2 MID domain contain two sulphate ions. The first sulphate (sulphate I) is coordinated by the highly conserved amino acids Tyr 595, Lys 599 and Lys 638, and is in the same position as the 5′-phosphate of UMP observed in the human AGO2 MID domain structure (Fig 1A). These interactions are similar to those observed for the 5′-phosphate of the guide strand of the previously solved archaeal and eubacterial structures (Ma et al, 2005; Parker et al, 2005; Wang et al, 2008). Thus, sulphate I bound in the QDE-2 MID domain structure likely represents the 5′-nucleotide-binding site. Most intriguing is the position of the second sulphate (sulphate II), located in an adjacent but partly overlapping binding site with sulphate I. Sulphate II is 6.3 Å from sulphate I, shares coordination with Lys 599 and Lys 638, and is further coordinated by Thr 610. Sulphate II can be excluded from representing the phosphate backbone of a microRNA (miRNA) or target because it is bound in the side of the MID domain opposite from where the guide RNA extends from the 5′-nucleotide-binding site. Although the presence of sulphate II does not guarantee a biologically relevant ligand-binding site, it is tempting to speculate, in the light of a recent study by Djuranovic et al, that sulphate II occupies an allosteric ligand-binding site.Open in a separate windowFigure 1Structural comparison of Neurospora crassa QDE-2 MID domain and human Argonaute 2 MID domain. (A) The N. crassa QDE-2 MID domain structure (green ribbon). UMP is modelled from a superposition of the human AGO2 MID domain structure in a complex with UMP (Protein Data Bank code 3LUJ). Sulphate I and II (S I and S II) are shown as observed in the QDE-2 structure. Conserved QDE-2 amino acids involved in binding sulphate I and II are shown as sticks. (B) Human AGO2 MID domain structure (blue ribbon) in complex with UMP (sticks). The nucleotide specificity loop is coloured in yellow. Sulphate I and II are modelled from a superposition of the N. crassa QDE-2 MID domain. AGO, Argonaute; MID, middle.Djuranovic et al describe a second ligand-binding site in the Drosophila melanogaster AGO1 MID domain that is separate and distinct from the 5′-nucleotide-binding site. They demonstrate that free nucleotides, including the cap analogue m⁷GpppG, bind to an allosteric site, which in turn enhances the binding of miRNA. Cap binding was reported previously for human AGO2 (Kiriakidou et al, 2007), leading to the proposal that two phenylalanine residues in the MID domain make stacking interactions with the m⁷GpppG cap structure, analogous to eukaryotic initiation factor 4E. However, in the human AGO2 MID domain structure it is clear that these phenylalanine residues are on opposite sides of the MID domain and are located in the hydrophobic core. In the QDE-2 MID domain only one of these phenylalanines is conserved, but a similar conclusion is drawn on the basis of the positions of the two residues being more than 25 Å apart. This strongly argues that an alternative mechanism exists for cap binding by eukaryotic AGO proteins.The data presented by Djuranovic et al might be explained by the structure of the QDE-2 MID domain, with sulphate I representing the 5′-binding site of a miRNA and sulphate II representing the allosteric site. This argument is strengthened by the fact that the two binding sites are partly shared, namely by interactions with the side chains of Lys 599 and Lys 638, so it would not be surprising that binding of a ligand to one site would have a positive effect on ligand binding at the other site. To identify the location of the potential allosteric site, Djuranovic et al mutated Asp 627—a conserved residue located in a loop 15 Å away from the 5′-nucleotide binding site—to a lysine in D. melanogaster AGO1. The D627K mutant failed to bind cap analogues, indicating the importance of Asp 627 for binding ligands in the allosteric site. When mapped onto the new structures of the QDE-2 and human AGO2 MID domains, this loop and Asp 627 (Asp 603 in QDE-2 and Asp 537 in human AGO2) are in the vicinity of sulphate II, thus Asp 627 is probably a part of the allosteric binding site (Fig 1A,B). The most significant finding in the Djuranovic et al study is that the D627K mutant located in the allosteric site fails to bind to miRNA in the 5′-nucleotide-binding site and no longer associates with GW182, an essential factor in miRNA-induced gene silencing.Almost all miRNA sequences and RNA sequencing data obtained from immunopurified AGO proteins show a marked bias for uridine and adenosine nucleotides at the 5′-end of miRNA guide strands. The structure of human AGO2 MID domain alone and in a complex with UMP, AMP, GMP and CMP provides the first explanation for the observed 5′-nucleotide bias in eukaryotic AGO proteins (Frank et al, 2010). There is little movement induced on nucleotide binding in the overall fold of the MID domain. Electron density is observed for the entire nucleotide in the case of UMP and AMP. The 5′-phosphate in the UMP and AMP complexes is hydrogen bonded to the highly conserved side chains of Tyr 529, Lys 533, Gln 545 and Lys 570. The base of each nucleotide stacks with Tyr 529, completing a nonspecific recognition pocket for the 5′-nucleotide (Fig 1B). A similar pocket is formed in the N. crassa MID domain structure to recognize the 5′-nucleotide. Interestingly, clear electron density for only the phosphate and ribose is observed for GMP and CMP, with density for the GMP and CMP bases missing. These results are consistent with the preference for UMP and AMP binding to human AGO2, but where does this specificity originate?A closer look at the UMP and AMP complex structures show that base-specific contacts are formed with backbone atoms of a loop spanning residues Pro 523 through Pro 527, appropriately termed the nucleotide specificity loop. In the case of GMP and CMP, the hydrogen-bonding partners are in the opposite orientation, resulting in charge repulsion from backbone atoms in the nucleotide specificity loop, thus explaining the observed bias in the 5′-position of the guide strand. In the nucleotide-free structure, the conformation of the nucleotide specificity loop is merely unchanged, suggesting that the loop is rather rigid. When the length of the nucleotide specificity loop is increased by the insertion of a single glycine residue, the specificity for uridine and adenosine nucleotides is lost, further endorsing the idea that the particular conformation and rigid nature of the loop is essential for specific base recognition. Interestingly, the QDE-2 MID domain deviates from the human AGO2 MID domain in the nucleotide specificity loop. An insertion of an aspartate residue in QDE-2 makes the nucleotide specificity loop one amino acid longer, suggesting that QDE-2 might have lost its specificity for nucleotides at the 5′-end of miRNAs, although this is yet to be tested.A complete understanding of miRNA loading and the allosteric mechanism will have to await structures of full-length eukaryotic AGO proteins, as the PIWI domain contributes numerous contacts with the MID domain, encompassing both the 5′-nucleotide-binding site and the putative allosteric site. However, the structures of N. crassa QDE-2 MID and human AGO2 MID domains together are important pieces of the puzzle in our understanding of the mechanism of RNA interference. Specific recognition of the 5′-nucleotide of the guide strand might be a quality control mechanism for some eukaryotic AGOs, ensuring that after primary processing the correct miRNA guide sequence is loaded. Once loaded with a proper guide strand, AGO might trigger the adjacent allosteric site to bind to m⁷GpppG-capped mRNA, GW182 or other unknown ligands. Together, these events ultimately lead to effective gene silencing.     

The GW/WG repeats of Drosophila GW182 function as effector motifs for miRNA-mediated repression

The control of messenger RNA (mRNA) function by micro RNAs (miRNAs) in animal cells requires the GW182 protein. GW182 is recruited to the miRNA repression complex via interaction with Argonaute protein, and functions downstream to repress protein synthesis. Interaction with Argonaute is mediated by GW/WG repeats, which are conserved in many Argonaute-binding proteins involved in RNA interference and miRNA silencing, from fission yeast to mammals. GW182 contains at least three effector domains that function to repress target mRNA. Here, we analyze the functions of the N-terminal GW182 domain in repression and Argonaute1 binding, using tethering and immunoprecipitation assays in Drosophila cultured cells. We demonstrate that its function in repression requires intact GW/WG repeats, but does not involve interaction with the Argonaute1 protein, and is independent of the mRNA polyadenylation status. These results demonstrate a novel role for the GW/WG repeats as effector motifs in miRNA-mediated repression.     

Crystal structure and ligand binding of the MID domain of a eukaryotic Argonaute protein

Argonaute (AGO) proteins are core components of RNA‐induced silencing complexes and have essential roles in RNA‐mediated gene silencing. They are characterized by a bilobal architecture, consisting of one lobe containing the amino‐terminal and PAZ domains and another containing the MID and PIWI domains. Except for the PAZ domain, structural information on eukaryotic AGO domains is not yet available. In this study, we report the crystal structure of the MID domain of the eukaryotic AGO protein QDE‐2 from Neurospora crassa. This domain adopts a Rossmann‐like fold and recognizes the 5′‐terminal nucleotide of a guide RNA in a manner similar to its prokaryotic counterparts. The 5′‐nucleotide‐binding site shares common residues with a second, adjacent ligand‐binding site, suggesting a mechanism for the cooperative binding of ligands to the MID domain of eukaryotic AGOs.     

Argonaute identity defines the length of mature mammalian microRNAs

MicroRNAs (miRNAs) are 19- to 25-nt-long non-coding RNAs that regulate gene expression by base-pairing with target mRNAs and reducing their stability or translational efficiency. Mammalian miRNAs function in association with four closely related Argonaute proteins, AGO1-4. All four proteins contain the PAZ and the MID domains interacting with the miRNA 3' and 5' termini, respectively, as well as the PIWI domain comprising an mRNA 'slicing' activity in the case of AGO2 but not AGO1, AGO3 and AGO4. However, the slicing mode of the miRNA-programmed AGO2 is rarely realized in vivo and the four Argonautes are thought to play largely overlapping roles in the mammalian miRNA pathway. Here, we show that the average length of many miRNAs is diminished during nervous system development as a result of progressive shortening of the miRNA 3' ends. We link this modification with an increase in the fractional abundance of Ago2 in the adult brain and identify a specific structural motif within the PAZ domain that enables efficient trimming of miRNAs associated with this but not the other three Argonautes. Taken together, our data suggest that mammalian Argonautes may define the length and possibly biological activity of mature mammalian miRNAs in a developmentally controlled manner.     

Local and global effects of Mg2+ on Ago and miRNA-target interactions

Three magnesium ions (Mg(2+)), named Mg1 (in Mid domain), Mg2 and Mg3 (both in PIWI domain), located at the small RNA binding domain of Argonaute (Ago) protein, are important for sequence-specific miRNA-target interactions. Such conjunction between the Ago protein and miRNA raises the question: How do Mg(2+) ions participate in the recognition process of miRNA by Ago or its target. Furthermore, it is still unclear whether the Mg(2+) ions contribute to the local or global stability of the miRNA complex. In this work, we have performed a series of 16 independent molecular dynamic simulations (MD) to characterize the functions of Mg(2+), hydration patterns and the conformational events involved in the miRNA-target interactions. The cross correlation analysis shows that Mg1 and Mg2 significantly enhance a locally cooperated movement of the PAZ, PIWI and Mid domains with the average correlation coefficient of ～0.65, producing an "open-closed" motion (rotation Angle, 46.5°) between the PAZ and PIWI domains. Binding of Mg3 can globally stabilize the whole Ago protein with the average RMSD of ～0.34 ?, compared with the systems in absence of Mg3 (average RMSD?=?～0.43 ?). Three structural water molecules surrounding the Mg(2+)-binding regions also stabilize these ions, thus facilitating the recognition of miRNA to its target. In addition, the thermodynamic analysis also verifies the positive contribution of all three Mg(2+) to the binding of miRNA to Ago, as well as the importance Mg2 plays in the cleavage of the miRNA targets.     

Bioenergetics and Gene Silencing Approaches for Unraveling Nucleotide Recognition by the Human EIF2C2/Ago2 PAZ Domain

Gene silencing and RNA interference are major cellular processes that control gene expression via the cleavage of target mRNA. Eukaryotic translation initiation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2) is considered to be the major player of RNAi as it is the core component of RISC complexes. While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized. Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has an inherent lack of binding to adenine nucleotides, a feature that highlights the poor binding of 3′-adenylated RNAs with the PAZ domain as well as the selective high trimming of the 3′-ends of miRNA containing adenine nucleotides. We further show that the PAZ domain selectively binds all ribonucleotides (except adenosine), whereas it poorly recognizes deoxyribonucleotides. In this context, the modification of dTMP to its ribonucleotide analogue gave a drastic improvement of binding enthalpy and, hence, binding affinity. Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders. These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain.     

Seq and CLIP through the miRNA world

High-throughput sequencing of RNAs crosslinked to Argonaute proteins reveals not only a multitude of atypical miRNA binding sites but also of miRNA targets with atypical functions, and can be used to infer quantitative models of miRNA-target interaction strength.     

Altered Gene Expression Associated with microRNA Binding Site Polymorphisms

Allele-specific gene expression associated with genetic variation in regulatory regions can play an important role in the development of complex traits. We hypothesized that polymorphisms in microRNA (miRNA) response elements (MRE-SNPs) that either disrupt a miRNA binding site or create a new miRNA binding site can affect the allele-specific expression of target genes. By integrating public expression quantitative trait locus (eQTL) data, miRNA binding site predictions, small RNA sequencing, and Argonaute crosslinking immunoprecipitation (AGO-CLIP) datasets, we identified genetic variants that can affect gene expression by modulating miRNA binding efficiency. We also identified MRE-SNPs located in regions associated with complex traits, indicating possible causative mechanisms associated with these loci. The results of this study expand the current understanding of gene expression regulation and help to interpret the mechanisms underlying eQTL effects.     

A C-terminal silencing domain in GW182 is essential for miRNA function

Proteins of the GW182 family are essential for miRNA-mediated gene silencing in animal cells; they interact with Argonaute proteins (AGOs) and are required for both the translational repression and mRNA degradation mediated by miRNAs. To gain insight into the role of the GW182–AGO1 interaction in silencing, we generated protein mutants that do not interact and tested them in complementation assays. We show that silencing of miRNA targets requires the N-terminal domain of GW182, which interacts with AGO1 through multiple glycine–tryptophan (GW)-repeats. Indeed, a GW182 mutant that does not interact with AGO1 cannot rescue silencing in cells depleted of endogenous GW182. Conversely, silencing is impaired by mutations in AGO1 that strongly reduce the interaction with GW182 but not with miRNAs. We further show that a GW182 mutant that does not localize to P-bodies but interacts with AGO1 rescues silencing in GW182-depleted cells, even though in these cells, AGO1 also fails to localize to P-bodies. Finally, we show that in addition to the N-terminal AGO1-binding domain, the middle and C-terminal regions of GW182 (referred to as the bipartite silencing domain) are essential for silencing. Together our results indicate that miRNA silencing in animal cells is mediated by AGO1 in complex with GW182, and that P-body localization is not required for silencing.     

Structural and functional analysis of methylation and 5'-RNA sequence requirements of short capped RNAs by the methyltransferase domain of dengue virus NS5

The N-terminal 33 kDa domain of non-structural protein 5 (NS5) of dengue virus (DV), named NS5MTase(DV), is involved in two of four steps required for the formation of the viral mRNA cap (7Me)GpppA(2'OMe), the guanine-N7 and the adenosine-2'O methylation. Its S-adenosyl-l-methionine (AdoMet) dependent 2'O-methyltransferase (MTase) activity has been shown on capped (7Me+/-)GpppAC(n) RNAs. Here we report structural and binding studies using cap analogues and capped RNAs. We have solved five crystal structures at 1.8 A to 2.8 A resolution of NS5MTase(DV) in complex with cap analogues and the co-product of methylation S-adenosyl-l-homocysteine (AdoHcy). The cap analogues can adopt several conformations. The guanosine moiety of all cap analogues occupies a GTP-binding site identified earlier, indicating that GTP and cap share the same binding site. Accordingly, we show that binding of (7Me)GpppAC(4) and (7Me)GpppAC(5) RNAs is inhibited in the presence of GTP, (7Me)GTP and (7Me)GpppA but not by ATP. This particular position of the cap is in accordance with the 2'O-methylation step. A model was generated of a ternary 2'O-methylation complex of NS5MTase(DV), (7Me)GpppA and AdoMet. RNA-binding increased when (7Me+/-)GpppAGC(n-1) starting with the consensus sequence GpppAG, was used instead of (7Me+/-)GpppAC(n). In the NS5MTase(DV)-GpppA complex the cap analogue adopts a folded, stacked conformation uniquely possible when adenine is the first transcribed nucleotide at the 5' end of nascent RNA, as it is the case in all flaviviruses. This conformation cannot be a functional intermediate of methylation, since both the guanine-N7 and adenosine-2'O positions are too far away from AdoMet. We hypothesize that this conformation mimics the reaction product of a yet-to-be-demonstrated guanylyltransferase activity. A putative Flavivirus RNA capping pathway is proposed combining the different steps where the NS5MTase domain is involved.     

Structural evolution and functional diversification analyses of argonaute protein

Argonaute (AGO) proteins are highly specialized small-RNA-binding modules and small RNAs are anchored to their specific binding pockets guiding AGO proteins to target mRNA molecules for silencing or destruction. The 135 full-length AGO protein sequences derived from 36 species covering prokaryote, archaea, and eukaryote are chosen for structural and functional analyses. The results show that bacteria and archaeal AGO proteins are clustered in the same clade and there exist multiple AGO proteins in most eukaryotic species, demonstrating that the increase of AGO gene copy number and horizontal gene transfer (HGT) have been the main evolutionary driving forces for adaptability and biodiversity. And the emergence of PAZ domain in AGO proteins is the unique evolutionary event. The analysis of middle domain (MID)-nucleotide contaction shows that either the position of sulfate I bond in Nc_QDE2 or the site of phosphate I bond in Hs_AGO2 represents the 5'-nucleotide binding site of miRNA. Also, H334, T335, and Y336 of Hs_AGO1 can form hydrogen bonds with 3'-overhanging ends of miRNAs and the same situation exists in Hs_AGO2, Hs_AGO3, Hs_AGO4, Dm_AGO1, and Ce_Alg1. Some PIWI domains containing conserved DDH motif have no slicer activity, and post-translational modifications may be associated with the endonucleolytic activities of AGOs. With the numbers of AGO genes increasing and fewer crystal structures available, the evolutionary and functional analyses of AGO proteins can help clarify the molecular mechanism of function diversification in response to environmental changes, and solve major issues including host defense mechanism against virus infection and molecular basis of disease.     

Arabidopsis Argonaute MID domains use their nucleotide specificity loop to sort small RNAs

The 5'-nucleotide of small RNAs associates directly with the MID domain of Argonaute (AGO) proteins. In humans, the identity of the 5'-base is sensed by the MID domain nucleotide specificity loop and regulates the integrity of miRNAs. In Arabidopsis thaliana, the 5'-nucleotide also controls sorting of small RNAs into the appropriate member of the AGO family; however, the structural basis for this mechanism is unknown. Here, we present crystal structures of the MID domain from three Arabidopsis AGOs, AtAGO1, AtAGO2 and AtAGO5, and characterize their interactions with nucleoside monophosphates (NMPs). In AtAGOs, the nucleotide specificity loop also senses the identity of the 5'-nucleotide but uses more diverse modes of recognition owing to the greater complexity of small RNAs found in plants. Binding analyses of these interactions reveal a strong correlation between their affinities and evolutionary conservation.     

The GW182 protein family in animal cells: New insights into domains required for miRNA-mediated gene silencing

GW182 family proteins interact directly with Argonaute proteins and are required for miRNA-mediated gene silencing in animal cells. The domains of the GW182 proteins have recently been studied to determine their role in silencing. These studies revealed that the middle and C-terminal regions function as an autonomous domain with a repressive function that is independent of both the interaction with Argonaute proteins and of P-body localization. Such findings reinforce the idea that GW182 proteins are key components of miRNA repressor complexes in metazoa.     

On Measuring miRNAs after Transient Transfection of Mimics or Antisense Inhibitors

The ability to alter microRNA (miRNA) abundance is crucial for studying miRNA function. To achieve this there is widespread use of both exogenous double-stranded miRNA mimics for transient over-expression, and single stranded antisense RNAs (antimiRs) for miRNA inhibition. The success of these manipulations is often assessed using qPCR, but this does not accurately report the level of functional miRNA. Here, we draw attention to this discrepancy, which is overlooked in many published reports. We measured the functionality of exogenous miRNA by comparing the total level of transfected miRNA in whole cell extracts to the level of miRNA bound to Argonaute following transfection and show that the supraphysiological levels of transfected miRNA frequently seen using qPCR do not represent the functional levels, because the majority of transfected RNA that is detected is vesicular and not accessible for loading into Argonaute as functionally active miRNAs. In the case of microRNA inhibition by transient transfection with antisense inhibitors, there is also the potential for discrepancy, because following cell lysis the abundant inhibitor levels from cellular vesicles can directly interfere with the PCR reaction used to measure miRNA level.