Anti-Adenoviral Effect of Human Argonaute 2 Alone and in Combination with Artificial microRNAs.

During infection, adenoviruses inhibit the cellular RNA interference (RNAi) machinery by saturating the RNA-induced silencing complex (RISC) of the host cells with large amounts of virus-derived microRNAs (mivaRNAs) that bind to the key component of the complex, Argonaute 2 (AGO2). In the present study, we investigated AGO2 as a prominent player at the intersection between human adenovirus 5 (HAdV-5) and host cells because of its ability to interfere with the HAdV-5 life cycle. First, the ectopic expression of AGO2 had a detrimental effect on the ability of the virus to replicate. In addition, in silico and in vitro analyses suggested that endogenous microRNAs (miRNAs), particularly hsa-miR-7-5p, have similar effects. This miRNA was found to be able to target the HAdV-5 DNA polymerase mRNA. The inhibitory effect became more pronounced upon overexpression of AGO2, likely due to elevated AGO2 levels, which abolished the competition between cellular miRNAs and mivaRNAs for RISC incorporation. Collectively, our data suggest that endogenous miRNAs would be capable of significantly inhibiting viral replication if adenoviruses had not developed a mechanism to counteract this function. Eventually, AGO2 overexpression-mediated relief of the RISC-saturating action of mivaRNAs strongly enhanced the effectiveness of artificial miRNAs (amiRNAs) directed against the HAdV-5 preterminal protein (pTP) mRNA, suggesting a substantial benefit of co-expressing amiRNAs and AGO2 in RNAi-based strategies for the therapeutic inhibition of adenoviruses.

Induction of necrosis symptoms by potato virus X in AGO2-silenced tomato plants associates with reduced transcript accumulation of copper chaperon for superoxide dismutase gene.

RNA silencing is a prominent antiviral defense mechanism in plants. When infected with a virus, RNA silencing-deficient plants tend to show exacerbated symptoms along with increased virus accumulation. However, how symptoms are exacerbated is little understood. Here, we investigated the role of the copper chaperon for superoxide dismutase (CCS) 1, in systemic necrosis observed in Argonaute (AGO)2-silenced tomato plants infected with potato virus X (PVX). While infection with the UK3 strain of PVX induced mosaic symptoms in tomato plants, systemic necrosis occurred when AGO2 was silenced. The CCS1 mRNA level was reduced and micro RNA398 (miR398), which potentially target CCS1, was increased in AGO2-knockdown tomato plants infected with PVX-UK3. Ectopic expression of CCS1 using recombinant PVX attenuated necrosis, suggesting that CCS1 alleviates systemic necrosis by activating superoxide dismutases to scavenge reactive oxygen species. Previous reports have indicated a decrease in the levels of CCS1 and superoxide dismutases along with an increased level of miR398 in plants infected with other viruses and viroids, and thus might represent shared regulatory mechanisms that exacerbate symptoms in these plants.

m6A modification inhibits miRNAs' intracellular function, favoring their extracellular export for intercellular communication.

Epitranscriptomics represents a further layer of gene expression regulation. Specifically, N6-methyladenosine (m6A) regulates RNA maturation, stability, degradation, and translation. Regarding microRNAs (miRNAs), while it has been reported that m6A impacts their biogenesis, the functional effects on mature miRNAs remain unclear. Here, we show that m6A modification on specific miRNAs weakens their coupling to AGO2, impairs their function on target mRNAs, determines their delivery into extracellular vesicles (EVs), and provides functional information to receiving cells. Mechanistically, the intracellular functional impairment is caused by m6A-mediated inhibition of AGO2/miRNA interaction, the EV loading is favored by m6A-mediated recognition by the RNA-binding protein (RBP) hnRNPA2B1, and the EV-miRNA function in the receiving cell requires their FTO-mediated demethylation. Consequently, cells express specific miRNAs that do not impact endogenous transcripts but provide regulatory information for cell-to-cell communication. This highlights that a further level of complexity should be considered when relating cellular dynamics to specific miRNAs.

Structure and Stability of Ago2 MID-Nucleotide Complexes: All-in-One (Drop) His6-SUMO Tag Removal, Nucleotide Binding, and Crystal Growth.

The middle (MID) domain of eukaryotic Argonaute (Ago) proteins and archaeal and bacterial homologues mediates the interaction with the 5'-terminal nucleotide of miRNA and siRNA guide strands. The MID domain of human Ago2 (hAgo2) is comprised of 139 amino acids with a molecular weight of 15.56 kDa. MID adopts a Rossman-like beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-alpha4 fold with a nucleotide specificity loop between beta3 and alpha3. Multiple crystal structures of nucleotides bound to hAgo2 MID have been reported, whereby complexes were obtained by soaking ligands into crystals of MID domain alone. This protocol describes a simplified one-step approach to grow well-diffracting crystals of hAgo2 MID-nucleotide complexes by mixing purified His6-SUMO-MID fusion protein, Ulp1 protease, and excess nucleotide in the presence of buffer and precipitant. The crystal structures of MID complexes with UMP, UTP and 2'-3' linked α-L-threofuranosyl thymidine-3'-triphosphate (tTTP) are presented. This article also describes fluorescence-based assays to measure dissociation constants (Kd) of MID-nucleotide interactions for nucleoside 5'-monophosphates and nucleoside 3',5'-bisphosphates. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Crystallization of Ago2 MID-nucleotide complexes Basic Protocol 2: Measurement of dissociation constant Kd between Ago2 MID and nucleotides.

The guide RNA sequence dictates the slicing kinetics and conformational dynamics of the Argonaute silencing complex.

The RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that for different guide-RNA sequences, slicing rates of perfectly complementary, bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6-7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3'-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3' complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action.

AGO1 controls protein folding in mouse embryonic stem cell fate decisions.

Argonaute (AGO) proteins are evolutionarily conserved RNA-binding proteins that control gene expression through the small RNAs they interact with. Whether AGOs have regulatory roles independent of RNAs, however, is unknown. Here, we show that AGO1 controls cell fate decisions through facilitating protein folding. We found that in mouse embryonic stem cells (mESCs), while AGO2 facilitates differentiation via the microRNA (miRNA) pathway, AGO1 controls stemness independently of its binding to small RNAs. We determined that AGO1 specifically interacts with HOP, a co-chaperone for the HSP70 and HSP90 chaperones, and enhances the folding of a set of HOP client proteins with intrinsically disordered regions. This AGO1-mediated facilitation of protein folding is important for maintaining stemness in mESCs. Our results demonstrate divergent functions between AGO1 and AGO2 in controlling cellular states and identify an RNA-independent function of AGO1 in controlling gene expression and cell fate decisions.

Nuclear AGO2 promotes myocardial remodeling by activating ANKRD1 transcription in failing hearts.

Heart failure (HF) is manifested by transcriptional and posttranscriptional reprogramming of critical genes. Multiple studies have revealed that microRNAs could translocate into subcellular organelles such as the nucleus to modify gene expression. However, the functional property of subcellular Argonaute2 (AGO2), the core member of the microRNA machinery, has remained elusive in HF. AGO2 was found to be localized in both the cytoplasm and nucleus of cardiomyocytes, and robustly increased in the failing hearts of patients and animal models. We demonstrated that nuclear AGO2 rather than cytosolic AGO2 overexpression by recombinant adeno-associated virus (serotype 9) with cardiomyocyte-specific troponin T promoter exacerbated the cardiac dysfunction in transverse aortic constriction (TAC)-operated mice. Mechanistically, nuclear AGO2 activates the transcription of ANKRD1, encoding ankyrin repeat domain-containing protein 1 (ANKRD1), which also has a dual function in the cytoplasm as part of the I-band of the sarcomere and in the nucleus as a transcriptional cofactor. Overexpression of nuclear ANKRD1 recaptured some key features of cardiac remodeling by inducing pathological MYH7 activation, whereas cytosolic ANKRD1 seemed cardioprotective. For clinical practice, we found ivermectin, an antiparasite drug, and ANPep, an ANKRD1 nuclear location signal mimetic peptide, were able to prevent ANKRD1 nuclear import, resulting in the improvement of cardiac performance in TAC-induced HF.

MicroRNA-320a enhances LRWD1 expression through the AGO2/FXR1-dependent pathway to affect cell behaviors and the oxidative stress response in human testicular embryonic carcinoma cells.

BACKGROUND: Testicular cancer is fairly rare but can affect fertility in adult males. Leucine-rich repeats- and WD repeat domain-containing protein 1 (LRWD1) is a sperm-specific marker that mainly affects sperm motility in reproduction. Our previous study demonstrated the impact of LRWD1 on testicular cancer development; however, the underlying mechanisms remain unclear. METHODS: In this study, various plasmids associated with LRWD1 and miR-320a manipulation were used to explore the roles and regulatory effects of these molecules in NT2D1 cellular processes. A Dual-Glo luciferin-luciferase system was used to investigate LRWD1 transcriptional activity, and qRT-PCR and western blotting were used to determine gene and protein expression. RESULTS: The results suggested that miR-320a positively regulated LRWD1 and positively correlated with NT2D1 cell proliferation but negatively correlated with cell migration and invasion ability. In addition, the miRNA-ribonucleoprotein complex AGO2/FXR1 was shown to be essential in the mechanism by which miR-320a regulates LRWD1 mRNA expression. As miR-320a was required to regulate LRWD1 expression through the AGO2 and FXR1 complex, eEF2 and eLF4E were also found to be involved in miR-320a increasing LRWD1 expression. Furthermore, miR-320a and LRWD1 were responsive to oxidative stress, and NRF2 was affected by the presence of miR-320a in response to ROS stimulation. CONCLUSIONS: This is the first study showing the role of miR-320a in upregulating the testicular cancer-specific regulator LRWD1 and the importance of the AGO2/FXR1 complex in miR-320a-mediated upregulation of LRWD1 during testicular cancer progression.

Phosphorylation of AGO2 by TBK1 Promotes the Formation of Oncogenic miRISC in NSCLC.

Non-small-cell lung cancer (NSCLC) is a highly lethal tumor that often develops resistance to targeted therapy. It is shown that Tank-binding kinase 1 (TBK1) phosphorylates AGO2 at S417 (pS417-AGO2), which promotes NSCLC progression by increasing the formation of microRNA-induced silencing complex (miRISC). High levels of pS417-AGO2 in clinical NSCLC specimens are positively associated with poor prognosis. Interestingly, the treatment with EGFR inhibitor Gefitinib can significantly induce pS417-AGO2, thereby increasing the formation and activity of oncogenic miRISC, which may contribute to NSCLC resistance to Gefitinib. Based on these, two therapeutic strategies is developed. One is jointly to antagonize multiple oncogenic miRNAs highly expressed in NSCLC and use TBK1 inhibitor Amlexanox reducing the formation of oncogenic miRISC. Another approach is to combine Gefitinib with Amlexanox to inhibit the progression of Gefitinib-resistant NSCLC. This findings reveal a novel mechanism of oncogenic miRISC regulation by TBK1-mediated pS417-AGO2 and suggest potential therapeutic approaches for NSCLC.

Mature microRNA-binding protein QKI promotes microRNA-mediated gene silencing.

Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.

Pycard deficiency inhibits microRNA maturation and prevents neointima formation by promoting chaperone-mediated autophagic degradation of AGO2/argonaute 2 in adipose tissue.

PYCARD (PYD and CARD domain containing), a pivotal adaptor protein in inflammasome assembly and activation, contributes to innate immunity, and plays an essential role in the pathogenesis of atherosclerosis and restenosis. However, its roles in microRNA biogenesis remain unknown. Therefore, this study aimed to investigate the roles of PYCARD in miRNA biogenesis and neointima formation using pycard knockout (pycard-/-) mice. Deficiency of Pycard reduced circulating miRNA profile and inhibited Mir17 seed family maturation. The systemic pycard knockout also selectively reduced the expression of AGO2 (argonaute RISC catalytic subunit 2), an important enzyme in regulating miRNA biogenesis, by promoting chaperone-mediated autophagy (CMA)-mediated degradation of AGO2, specifically in adipose tissue. Mechanistically, pycard knockout increased PRMT8 (protein arginine N-methyltransferase 8) expression in adipose tissue, which enhanced AGO2 methylation, and subsequently promoted its binding to HSPA8 (heat shock protein family A (Hsp70) member 8) that targeted AGO2 for lysosome degradation through chaperone-mediated autophagy. Finally, the reduction of AGO2 and Mir17 family expression prevented vascular injury-induced neointima formation in Pycard-deficient conditions. Overexpression of AGO2 or administration of mimic of Mir106b (a major member of the Mir17 family) prevented Pycard deficiency-mediated inhibition of neointima formation in response to vascular injury. These data demonstrate that PYCARD inhibits CMA-mediated degradation of AGO2, which promotes microRNA maturation, thereby playing a critical role in regulating neointima formation in response to vascular injury independently of inflammasome activity and suggest that modulating PYCARD expression and function may represent a powerful therapeutic strategy for neointima formation.Abbreviations: 6-AN: 6-aminonicotinamide; ACTB: actin, beta; aDMA: asymmetric dimethylarginine; AGO2: argonaute RISC catalytic subunit 2; CAL: carotid artery ligation; CALCOCO2: calcium binding and coiled-coil domain 2; CMA: chaperone-mediated autophagy; CTSB: cathepsin B; CTSD: cathepsin D; DGCR8: DGCR8 microprocessor complex subunit; DOCK2: dedicator of cyto-kinesis 2; EpiAdi: epididymal adipose tissue; HSPA8: heat shock protein family A (Hsp70) member 8; IHC: immunohistochemical; ISR: in-stent restenosis; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; miRNA: microRNA; NLRP3: NLR family pyrin domain containing 3; N/L: ammonium chloride combined with leupeptin; PRMT: protein arginine methyltransferase; PVAT: peri-vascular adipose tissues; PYCARD: PYD and CARD domain containing; sDMA: symmetric dimethylarginine; ULK1: unc-51 like kinase 1; VSMCs: vascular smooth muscle cells; WT: wild-type.

Identification of AGO2 and PLEC genes polymorphisms in Hu sheep and their relationship with body size traits.

The body size traits are major traits in livestock, which intuitively displays the development of the animal's bones and muscles. This study used PCR amplification, Sanger sequencing, KASPar genotyping, and quantitative real-time reverse transcription PCR (qRT-PCR) to analyze the Single-nucleotide polymorphism and expression characteristics of Argonaute RISC catalytic component 2 (AGO2) and Plectin (PLEC) genes in Hu sheep. Two intron mutations were found in Hu sheep, which were AGO2 g.51700 A > C and PLEC g.23157 C > T, respectively. Through association analysis of two mutation sites and body size traits, it was found that AGO2 g.51700 A > C mainly affects the chest and cannon circumference of Hu sheep of while PLEC g.23157 C mainly affects body height and body length. The combined genotypes of AGO2 and PLEC genes with body size traits showed SNPs at the AGO2 g.51700 A > C and PLEC g.23157 C > T loci significantly improved the body size traits of Hu sheep. In addition, the AGO2 gene has the highest expression levels in the heart, rumen, and tail fat, and the PLEC gene is highly expressed in the heart. These two loci can provide new research ideas for improving the body size traits of Hu sheep.

Cutting through the stress: RNA decay pathways at the endoplasmic reticulum.

The endoplasmic reticulum (ER) is central to the processing of luminal, transmembrane, and secretory proteins, and maintaining a functional ER is essential for organismal physiology and health. Increased protein-folding load on the ER causes ER stress, which activates quality control mechanisms to restore ER function and protein homeostasis. Beyond protein quality control, mRNA decay pathways have emerged as potent ER fidelity regulators, but their mechanistic roles in ER quality control and their interrelationships remain incompletely understood. Herein, we review ER-associated RNA decay pathways - including regulated inositol-requiring enzyme 1α (IRE1α)-dependent mRNA decay (RIDD), nonsense-mediated mRNA decay (NMD), and Argonaute-dependent RNA silencing - in ER homeostasis, and highlight the intricate coordination of ER-targeted RNA and protein decay mechanisms and their association with antiviral defense.

Identification of RNA-binding proteins' direct effects on gene expression via the degradation tag system.

RNA-binding proteins (RBPs) are critical regulators of gene expression. An RBP typically binds to multiple mRNAs and modulates their expression. Although loss-of-function experiments on an RBP can infer how it regulates a specific target mRNA, the results are confounded by potential secondary effects due to the attenuation of all other interactions of the target RBP. For example, regarding the interaction between Trim71, an evolutionarily conserved RBP, and Ago2 mRNA, although Trim71 binds to Ago2 mRNA and overexpression of Trim71 represses Ago2 mRNA translation, it is puzzling that AGO2 protein levels are not altered in the Trim71 knockdown/knockout cells. To address this, we adapted the dTAG (degradation tag) system for determining the direct effects of the endogenous Trim71. Specifically, we knocked in the dTAG to the Trim71 locus, enabling inducible rapid Trim71 protein degradation. We observed that following the induction of Trim71 degradation, Ago2 protein levels first increased, confirming the Trim71-mediated repression, and then returned to the original levels after 24 h post-induction, revealing that the secondary effects from the Trim71 knockdown/knockout counteracted its direct effects on Ago2 mRNA. These results highlight a caveat in interpreting the results from loss-of-function studies on RBPs and provide a method to determine the primary effect(s) of RBPs on their target mRNAs.

Oncogenic K-Ras suppresses global miRNA function.

K-Ras frequently acquires gain-of-function mutations (K-RasG12D being the most common) that trigger significant transcriptomic and proteomic changes to drive tumorigenesis. Nevertheless, oncogenic K-Ras-induced dysregulation of post-transcriptional regulators such as microRNAs (miRNAs) during oncogenesis is poorly understood. Here, we report that K-RasG12D promotes global suppression of miRNA activity, resulting in the upregulation of hundreds of targets. We constructed a comprehensive profile of physiological miRNA targets in mouse colonic epithelium and tumors expressing K-RasG12D using Halo-enhanced Argonaute pull-down. Combining this with parallel datasets of chromatin accessibility, transcriptome, and proteome, we uncovered that K-RasG12D suppressed the expression of Csnk1a1 and Csnk2a1, subsequently decreasing Ago2 phosphorylation at Ser825/829/832/835. Hypo-phosphorylated Ago2 increased binding to mRNAs while reducing its activity to repress miRNA targets. Our findings connect a potent regulatory mechanism of global miRNA activity to K-Ras in a pathophysiological context and provide a mechanistic link between oncogenic K-Ras and the post-transcriptional upregulation of miRNA targets.

The RNA Interference Effector Protein Argonaute 2 Functions as a Restriction Factor Against SARS-CoV-2.

Argonaute 2 (Ago2) is a key component of the RNA interference (RNAi) pathway, a gene-regulatory system that is present in most eukaryotes. Ago2 uses microRNAs (miRNAs) and small interfering RNAs (siRNAs) for targeting to homologous mRNAs which are then degraded or translationally suppressed. In plants and invertebrates, the RNAi pathway has well-described roles in antiviral defense, but its function in limiting viral infections in mammalian cells is less well understood. Here, we examined the role of Ago2 in replication of the betacoronavirus SARS-CoV-2, the etiologic agent of COVID-19. Microscopic analyses of infected cells revealed that a pool of Ago2 closely associates with viral replication sites and gene ablation studies showed that loss of Ago2 resulted in over 1,000-fold increase in peak viral titers. Replication of the alphacoronavirus 229E was also significantly increased in cells lacking Ago2. The antiviral activity of Ago2 was dependent on both its ability to bind small RNAs and its endonuclease function. Interestingly, in cells lacking Dicer, an upstream component of the RNAi pathway, viral replication was the same as in parental cells. This suggests that the antiviral activity of Ago2 is independent of Dicer processed miRNAs. Deep sequencing of infected cells by other groups identified several SARS-CoV-2-derived small RNAs that bind to Ago2. A mutant virus lacking the most abundant ORF7A-derived viral miRNA was found to be significantly less sensitive to Ago2-mediated restriction. This combined with our findings that endonuclease and small RNA-binding functions of Ago2 are required for its antiviral function, suggests that Ago2-small viral RNA complexes target nascent viral RNA produced at replication sites for cleavage. Further studies are required to elucidate the processing mechanism of the viral small RNAs that are used by Ago2 to limit coronavirus replication.

Genes Involved in miRNA Biogenesis Are Not Downregulated in SARS-CoV-2 Infection.

miRNAs, small non-coding RNAs that regulate gene expression, are involved in various pathological processes, including viral infections. Virus infections may interfere with the miRNA pathway through the inhibition of genes involved in miRNA biogenesis. A reduction in the number and the levels of miRNAs expressed in nasopharyngeal swabs of patients with severe COVID-19 was lately observed by us, pointing towards the potential of miRNAs as possible diagnostic or prognostic biomarkers for predicting outcomes among patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The objective of the present study was to investigate whether SARS-CoV-2 infection influences the expression levels of messenger RNAs (mRNAs) of key genes involved in miRNA biogenesis. mRNA levels of AGO2, DICER1, DGCR8, DROSHA, and Exportin-5 (XPO5) were measured by quantitative reverse-transcription polymerase chain reaction (RT-qPCR) in nasopharyngeal swab specimens from patients with COVID-19 and controls, as well as in cells infected with SARS-CoV-2 in vitro. Our data showed that the mRNA expression levels of AGO2, DICER1, DGCR8, DROSHA, and XPO5 were not significantly different in patients with severe COVID-19 when compared to patients with non-severe COVID-19 and controls. Similarly, the mRNA expression of these genes was not affected by SARS-CoV-2 infection in NHBE and Calu-3 cells. However, in Vero E6 cells, AGO2, DICER1, DGCR8, and XPO5 mRNA levels were slightly upregulated 24 h after infection with SARS-CoV-2. In conclusion, we did not find evidence for downregulation of mRNA levels of miRNA biogenesis genes during SARS-CoV-2 infection, neither ex vivo nor in vitro.

Identify MicroRNA Targets Using AGO2-CLASH (Cross-linking, Ligation, and Sequencing of Hybrids) and AGO2-CLIP (Cross-Linking and Immuno-Precipitation) in Cells with or Without the MicroRNA of Interest Depleted.

MicroRNAs (miRNAs) are short noncoding RNAs and important players in the regulation of gene expression through post-transcriptional mechanisms. MicroRNAs regulate many cellular processes and are involved in disease progression. Identification of novel miRNA-to-target RNA connections can fill the gaps in the signaling pathways and suggest new therapeutic targets. MiRNA targets are often predicted by base-complementarity of their seed and flanking sequences with target sequences. Direct targets can also be identified by the physical interaction between the miRNA and the target RNA using immunoprecipitation of the Argonaute (AGO) protein, a component of the RNA-induced silencing complex, followed by ligation of AGO-associated miRNA and target RNA and next generation sequencing (CLASH). Databases describing these miRNA-RNA interactions have been generated from cells commonly studied or used. However, because the regulation by miRNAs varies among organs, tissues, cell types and species, identifying relevant targets in specific cells under conditions of interest may not be available. Here, the author describes simplified methods of AGO2-CLASH and AGO2-CLIP to identify miRNA targets by comparing primary cells derived from wild-type mice and those from specific miRNA knockout mice.

Dosage sensitivity to Pumilio1 variants in the mouse brain reflects distinct molecular mechanisms.

Different mutations in the RNA-binding protein Pumilio1 (PUM1) cause divergent phenotypes whose severity tracks with dosage: a mutation that reduces PUM1 levels by 25% causes late-onset ataxia, whereas haploinsufficiency causes developmental delay and seizures. Yet PUM1 targets are derepressed to equal degrees in both cases, and the more severe mutation does not hinder PUM1's RNA-binding ability. We therefore considered the possibility that the severe mutation might disrupt PUM1 interactions, and identified PUM1 interactors in the murine brain. We find that mild PUM1 loss derepresses PUM1-specific targets, but the severe mutation disrupts interactions with several RNA-binding proteins and the regulation of their targets. In patient-derived cell lines, restoring PUM1 levels restores these interactors and their targets to normal levels. Our results demonstrate that dosage sensitivity does not always signify a linear relationship with protein abundance but can involve distinct mechanisms. We propose that to understand the functions of RNA-binding proteins in a physiological context will require studying their interactions as well as their targets.