miRNA-induced Silencing Complex Research Articles

Argonaute proteins in cardiac tissue contribute to the heart injury during viral myocarditis.

MicroRNAs (miRNAs) are a group of short, noncoding, regulatory RNA molecules the dysregulation of which contributes to the pathogenesis of myocarditis. Argonaute proteins are essential components of miRNA-induced silencing complex and play important roles during miRNA biogenesis and function. However, the expression pattern of four AGO family members has not yet been detected in the coxsackievirus B3 (CVB3)-induced myocarditis tissue samples. In this study, we detected the expression of four AGOs in the CVB3-infected mouse heart tissues and found that AGO1 and AGO3 up-regulated significantly at 4 and 8h after CVB3 infection. Further in vitro research indicated that up-regulated AGO1 and AGO3 are related to the down-regulated TNFAIP3, which is a negative regulator of NF-κB pathway. Subsequently, we confirmed that TNFAIP3 is a direct target of miR-19a/b, and during CVB3 infection, the expression of miR-19a/b and miR-125a/b is not significantly changed. TNFAIP3 level is mainly reduced by up-regulated AGO1 and AGO3. This research sheds light on the relationship between overexpressed AGO proteins and CVB3-induced myocarditis, and this provides potential therapeutic target for viral myocarditis.

Assembly and analysis of eukaryotic Argonaute-RNA complexes in microRNA-target recognition.

Experimental studies have uncovered a variety of microRNA (miRNA)–target duplex structures that include perfect, imperfect and seedless duplexes. However, non-canonical binding modes from imperfect/seedless duplexes are not well predicted by computational approaches, which rely primarily on sequence and secondary structural features, nor have their tertiary structures been characterized because solved structures to date are limited to near perfect, straight duplexes in Argonautes (Agos). Here, we use structural modeling to examine the role of Ago dynamics in assembling viable eukaryotic miRNA-induced silencing complexes (miRISCs). We show that combinations of low-frequency, global modes of motion of Ago domains are required to accommodate RNA duplexes in model human and C. elegans Ago structures. Models of viable miRISCs imply that Ago adopts variable conformations at distinct target sites that generate distorted, imperfect miRNA-target duplexes. Ago's ability to accommodate a duplex is dependent on the region where structural distortions occur: distortions in solvent-exposed seed and 3′-end regions are less likely to produce steric clashes than those in the central duplex region. Energetic analyses of assembled miRISCs indicate that target recognition is also driven by favorable Ago-duplex interactions. Such structural insights into Ago loading and target recognition mechanisms may provide a more accurate assessment of miRNA function.

PARN deadenylase is involved in miRNA-dependent degradation of TP53 mRNA in mammalian cells.

mRNA deadenylation is under the control of cis-acting regulatory elements, which include AU-rich elements (AREs) and microRNA (miRNA) targeting sites, within the 3′ untranslated region (3′ UTRs) of eukaryotic mRNAs. Deadenylases promote miRNA-induced mRNA decay through their interaction with miRNA-induced silencing complex (miRISC). However, the role of poly(A) specific ribonuclease (PARN) deadenylase in miRNA-dependent mRNA degradation has not been elucidated. Here, we present evidence that not only ARE- but also miRNA-mediated pathways are involved in PARN-mediated regulation of the steady state levels of TP53 mRNA, which encodes the tumor suppressor p53. Supporting this, Argonaute-2 (Ago-2), the core component of miRISC, can coexist in complexes with PARN resulting in the activation of its deadenylase activity. PARN regulates TP53 mRNA stability through not only an ARE but also an adjacent miR-504/miR-125b-targeting site in the 3′ UTR. More importantly, we found that miR-125b-loaded miRISC contributes to the specific recruitment of PARN to TP53 mRNA, and that can be reverted by the ARE-binding protein HuR. Together, our studies provide new insights into the role of PARN in miRNA-dependent control of mRNA decay and into the mechanisms behind the regulation of p53 expression.

A large‐scale functional screen identifies N ova1 and N coa3 as regulators of neuronal mi RNA function

MicroRNAs (miRNAs) are important regulators of neuronal development, network connectivity, and synaptic plasticity. While many neuronal miRNAs were previously shown to modulate neuronal morphogenesis, little is known regarding the regulation of miRNA function. In a large-scale functional screen, we identified two novel regulators of neuronal miRNA function, Nova1 and Ncoa3. Both proteins are expressed in the nucleus and the cytoplasm of developing hippocampal neurons. We found that Nova1 and Ncoa3 stimulate miRNA function by different mechanisms that converge on Argonaute (Ago) proteins, core components of the miRNA-induced silencing complex (miRISC). While Nova1 physically interacts with Ago proteins, Ncoa3 selectively promotes the expression of Ago2 at the transcriptional level. We further show that Ncoa3 regulates dendritic complexity and dendritic spine maturation of hippocampal neurons in a miRNA-dependent fashion. Importantly, both the loss of miRNA activity and increased dendrite complexity upon Ncoa3 knockdown were rescued by Ago2 overexpression. Together, we uncovered two novel factors that control neuronal miRISC function at the level of Ago proteins, with possible implications for the regulation of synapse development and plasticity.

Towards a molecular understanding of microRNA-mediated gene silencing.

MicroRNAs (miRNAs) are a conserved class of small non-coding RNAs that assemble with Argonaute proteins into miRNA-induced silencing complexes (miRISCs) to direct post-transcriptional silencing of complementary mRNA targets. Silencing is accomplished through a combination of translational repression and mRNA destabilization, with the latter contributing to most of the steady-state repression in animal cell cultures. Degradation of the mRNA target is initiated by deadenylation, which is followed by decapping and 5'-to-3' exonucleolytic decay. Recent work has enhanced our understanding of the mechanisms of silencing, making it possible to describe in molecular terms a continuum of direct interactions from miRNA target recognition to mRNA deadenylation, decapping and 5'-to-3' degradation. Furthermore, an intricate interplay between translational repression and mRNA degradation is emerging.

Exploring the Requirement for the CCR4‐NOT Deadenylase Complex Subunit Regena/NOT2 in MicroRNA‐mediated Gene Silencing

A forward genetic screen was carried out using Drosophila melanogaster to determine the cellular components and molecular mechanisms that regulate microRNA (miRNA)‐mediated gene silencing. MiRNAs are non‐coding RNA molecules that interact with target‐gene messenger RNA transcripts via complementary base pairing, and silence target gene expression via interaction with the miRNA‐induced silencing complex (miRISC). Our screen utilized a Green Fluorescent Protein (GFP)‐based reporter to visualize miRNA‐mediated gene silencing. The GFP transgene contains miRNA binding sites, and is regulated by endogenous miRNAs. MiRNA‐mediated silencing is accomplished through translation block, mRNA degradation, or a combination of these mechanisms by miRISC. A requirement for the CCR4‐NOT complex subunit Regena/NOT2 in miRNA‐mediated gene silencing was discovered through genetic analysis of an isolated mutant. We are exploring the specific role of Regena/NOT2 in silencing by determining whether this CCR4‐NOT complex component is required for translational block, transcript degradation, or both. We are further characterizing a putative role for Regena/NOT2 in miRNA biogenesis and maturation, as has been demonstrated in Arabidopsis. Ongoing studies of additional mutants generated in the screen aim to identify the components of miRISC in different cellular contexts and with respect to different miRNA targets, to elucidate the complex molecular mechanisms of miRNA‐mediated silencing.

MicroRNAs: heralds of the noncoding RNA revolution.

MicroRNAs: heralds of the noncoding RNA revolution.

HOS1 regulates Argonaute1 by promoting transcription of the microRNA gene MIR168b in Arabidopsis

Proper accumulation and function of miRNAs is essential for plant growth and development. While core components of the miRNA biogenesis pathway and miRNA-induced silencing complex have been well characterized, cellular regulators of miRNAs remain to be fully explored. Here we report that High Expression Of Osmotically Responsive Genes1 (HOS1) is a regulator of an important miRNA, mi168a/b, that targets the Argonaute1 (AGO1) gene in Arabidopsis. HOS1 functions as an ubiquitin E3 ligase to regulate plant cold-stress responses, associates with the nuclear pores to regulate mRNA export, and regulates the circadian clock and flowering time by binding to chromatin of the flowering regulator gene Flowering Locus C (FLC). In a genetic screen for enhancers of sic-1, we isolated a loss-of-function Arabidopsis mutant of HOS1 that is defective in miRNA biogenesis. Like other hos1 mutant alleles, the hos1-7 mutant flowered early and was smaller in stature than the wild-type. Dysfunction in HOS1 reduced the abundance of miR168a/b but not of other miRNAs. In hos1 mutants, pri-MIR168b and pre-MIR168b levels were decreased, and RNA polymerase II occupancy was reduced at the promoter of MIR168b but not that of MIR168a. Chromatin immunoprecipitation assays revealed that HOS1 protein is enriched at the chromatin of the MIR168b promoter. The reduced miR168a/b level in hos1 mutants results in an increase in the mRNA and protein levels of its target gene, AGO1. Our results reveal that HOS1 regulates miR168a/b and AGO1 levels in Arabidopsis by maintaining proper transcription of MIR168b.

Gene silencing in vitro and in vivo using intronic microRNAs.

MicroRNAs (miRNAs) are small, single-stranded noncoding RNAs important in many biological processes through posttranscriptional modification of complementary intracellular messenger RNAs (mRNAs). MiRNAs have been reported to induce RNA interference (RNAi), by utilizing the miRNA-induced silencing complex (miRISC) to target mRNAs. They were first discovered in Caenorhabditis elegans as native RNA fragments that modulate a wide range of genetic regulatory pathways during embryonic development, and are now recognized as small gene silencers transcribed from the noncoding regions of a genome. In humans, nearly 97 % of the genome is noncoding DNA and changes in these sequences are frequently noted to manifest in clinical and circumstantial malfunction; for example, type 2 myotonic dystrophy and fragile X syndrome were found to be associated with miRNAs derived from introns. Intronic miRNA (mirtrons) is a class of miRNAs derived from the processing of non-protein-coding regions of gene transcripts. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of RNA polymerase (Pol)-II and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in C. elegans, mouse, and human cells; however, their functions and applications have not been reported. It is notable that there are different, but still highly conserved, mirtrons in mammalian than in invertebrates, and could be an indication that mirtrons are an evolutionary precursor to existing miRNA biogenesis pathways. Here, we show that intron-derived miRNA is not only able to induce RNAi in mammalian cells but also in fish, chicken embryos, and adult mice cells, demonstrating the evolutionary preservation of this gene regulation system in vivo. These miRNA-mediated animal models provide artificial means to reproduce the mechanisms of miRNA-induced disease in vivo and will shed further light on miRNA-related therapies.

Anti-PABPC1 co-immunoprecipitation for examining the miRNAs directly targeting the 3'-UTR of EED mRNA.

MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate post-transcriptional gene expression by base pairing with partially complementary sequences within target messenger RNAs (mRNAs). Although the target genes and the precise biological functions of individual miRNAs remain largely unknown, miRNAs have been implicated in diverse biological processes, including both normal and pathological states. As a single stranded mRNA can be directly targeted by multiple miRNAs, and as the target sites may exist in the 3′-untranslated region (UTR), 5′-UTR, or the coding regions, it is essential to develop an effective method to identify the full-scale miRNA regulatory pattern of each particular gene. In this study, we employed a biochemical approach to identify the miRNA profiles that regulate the expression of embryonic ectoderm development (EED) protein by using anti-PABPC1 ribonucleoprotein (RNP) co-immunoprecipitation (Co-IP). The full length EED mRNA was subcloned into an expression vector and transiently transfected into a Flag-PABPC1 stable expression cell line. Subsequent to cross-linking and an anti-Flag Co-IP, the miRNAs that directly targeted EED were identified. We found that the best time point to distinguish the positive miRNAs from the background was 18 hours after the plasmid transfection. As expected, the miRNAs that directly target EED were found to interact with EED mRNA through the miRNA-induced silencing complex (miRISC). Meanwhile, the EED mRNA was bound by Flag-PABPC1. This method depends on the integrity of the miRISC complex and achieves greater efficiency when ultraviolet irradiation is used for the process of cross-linking. By using anti-PABPC1 RIP, we identified EED to be a new target gene of miR-16; a finding further confirmed using a dual-luciferase assay. In summary, our data indicate that anti-PABPC1 RIP is a validated and direct biochemical method to provide data about specific miRNA-mRNA interactions, as well as global miRNA patterns regulating the mRNAs.

Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1.

MicroRNAs (miRNAs) play critical roles in a variety of biological processes through widespread effects on protein synthesis. Upon association with the miRNA-induced silencing complex (miRISC), miRNAs repress target mRNA translation and accelerate mRNA decay. Degradation of the mRNA is initiated by shortening of the poly(A) tail by the CCR4-NOT deadenylase complex followed by the removal of the 5' cap structure and exonucleolytic decay of the mRNA. Here, we report a direct interaction between the large scaffolding subunit of CCR4-NOT, CNOT1, with the translational repressor and decapping activator protein, DDX6. DDX6 binds to a conserved CNOT1 subdomain in a manner resembling the interaction of the translation initiation factor eIF4A with eIF4G. Importantly, mutations that disrupt the DDX6-CNOT1 interaction impair miRISC-mediated gene silencing in human cells. Thus, CNOT1 facilitates recruitment of DDX6 to miRNA-targeted mRNAs, placing DDX6 as a downstream effector in the miRNA silencing pathway.

Novel roles of the multi-functional CCR4-NOT complex in post-transcriptional regulation.

The CCR4-NOT complex is a highly conserved specific gene silencer that also serves more general post-transcriptional functions. Specific regulatory proteins including the miRNA-induced silencing complex and its associated proteins, bind to 3’-UTR elements of mRNA and recruit the CCR4-NOT complex thereby promoting poly(A) shortening and repressing translation and/or mRNA degradation. Recent studies have shown that the CCR4-NOT complex that is tethered to mRNA by such regulator(s) represses translation and facilitates mRNA decay independent of a poly(A) tail and its shortening. In addition to deadenylase activity, the CCR4-NOT complex also has an E3 ubiquitin ligase activity and is involved in a novel protein quality control system, i.e., co-translational proteasomal-degradation of aberrant proteins. In this review, we describe recent progress in elucidation of novel roles of the multi-functional complex CCR4-NOT in post-transcriptional regulation.

Loss of miR-100 enhances migration, invasion, epithelial-mesenchymal transition and stemness properties in prostate cancer cells through targeting Argonaute 2.

Evidence in literature has demonstrated that some microRNAs (miRNAs) play a pivotal role in most solid tumor metastasis. Previous studies have showed that miR-100 is downregulated in human prostate cancer tissue compared to normal prostate and also significantly decreased in bone metastatic prostate cancer samples compared with primary prostate cancer. Argonaute2 (AGO2) is the core effector protein of the miRNA-induced silencing complex and overexpression of AGO2 might enhance tumor metastasis. However, it is unknown whether and how miR-100 and AGO2 regulates metastasis of prostate cancer. Here, we report that miR-100 negatively regulated migration, invasion, epithelial-mesenchymal transition (EMT), colony formation, spheroid formation and expression of the stemness factors c-Myc, Oct4 and Klf4 in PC-3 and DU145 cells. Furthermore, miR-100 expression was negatively correlated with bone metastasis of prostate cancer patients. Notably, luciferase assay showed that AGO2 was a direct target of miR-100. Downregulation of AGO2 repressed migration, invasion, EMT and stemness of prostate cancer cells, and reversed the effects seen with miR-100 downregulation. Downregulation of AGO2 enhanced expression of miR-34a and miR-125b which can suppress migration, invasion, EMT and stemness of cancer cells. Taken together, our findings indicate that loss of miR-100 promotes the metastatic ability of prostate cancer cells at least partially by upregulating AGO2 expression through modulating migration, invasion, EMT and stemness of cancer cells, and suggest that miR-100/AGO2 may play an important role in regulating the metastasis of prostate cancer and is a potential target of prevention and therapy.

RACK-1 regulates let-7 microRNA expression and terminal cell differentiation in Caenorhabditis elegans

The let-7 microRNA (miRNA) regulates cell cycle exit and terminal differentiation in the C. elegans heterochronic gene pathway. Low expression of let-7 results in retarded vulva and hypodermal cell development in C. elegans and has been associated with several human cancers. Previously, the versatile scaffold protein receptor for activated C kinase 1 (RACK1) was proposed to facilitate recruitment of the miRNA-induced silencing complex (miRISC) to the polysome and to be required for miRNA function in C. elegans and humans. Here, we show that depletion of C. elegans RACK-1 by RNAi increases let-7 miRNA levels and suppresses the retarded terminal differentiation of lateral hypodermal seam cells in mutants carrying the hypomorphic let-7(n2853) allele or lacking the let-7 family miRNA genes mir-48 and mir-241. Depletion of RACK-1 also increases the levels of precursor let-7 miRNA. When Dicer is knocked down and pre-miRNA processing is inhibited, depletion of RACK-1 still leads to increased levels of pre-let-7, suggesting that RACK-1 affects a biogenesis mechanism upstream of Dicer. No changes in the activity of the let-7 promoter or the levels of primary let-7 miRNA are associated with depletion of RACK-1, suggesting that RACK-1 affects let-7 miRNA biogenesis at the post-transcriptional level. Interestingly, rack-1 knockdown also increases the levels of a few other precursor miRNAs. Our results reveal that RACK-1 controls the biogenesis of a subset of miRNAs, including let-7, and in this way plays a role in the heterochronic gene pathway during C. elegans development.

Post-Transcriptional Regulation of Interferons and Their Signaling Pathways

Interferons (IFNs) are low molecular weight cell-derived proteins that include the type I, II, and III IFN families. IFNs are critical for an optimal immune response during microbial infections while dysregulated expression can lead to autoimmune diseases. Given its role in disease, it is important to understand cellular mechanisms of IFN regulation. 3' untranslated regions (3' UTRs) have emerged as potent regulators of mRNA and protein dosage and are controlled through multiple regulatory elements including adenylate uridylate (AU)-rich elements (AREs) and microRNA (miRNA) recognition elements. These AREs are targeted by RNA-binding proteins (ARE-BPs) for degradation and/or stabilization through an ARE-mediated decay process. miRNA are endogenous, single-stranded RNA molecules ~22 nucleotides in length that regulate mRNA translation through the miRNA-induced silencing complex. IFN transcripts, like other labile mRNAs, harbor AREs in their 3' UTRs that dictate the turnover of mRNA. This review is a survey of the literature related to IFN regulation by miRNA, ARE-BPs, and how these complexes interact dynamically on the 3' UTR. Additionally, downstream effects of these post-transcriptional regulators on the immune response will be discussed. Review topics include past studies, current understanding, and future challenges in the study of post-transcriptional regulation affecting IFN responses.

An in vivo approach to the discovery and characterization of cellular mechanisms regulating microRNA‐mediated gene silencing (744.3)

Our goal is to determine the cellular mechanisms that regulate microRNA (miRNA)‐mediated gene silencing. MiRNAs are non‐coding RNA molecules that interact with target‐gene messenger RNA transcripts via complementary base pairing, and silence target gene expression via interaction with the miRNA‐induced silencing complex (miRISC). Silencing is accomplished through translation block, mRNA degradation, or a combination of these mechanisms. A forward genetic screen was carried out to identify requirements for miRNA‐mediated silencing in Drosophila melanogaster. miRNA function was assayed using Green Fluorescent Protein (GFP)‐based reporters of gene silencing. The GFP transgenes contain miRNA binding sites, and are regulated by endogenous miRNAs. We have genetic evidence demonstrating roles for both Regena (NOT2), a subunit of the CCR4‐NOT deadenylase complex, and the protein kinase dAkt1, in miRNA‐mediated gene silencing. Further studies have implicated insulin signaling in general and specific components of the insulin‐signaling pathway in particular in the regulation of miRNA activity. We are investigating the necessity of these genes in miRNA‐mediated gene silencing at various stages of Drosophila development. Investigation of the composition, abundance, and localization of known components of the silencing machinery is revealing distinct mechanisms for the regulation of silencing.

MicroRNAs in planarian regeneration (LB14)

Planarian, with the robust regeneration ability, has been used as a model organism for stem cell research in recent years. Regeneration in planarian is mediated by adult stem cells, called neoblasts, which can proliferate and differentiate into almost all cell types for replacing the whole body. MicroRNAs are 20‐24nt small non‐coding RNAs that are associated with Argonaut proteins and form miRNA‐induced silencing complex (miRISC) to regulate gene expression at post‐transcriptional level. Our preliminary study showed that a certain population of miRNAs are highly expressed in regenerating tissues, implicates the potential important roles of miRNAs in neoblasts during regeneration. Interestingly, depeletion of planarian Argonaute 2 (DjAgo2) results in a dramatic effect of head degeneration, suggesting that miRNAs are also required for planarian homeostasis. In this study, we further examined the roles of specific miRNAs in planarian. We developed a method to specifically block the function of miRNA. Successful inhibition of several candidate miRNAs results in a degeneration phenotype in planarian treated with miRNA inhibitors. This result proved our hypothesis that miRNAs indeed participate in self‐renewal of stem cells and the differentiation process in planarian.

NMDA receptor-dependent regulation of miRNA expression and association with Argonaute during LTP in vivo

microRNAs (miRNAs) are major regulators of protein synthesis in the brain. A major goal is to identify changes in miRNA expression underlying protein synthesis-dependent forms of synaptic plasticity such as long-term potentiation (LTP). Previous analyses focused on changes in miRNA levels in total lysate samples. Here, we asked whether changes in total miRNA accurately reflect changes in the amount of miRNA bound to Argonaute protein within the miRNA-induced silencing complex (miRISC). Ago2 immunoprecipitation was used to isolate RISC-associated miRNAs following high-frequency stimulation (HFS)-induced LTP in the dentate gyrus of anesthetized rats. Using locked-nucleic acid-based PCR cards for high-throughput screening and independent validation by quantitative TaqMan RT-PCR, we identified differential regulation of Ago2-associated and total miRNA expression. The ratio of Ago2/total miRNA expression was regulated bidirectionally in a miRNA-specific manner and was largely dependent on N-methyl-D-aspartate receptor (NMDA) activation during LTP induction. The present results identify miRNA association with Ago2 as a potential control point in activity-dependent synaptic plasticity in the adult brain. Finally, novel computational analysis for targets of the Ago2-associated miRNAs identifies 21 pathways that are enriched and differentially targeted by the miRNAs including axon guidance, mTOR, MAPK, Ras, and LTP.

You are what you eat: multifaceted functions of autophagy during C. elegans development.

Autophagy involves the sequestration of a portion of the cytosolic contents in an enclosed double-membrane autophagosomal structure and its subsequent delivery to lysosomes for degradation. Autophagy activity functions in multiple biological processes during Caenorhabditis elegans development. The basal level of autophagy in embryos removes aggregate-prone proteins, paternal mitochondria and spermatid-specific membranous organelles (MOs). Autophagy also contributes to the efficient removal of embryonic apoptotic cell corpses by promoting phagosome maturation. During larval development, autophagy modulates miRNA-mediated gene silencing by selectively degrading AIN-1, a component of miRNA-induced silencing complex, and thus participates in the specification of multiple cell fates controlled by miRNAs. During development of the hermaphrodite germline, autophagy acts coordinately with the core apoptotic machinery to execute genotoxic stress-induced germline cell death and also cell death when caspase activity is partially compromised. Autophagy is also involved in the utilization of lipid droplets in the aging process in adult animals. Studies in C. elegans provide valuable insights into the physiological functions of autophagy in the development of multicellular organisms.

Functionally Diverse MicroRNA Effector Complexes Are Regulated by Extracellular Signaling

Because microRNAs (miRNAs) influence the expression of many genes in cells, discovering how the miRNA pathway is regulated is an important area of investigation. We found that the Drosophila miRNA-induced silencing complex (miRISC) exists in multiple forms. A constitutive form, called G-miRISC, is comprised of Ago1, miRNA, and GW182. Two distinct miRISC complexes that lack GW182 are regulated by mitogenic signaling. Exposure of cells to serum, lipids, or the tumor promoter PMA suppressed formation of these complexes. P-miRISC is comprised of Ago1, miRNA, and Loqs-PB, and it associates with mRNAs assembled into polysomes. The other regulated Ago1 complex associates with membranous organelles and is likely an intermediate in miRISC recycling. The formation of these complexes is correlated with a 5- to 10-fold stronger repression of target gene expression inside cells. Taken together, these results indicate that mitogenic signaling regulates the miRNA effector machinery to attenuate its repressive activities.