Component Of RNA-induced Silencing Complex Research Articles

Mature microRNA-binding protein QKI suppresses extracellular microRNA let-7b release.

Argonaute (AGO), a component of RNA-induced silencing complexes (RISCs), is a representative RNA-binding protein (RBP) known to bind with mature microRNA (miRNA) and is directly involved in post-transcriptional gene silencing. However, despite the biological significance of miRNA, the roles of other micro RNA-binding proteins (miRBPs) remain unclear in regulation of miRNA loading, dissociation from RISC, and extracellular release. In this study, we perform protein arrays to profile miRBPs and identify 118 RNA-binding proteins directly binding with miRNAs. Among those proteins, RBP quaking (QKI) inhibits extracellular release of mature microRNA let-7b by controlling the loading of let-7b into extracellular vesicles via additional miRBPs such as hnRNPD/AUF1 and hnRNPK. The enhanced extracellular release of let-7b after QKI depletion activates the Toll-like Receptor 7 (TLR7) and promotes the production of proinflammatory cytokines in recipient cells, leading to brain inflammation in mouse cortex. Thus, this study reveals contribution of QKI to the inhibition of brain inflammation via regulation of extracellular let-7b release.

The subgenomic flaviviral RNA suppresses RNA interference through competing with siRNAs for binding RISC components.

During the life cycle of mosquito-borne flaviviruses, substantial subgenomic flaviviral RNA (sfRNA) is produced via incomplete degradation of viral genomic RNA by host XRN1. Zika virus (ZIKV) sfRNA has been detected in mosquito and mammalian somatic cells. Human neural progenitor cells (hNPCs) in the developing brain are the major target cells of ZIKV, and antiviral RNA interference (RNAi) plays a critical role in hNPCs. However, whether ZIKV sfRNA was produced in ZIKV-infected hNPCs as well as its function remains not known. In this study, we demonstrate that abundant sfRNA was produced in ZIKV-infected hNPCs. RNA pulldown and mass spectrum assays showed ZIKV sfRNA interacted with host proteins RHA and PACT, both of which are RNA-induced silencing complex (RISC) components. Functionally, ZIKV sfRNA can antagonize RNAi by outcompeting small interfering RNAs (siRNAs) in binding to RHA and PACT. Furthermore, the 3' stem loop (3'SL) of sfRNA was responsible for RISC components binding and RNAi inhibition, and 3'SL can enhance the replication of a viral suppressor of RNAi (VSR)-deficient virus in a RHA- and PACT-dependent manner. More importantly, the ability of binding to RISC components is conversed among multiple flaviviral 3'SLs. Together, our results identified flavivirus 3'SL as a potent VSR in RNA format, highlighting the complexity in virus-host interaction during flavivirus infection.IMPORTANCEZika virus (ZIKV) infection mainly targets human neural progenitor cells (hNPCs) and induces cell death and dysregulated cell-cycle progression, leading to microcephaly and other central nervous system abnormalities. RNA interference (RNAi) plays critical roles during ZIKV infections in hNPCs, and ZIKV has evolved to encode specific viral proteins to antagonize RNAi. Herein, we first show that abundant sfRNA was produced in ZIKV-infected hNPCs in a similar pattern to that in other cells. Importantly, ZIKV sfRNA acts as a potent viral suppressor of RNAi (VSR) by competing with siRNAs for binding RISC components, RHA and PACT. The 3'SL of sfRNA is responsible for binding RISC components, which is a conserved feature among mosquito-borne flaviviruses. As most known VSRs are viral proteins, our findings highlight the importance of viral non-coding RNAs during the antagonism of host RNAi-based antiviral innate immunity.

Interaction between Bombyx mori Cytoplasmic Polyhedrosis Virus NSP8 and BmAgo2 Inhibits RNA Interference and Enhances Virus Proliferation.

Some insect viruses encode suppressors of RNA interference (RNAi) to counteract the antiviral RNAi pathway. However, it is unknown whether Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) encodes an RNAi suppressor. In this study, the presence of viral small interfering RNA (vsiRNA) in BmN cells infected with BmCPV was confirmed by small RNA sequencing. The Dual-Luciferase reporter test demonstrated that BmCPV infection may prevent firefly luciferase (Luc) gene silencing caused by particular short RNA. It was also established that the inhibition relied on the nonstructural protein NSP8, which suggests that NSP8 was a possible RNAi suppressor. In cultured BmN cells, the expressions of viral structural protein 1 (vp1) and NSP9 were triggered by overexpression of nsp8, suggesting that BmCPV proliferation was enhanced by NSP8. A pulldown assay was conducted with BmCPV genomic double-stranded RNA (dsRNA) labeled with biotin. The mass spectral detection of NSP8 in the pulldown complex suggests that NSP8 is capable of direct binding to BmCPV genomic dsRNA. The colocalization of NSP8 and B. mori Argonaute 2 (BmAgo2) was detected by an immunofluorescence assay, leading to the hypothesis that NSP8 interacts with BmAgo2. Coimmunoprecipitation further supported the present investigation. Moreover, vasa intronic protein, a component of RNA-induced silencing complex (RISC), could be detected in the coprecipitation complex of NSP8 by mass spectrum analysis. NSP8 and the mRNA decapping protein (Dcp2) were also discovered to colocalize to processing bodies (P bodies) for RNAi-mediated gene silencing in Saccharomyces cerevisiae. These findings revealed that by interacting with BmAgo2 and suppressing RNAi, NSP8 promoted BmCPV growth. IMPORTANCE It has been reported that the RNAi pathway is inhibited by binding RNAi suppressors encoded by some insect-specific viruses belonging to Dicistroviridae, Nodaviridae, or Birnaviridae to dsRNAs to protect dsRNAs from being cut by Dicer-2. However, it is unknown whether BmCPV, belonging to Spinareoviridae, encodes an RNAi suppressor. In this study, we found that nonstructural protein NSP8 encoded by BmCPV inhibits small interfering RNA (siRNA)-induced RNAi and that NSP8, as an RNAi suppressor, can bind to viral dsRNAs and interact with BmAgo2. Moreover, vasa intronic protein, a component of RISC, was found to interact with NSP8. Heterologously expressed NSP8 and Dcp2 were colocalized to P bodies in yeast. These results indicated that NSP8 promoted BmCPV proliferation by binding itself to BmCPV genomic dsRNAs and interacting with BmAgo2 through suppression of siRNA-induced RNAi. Our findings deepen our understanding of the game between BmCPV and silkworm in regulating viral infection.

A conserved RNAi molecule Ago2 involved in antiviral immunity of oyster Crassostrea gigas

Argonaute (Ago) is the core component of RNA-induced silencing complex to play a crucial role in the antiviral immunity, which always cooperates with Dicer in RNA interference (RNAi) to silence the target genes. In the present study, an Ago homologue (CgAgo2) was identified in the Pacific oyster Crassostrea gigas. There were four classical functional domains in the predicted CgAgo2 protein, including an N-terminal domain, a PAZ domain, a Mid domain, and a PIWI domain. The deduced amino acid sequence of CgAgo2 shared 63.52%–84.27% identity with other Agos. Transcriptome analysis showed that CgAgo2 was highly expressed in embryonic period and gradually decreased from blastula to gastrula. The transcripts of CgAgo2 were detectable in all the examined tissues of adult oysters, with the highest expression in haemocytes (36.61-fold of that in adductor muscle, p < 0.001). The expression level of CgAgo2 mRNA in haemocytes increased significantly at 12 h after poly (I:C) and dsRNA stimulation, which were 2.71-fold (p < 0.05) and 58.00-fold (p < 0.001) of that in the control group respectively. Immunocytochemistry assay revealed that CgAgo2 proteins were mainly distributed in the cytoplasm and nucleus of haemocytes. The interaction between the recombinant CgAgo2 protein (rCgAgo2) and cleavage protein rCgDicer was observed in vitro by BLI and pull-down assays. These results indicated that CgAgo2 participated in the antiviral immunity of oyster by functioning as a component of RNA-induced silencing complex in RNAi.

The effect of epistatic interactions between genetic variants located in microRNA and silencing complex genes on prostate cancer progression risk

Previous studies conducted in Asian and European populations have provided evidence of the association between microRNA-related genetic variants and prostate cancer (PCa) risk and/or progression. Nevertheless, the results obtained in these studies are inconsistent, which could be explained by the limitations of single-locus main effect evaluations to detect joint effects of multiple genetic variants, reflected in statistical epistases. Therefore, we conducted the analysis of potential epistatic interactions between variants located in microRNA genes and in genes encoding the components of RNA-induced silencing complex (RISC) in relation with PCa risk/aggressiveness. Raw data on genotyping results from our previous studies involving four microRNA polymorphisms and five variants in RISC genes were subjected to the exclusion of samples based on missing data criterion, followed by the re-evaluation of Hardy-Weinberg equilibrium. Afterwards, these genotyping results were included in the Multifactor dimensionality reduction (MDR) analysis. Permutation testing was conducted in order to assess statistical significance of the best models from MDR tests. MDR tests on the risk of developing PCa yielded statistically insignificant results. Nevertheless, the MDR results for comparison of PCa patients with high and low cancer progression risk were statistically significant for the analysis that included rs11614913, with the 3-locus best model comprising this genetic variant, rs7813 and rs784567. We conclude that statistical epistasis between rs11614913 in hsa-miR-196a2, rs7813 in GEMIN4 and rs784567 in TARBP2 shows association with the invasiveness of PCa.

Rice miR168a-5p regulates seed length, nitrogen allocation and salt tolerance by targeting OsOFP3, OsNPF2.4 and OsAGO1a, respectively

Rice microRNA168a (osa-miR168a) plays important roles in mediating flowering time, grain yield and vigor, seeding growth, and immunity by targeting the RNA-induced silencing complex component Argonaute 1 (AGO1). However, the functions of miR168a exerted by targeting other genes require further clarification before it could be used in rice molecular breeding. In this study, we identified a new target gene of osa-miR168a-5p (miR168a-5p) in rice called OsOFP3 (ovate family protein 3) and investigated the roles of miR168a-5p in response to brassinosteroids (BRs), salt stress, and nitrogen allocation. Up- and downregulated miR168a-5p expression respectively decreased and increased the expression of the BR-negative regulator OsOPF3. The results of RNA ligase-mediated rapid amplification of cDNA ends (5′RLM-RACE) revealed cleavage sites in OsOPF3 and OsNPF2.4 mRNAs. The phenotype of miR168a-5p transgenic rice was BR-associated and included the lamina bending response to BR, short seeds, and low 1000-grain weight. MicroRNA 168a-5p also regulated the expression of the nitrate transporter, OsNPF2.4, which affected nitrogen allocation, and regulated OsAGO1a expression in response to salt stress. Taken together, rice miR168a-5p regulates BR-associated pathways, nitrogen transport, and stress by targeting OsOFP3, OsNPF2.4, and OsAGO1a, respectively, resulting in a series of important agronomic traits for rice breeding.

NF90 interacts with components of RISC and modulates association of Ago2 with mRNA

BackgroundNuclear factor 90 (NF90) is a double-stranded RNA-binding protein involved in a multitude of different cellular mechanisms such as transcription, translation, viral infection, and mRNA stability. Recent data suggest that NF90 might influence the abundance of target mRNAs in the cytoplasm through miRNA- and Argonaute 2 (Ago2)-dependent activity.ResultsHere, we identified the interactome of NF90 in the cytoplasm, which revealed several components of the RNA-induced silencing complex (RISC) and associated factors. Co-immunoprecipitation analysis confirmed the interaction of NF90 with the RISC-associated RNA helicase, Moloney leukemia virus 10 (MOV10), and other proteins involved in RISC-mediated silencing, including Ago2. Furthermore, NF90 association with MOV10 and Ago2 was found to be RNA-dependent. Glycerol gradient sedimentation of NF90 immune complexes indicates that these proteins occur in the same protein complex. At target RNAs predicted to bind both NF90 and MOV10 in their 3′ UTRs, NF90 association was increased upon loss of MOV10 and vice versa. Interestingly, loss of NF90 led to an increase in association of Ago2 as well as a decrease in the abundance of the target mRNA. Similarly, during hypoxia, the binding of Ago2 to vascular endothelial growth factor (VEGF) mRNA increased after loss of NF90, while the level of VEGF mRNA decreased.ConclusionsThese findings reveal that, in the cytoplasm, NF90 can associate with components of RISC such as Ago2 and MOV10. In addition, the data indicate that NF90 and MOV10 may compete for the binding of common target mRNAs, suggesting a role for NF90 in the regulation of RISC-mediated silencing by stabilizing target mRNAs, such as VEGF, during cancer-induced hypoxia.

Genome-Wide Identification and Evolutionary Analysis of Argonaute Genes in Hexaploid Bread Wheat.

Argonaute (AGO) proteins play a pivotal role in plant growth and development as the core components of RNA-induced silencing complex (RISC). However, no systematic characterization of AGO genes in wheat has been reported to date. In this study, a total number of 69 TaAGO genes in the hexaploid bread wheat (Triticum aestivum cv. Chinese Spring) genome, divided into 10 subfamilies, were identified. Compared to all wheat genes, TaAGOs showed a significantly lower evolutionary rate, which is consistent with their high conservation in eukaryotes. However, the homoeolog retention was remarkably higher than the average, implying the nonredundant biological importance of TaAGO genes in bread wheat. Further homoeologous gene expression bias analyses revealed that TaAGOs may have undergone neofunctionalization after polyploidization and duplication through the divergent expression of homoeologous gene copies, to provide new opportunities for the generation of adaptive traits. Moreover, quantitative real-time polymerase chain reaction (qRT-PCR) analyses indicated that TaAGO gene expression was involved in response to heat, drought, and salt stresses. Our results would provide a theoretical basis for future studies on the biological functions of TaAGO genes in wheat and other gramineous species.

Germline AGO2 mutations impair RNA interference and human neurological development

ARGONAUTE-2 and associated miRNAs form the RNA-induced silencing complex (RISC), which targets mRNAs for translational silencing and degradation as part of the RNA interference pathway. Despite the essential nature of this process for cellular function, there is little information on the role of RISC components in human development and organ function. We identify 13 heterozygous mutations in AGO2 in 21 patients affected by disturbances in neurological development. Each of the identified single amino acid mutations result in impaired shRNA-mediated silencing. We observe either impaired RISC formation or increased binding of AGO2 to mRNA targets as mutation specific functional consequences. The latter is supported by decreased phosphorylation of a C-terminal serine cluster involved in mRNA target release, increased formation of dendritic P-bodies in neurons and global transcriptome alterations in patient-derived primary fibroblasts. Our data emphasize the importance of gene expression regulation through the dynamic AGO2-RNA association for human neuronal development.

The Ago2-miRNA-co-IP Assay to Study TGF- β1 Mediated Recruitment of miRNA to the RISC in CFBE Cells.

Micro(mi)RNAs are short, non-coding RNAs that mediate the RNA interference (RNAi) by post-transcriptional mechanisms. Specific miRNAs are recruited to the cytoplasmic RNA induced silencing complex (RISC). Argonaute2 (Ago2), an essential component of RISC, facilitates binding of miRNA to the target-site on mRNA, followed by cleaving the miRNA-mRNA duplex with its endonuclease activity. RNAi is mediated by a specific pool of miRNAs recruited to RISC, and thus is referred to as the functional pool. The cellular levels of many miRNAs are affected by the cytokine Transforming Growth Factor-β1 (TGF-β1). However, little is known about whether the TGF-β1 affects the functional pools of these miRNAs. The Ago2-miRNA-co-IP assay, discussed in this manuscript, is designed to examine effects of TGF-β1 on the recruitment of miRNAs to RISC and it helps to determine whether changes in the cellular miRNA levels correlate with changes in the RISC-associated, functional pools. The general principles of the assay are as follows. Cultured cells treated with TGF-β1 or vehicle control are lysed and the endogenous Ago2 is immunoprecipitated with immobilized anti-Ago2 antibody, and the active miRNAs complexed with Ago2 are isolated with a RISC immunoprecipitation (RIP) assay kit. The miRNAs are identified with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) using miRNA-specific stem-looped primers during reverse transcription, followed by PCR using miRNA-specific forward and reverse primers, and TaqMan hydrolysis probes.

Starvation stress attenuates the miRNA-target interaction in suppressing breast cancer cell proliferation

BackgroundEmerging evidence has demonstrated the limited access to metabolic substrates as an effective approach to block cancer cell growth. The mechanisms remain unclear. Our previous work has revealed that miR-221/222 plays important role in regulating breast cancer development and progression through interaction with target gene p27.ResultsHerein, we determined the miRNA-mRNA interaction in breast cancer cells under induced stress status of starvation. Starvation stimulation attenuated the miR-221/222-p27 interaction in MDA-MB-231 cells, thereby increased p27 expression and suppressed cell proliferation. Through overexpression or knockdown of miR-221/222, we found that starvation-induced stress attenuated the negative regulation of p27 expression by miR-221/222. Similar patterns for miRNA-target mRNA interaction were observed between miR-17-5p and CyclinD1, and between mR-155 and Socs1. Expression of Ago2, one of the key components of RNA-induced silencing complex (RISC), was decreased under starvation-induced stress status, which took responsibility for the impaired miRNA-target interaction since addition of exogenous Ago2 into MDA-MB-231 cells restored the miR-221/222-p27 interaction in starvation condition.ConclusionsWe demonstrated the attenuated interaction between miR-221/222 and p27 by starvation-induced stress in MDA-MB-231 breast cancer cells. The findings add a new page to the general knowledge of negative regulation of gene expression by miRNAs, also demonstrate a novel mechanism through which limited access to nutrients suppresses cancer cell proliferation. These insights provide a basis for development of novel therapeutic options for breast cancer.

Predominant Distribution of the RNAi Machinery at Apical Adherens Junctions in Colonic Epithelia Is Disrupted in Cancer.

The RNA interference (RNAi) machinery is an essential component of the cell, regulating miRNA biogenesis and function. RNAi complexes were thought to localize either in the nucleus, such as the microprocessor, or in the cytoplasm, such as the RNA-induced silencing complex (RISC). We recently revealed that the core microprocessor components DROSHA and DGCR8, as well as the main components of RISC, including Ago2, also associate with the apical adherens junctions of well-differentiated cultured epithelial cells. Here, we demonstrate that the localization of the core RNAi components is specific and predominant at apical areas of cell-cell contact of human normal colon epithelial tissues and normal primary colon epithelial cells. Importantly, the apical junctional localization of RNAi proteins is disrupted or lost in human colon tumors and in poorly differentiated colon cancer cell lines, correlating with the dysregulation of the adherens junction component PLEKHA7. We show that the restoration of PLEKHA7 expression at adherens junctions of aggressively tumorigenic colon cancer cells restores the junctional localization of RNAi components and suppresses cancer cell growth in vitro and in vivo. In summary, this work identifies the apical junctional localization of the RNAi machinery as a key feature of the differentiated colonic epithelium, with a putative tumor suppressing function.

HEPATIC AGO2-MEDIATED RNA SILENCING REGULATES SYSTEMIC GLUCOSE METABOLISM.

Obesity causes various metabolic complications, including insulin resistance and T2DM. However, currently, we have a limited understanding of the pathophysiology in the development of these processes. It’s generally considered that obesity develops when energy intake chronically exceeds total energy expenditure. The liver is a major organ for energy consumption, then mRNA translation accounts for the majority of energy expenditure in liver. While RNA silencing regulates mRNA translation, it’s unclear if RNA silencing regulates glucose metabolism. To investigate the role of RNA silencing in glucose metabolism, we focused on Argonaute 2 (Ago2), which is the main component of RNA-induced silencing complex that carries out RNA silencing. By generating liver-specific Ago2-deficient (L-Ago2 KO) mice, we revealed Ago2 regulates the maturation process of metabolic disease related miRNAs (MD-miRNAs), that silence genes critical for glucose metabolism. In addition, Ago2-deletion enhances ATP consumption associated with mRNA translation. Consequently, inactivation of hepatic Ago2 protect from diet-induced glucose intolerance in mice. Then, to further investigate if these molecular mechanisms are still activated in “older mice”, we employed 34-week of age mice and analyzed. Around this age, despite having the similar body weight, L-Ago2 KO mice fed HFD exhibited lower blood glucose and serum insulin levels. Consistently, expression levels of MD-miRNAs were decreased in the liver of L-Ago2 KO mice fed HFD. These results suggest that hepatic Ago2 function is continuously activated in “older mice” fed HFD, leading to enhanced biogenesis of MD-miRNAs and reduction of their target mRNAs, and these alterations are associated with systemic glucose metabolism.

The RNA\u2013RNA base pairing potential of human Dicer and Ago2 proteins

The ribonuclease Dicer produces microRNAs (miRNAs) and small interfering RNAs that are handed over to Ago proteins to control gene expression by targeting complementary sequences within transcripts. Interestingly, a growing number of reports have demonstrated that the activity of Dicer may extend beyond the biogenesis of small regulatory RNAs. Among them, a report from our latest studies revealed that human Dicer facilitates base pairing of complementary sequences present in two nucleic acids, thus acting as a nucleic acid annealer. Accordingly, in this manuscript, we address how RNA structure influences the annealing activity of human Dicer. We show that Dicer supports hybridization between a small RNA and a complementary sequence of a longer RNA in vitro, even when both complementary sequences are trapped within secondary structures. Moreover, we show that under applied conditions, human Ago2, a core component of RNA-induced silencing complex, displays very limited annealing activity. Based on the available data from new-generation sequencing experiments regarding the RNA pool bound to Dicer in vivo, we show that multiple Dicer-binding sites within mRNAs also contain miRNA targets. Subsequently, we demonstrate in vitro that Dicer but not Ago2 can anneal miRNA to its target present within mRNA. We hypothesize that not all miRNA duplexes are handed over to Ago proteins. Instead, miRNA-Dicer complexes could target specific sequences within transcripts and either compete or cooperate for binding sites with miRNA-Ago complexes. Thus, not only Ago but also Dicer might be directly involved in the posttranscriptional control of gene expression.

MicroRNA Changes in Diabetic Cardiac Mitochondria: What are they doing there?

The mitochondrion has been found to harbor non‐coding RNA (ncRNA) that appear to be dynamically regulated in health and disease. Due to the tremendous energy demand of the heart and the well‐characterized cardiac dysfunction observed in type 2 diabetes mellitus (T2DM), we investigated whether mitochondrial microRNA (miR) were differentially expressed in human and mouse diabetic heart tissue. In isolated mitochondria from human right atrial appendages (n = 7 non‐diabetic and n = 7 T2DM), small ncRNA sequencing was performed. Libraries were constructed using the NEXTflex Small RNA‐Seq Kit v3 and sequenced on the HiSeq 2500 in Rapid Run mode using 2 lanes of single‐end (SE) 40 base pair reads. After identifying miRNA expression changes in human cardiac mitochondria between non‐diabetic and T2DM patients, we sought to determine the role of these nuclear‐encoded miRNA in the mitochondria. To accomplish this, we were interested in the binding of mitochondria‐localized RNA‐induced silencing complex (RISC) components to miRNA in cardiac mitochondria of a diabetic mouse model. In FVB (n = 5 pooled samples) and FVB‐db/db (n=5 pooled samples) mouse models, cross‐linked immunoprecipitation (CLIP) sequencing was performed for both argonaute 2 (Ago2) and fragile X mental retardation syndrome‐related protein 1 (Fxr1). Libraries were constructed using the TruSeq Small RNA Library Prep Kit and sequenced on the HiSeq 2500 in Rapid Run mode using 2 lanes of single‐end (SE) 40 base pair reads. Files were aligned to the reference genomes GRCh38 (human) and GRCm38 (mouse) release 94 through Bowtie running “best” and “strata.” Overall, we identified differential expression of 236 miR species in non‐diabetic and T2DM human cardiac mitochondria. Those increased greater than 3‐fold in T2DM cardiac mitochondria included miR‐3687‐1, miR‐3687‐2; and some of those decreased greater than 3‐fold included miR‐23A, miR‐23B, miR‐30A, miR‐30B, miR‐30C1, miR‐30C2, miR‐30D, miR‐30E, miR‐101‐1, miR‐191, miR‐340, miR‐361, and miR‐374A. Interestingly, we found overlap of some miRNA species found in human cardiac mitochondria with those found bound to Ago2 and FXR1 in diabetic mouse cardiac mitochondria. These included miRNA‐let7i (p=0.049), miRNA‐26a‐1 (p=0.00045), miRNA‐26a‐2 (p=0.00045), miRNA‐27a (p=0.0023), miRNA‐27b (p=0.0014), miRNA‐30a (p=3.13E‐05), and miRNA‐181a‐1 (p=0.0046). In conclusion, the association of RISC components with miRNA species found to have significant expression changes in the T2DM human heart suggests that not only is the FVB‐db/db a clinically relevant model of T2DM, but also that miRNA play a role in translational repression in the T2DM human heart.Support or Funding InformationThis work was supported by: R01 HL‐128485 (JMH), AHA‐17PRE33660333 (QAH), WVU Genomics Core Facility, and the Community Foundation for the Ohio Valley Whipkey Trust.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Consideration of the role of MALAT1 long noncoding RNA and catalytic component of RNA-induced silencing complex (Argonaute 2, AGO2) in autism spectrum disorders: Yes, or no?

Autism spectrum disorders (ASD) are complex neurodevelopmental impairments in which dysregulation of long noncoding RNAs (lncRNAs) has been indicated. LncRNAs tend to play role in constituting comprehensive networks of ribonucleoprotein complexes including argonautes. Here, we aimed to study the expressions of MALAT1, a highly conserved lncRNA, and AGO2 gene, encoding the catalytic component of the RNA-induced silencing complex (RISC), in ASD patients. In this case-control study, peripheral whole blood samples were gathered from 30 ASD patients and 41 healthy controls and expression level of genes were matured by quantitative TaqMan real-time PCR. we found an increase in MALAT1 expression, which was statistically insignificant. Moreover, the AGO2 expression revealed a decrease that did not reach a level of significance. There was a significant and direct correlation between expression levels of MALAT1 and AGO2 (r = 0.427, P < 0.0001). Eventually, MALAT1 and AGO2 correlations with patients' age did not show a significant difference. These findings provide clues that although we have indicated a significant direct correlation between MALAT1 lncRNA and AGO2, implying their interactive network, there should be a reconsideration regarding their role in ASD development based on whole blood samples because the altered expressions were not strong enough to be significant. Further investigations employing larger sample sizes and specific leukocytes subsets separately could profoundly strengthen these findings.

RNA interference may suppress stress granule formation by preventing argonaute 2 recruitment.

RNA-induced silencing complex (RISC) is formed during RNA interference (RNAi), whereas stress granules (SG) are assembled in response to cellular stress. Here, we demonstrate an interesting connection between RISC and SG that may involve argonaute 2 (Ago2), a core component of RISC. We analyzed SG induction by arsenite, the commonly used SG inducer. SG formation was suppressed in heat shock transcription factor 1 (Hsf1) or hypoxia-inducible factor-1α (Hif1α) shRNA-transfected cells but not in Hsf1 or Hif1α-knockout cells, suggesting that RNAi per se (rather than gene deficiency) may account for the suppressive effect on SG. In support, the suppressive effect of RNAi on SG formation was reversed by the RISC-loading inhibitor aurintricarboxylic acid. In non-RNAi cells, arsenite induced the accumulation of Ago2 in SGs as shown by its colocalization and coimmunoprecipitation with SG proteins, but Ago2 was not recruited to SG in the cells with RNAi. Consistently, arsenite induced the dissociation of Ago2 from RISC proteins in non-RNAi cells but not in RNAi cells. CRISPR-Cas9-medicated ablation of Ago2 attenuated SG formation during arsenite treatment, suggesting a critical role of Ago2 in SG assembly. Together, these results indicate that RISC and SG may compete for some key components, such as Ago2. In response to cellular stress, Ago2 is recruited for SG assembly; however, during RNAi, Ago2 is held in RISC, becoming unavailable for SG formation.

Farnesylated heat shock protein 40 is a component of membrane-bound RISC in Arabidopsis

ARGONAUTE1 (AGO1) binds directly to small regulatory RNA and is a key effector protein of post-transcriptional gene silencing mediated by microRNA (miRNA) and small interfering RNA (siRNA) in Arabidopsis. The formation of an RNA-induced silencing complex (RISC) of AGO1 and small RNA requires the function of the heat shock protein 70/90 chaperone system. Some functions of AGO1 occur in association with endomembranes, in particular the rough endoplasmic reticulum (RER), but proteins interacting with AGO1 in membrane fractions remain unidentified. In this study, we show that the farnesylated heat shock protein 40 homologs, J2 and J3, associate with AGO1 in membrane fractions in a manner that involves protein farnesylation. We also show that three changes in AGO1 function are detectable in mutants in protein farnesylation and J2/J3. First, perturbations of the HSP40/70/90 pathway by mutation of J3, HSP90, and farnesyl transferase affect the amounts of AGO1 associated with membranes. Second, miRNA association with membrane-bound polysomes is increased in farnesyl transferase and farnesylation-deficient J2/J3 mutants. Third, silencing by noncell autonomously acting short interfering RNAs is impaired. These observations highlight the involvement of farnesylated J2/J3 in small RNA-mediated gene regulation, and suggest that the importance of chaperone-AGO1 interaction is not limited to the RISC assembly process.

Argonaute-miRNA Complexes Silence Target mRNAs in the Nucleus of Mammalian Stem Cells

In mammals, gene silencing by the RNA-induced silencing complex (RISC) is a well-understood cytoplasmic posttranscriptional gene regulatory mechanism. Here, we show that embryonic stem cells (ESCs) contain high levels of nuclear AGO proteins and that in ESCs nuclear AGO protein activity allows for the onset of differentiation. In the nucleus, AGO proteins interact with core RISC components, including the TNRC6 proteins and the CCR4-NOT deadenylase complex. In contrast to cytoplasmic miRNA-mediated gene silencing that mainly operates on cis-acting elements in mRNA 3' untranslated (UTR) sequences, in the nucleus AGO binding in the coding sequence and potentially introns also contributed to post-transcriptional gene silencing. Thus, nuclear localization of AGO proteins in specific cell types leads to a previously unappreciated expansion of the miRNA-regulated transcriptome.

MiR-361-3p regulates ERK1/2-induced EMT via DUSP2 mRNA degradation in pancreatic ductal adenocarcinoma

Metastasis remains one of the most intractable challenges in pancreatic ductal adenocarcinoma (PDAC) biology, and epithelial-to-mesenchymal transition (EMT) is essential to the epithelium-originated solid tumor metastasis cascade. Emerging evidence demonstrates that aberrant miRNA expression is involved in pancreatic cancer progression. We found that miR-361-3p was associated with an advanced stage of PDAC and poor prognosis. Hence, the effect of miR-361-3p on metastasis of PDAC cells was evaluated using Transwell assay and wound healing assay in vitro as well as orthotopic and liver metastasis pancreatic cancer models in vivo. Overexpression of miR-361-3p promoted pancreatic cancer cell migration and invasion in vitro, and miR-361-3p-elevated PDAC cells were prone to generating metastatic nodules in vivo. However, miR-361-3p showed no significant effect on the proliferation of PDAC cells in vivo or in vitro. Further study demonstrated that miR-361-3p could enhance EMT and ERK pathway activation, and ERK inhibitor could attenuate miR-361-3p-induced EMT. Luciferase assays, qPCR, and western blot and Ago2 co-immunoprecipitation were performed to identify the direct target of miR-361-3p. Mechanistic investigations identified DUSP2 as a direct target of miR-361-3p, and DUSP2 was revealed to be involved in miR-361-3p-induced EMT by directly leading to the inactivation of the ERK pathway. Moreover, we found that miR-361-3p-induced EMT was dependent on Ago2, the core component of RNA-induced silencing complex, while enforced expression of Ago2 enhanced the miR-361-3p-induced effect by promoting interference efficacy and specificity rather than regulating miR-361-3p stability and biogenesis. Thus, this study revealed that miR-361-3p functions as an oncomiR for promoting metastasis and identified the miR-361-3p/DUSP2/ERK axis as a novel EMT axis dependent on Ago2 in PDAC.