RNA-induced Silencing Complex Research Articles

Detection of MicroRNAs Released from Argonautes.

The Argonaute (AGO) family of proteins plays an essential role in the process of microRNA (miRNA)-mediated gene silencing. More specifically, they are the only known proteins to associate directly with miRNAs within the RNA-induced silencing complex (RISC). Given the importance of miRNA regulation of the transcriptome and its vast implications for human disease, it is essential to understand the molecular underpinnings of miRNA-AGO interactions. Although there are methods available to investigate mature miRNA decay and loading onto AGO2, no feasible method exists to detail the opposite process: release of miRNA from associated AGO proteins. In this chapter, we describe in detail a methodology derived from biochemical approaches, which can be used to quantify the release of any given miRNA from AGOs.

Su(var)2-10 and the SUMO Pathway Link piRNA-Guided Target Recognition to Chromatin Silencing.

Regulation of transcription is the main mechanism responsible for precise control of gene expression. Whereas the majority of transcriptional regulation ismediated by DNA-binding transcription factors that bind to regulatory gene regions, an elegant alternative strategy employs small RNA guides, Piwi-interacting RNAs (piRNAs) to identify targets of transcriptional repression. Here, we show that inDrosophila the small ubiquitin-like protein SUMOand the SUMO E3 ligase Su(var)2-10 are required for piRNA-guided deposition of repressive chromatin marks and transcriptional silencing of piRNA targets. Su(var)2-10 links the piRNA-guided target recognition complex to the silencing effector by binding the piRNA/Piwi complex and inducing SUMO-dependent recruitment of the SetDB1/Wde histone methyltransferase effector. We propose that in Drosophila, the nuclear piRNA pathway has co-opted a conserved mechanism of SUMO-dependent recruitment of the SetDB1/Wde chromatin modifier to confer repression of genomic parasites.

CircCAMSAP1 Promotes Tumor Growth in Colorectal Cancer via the miR-328-5p/E2F1 Axis.

Increasing studies indicated that circular RNAs (circRNAs) play important roles in cancer progression. However, the roles of circRNAs in colorectal cancer (CRC) remain largely unknown. In this study, we determined the circRNA expression profile by next-generation RNA sequencing from eight CRC and paired non-cancerous matched tissues. circCAMSAP1 (originating from exon 2 to exon 3 of the CAMSAP1 gene, hsa_circ_0001900) was significantly upregulated in CRC tissues. Increased circCAMSAP1 expression was significantly correlated with advanced tumor/node/metastasis (TNM) stage and shortened overall survival. An elevation of circCAMSAP1 expression was detected via droplet digital PCR in the serum of CRC patients prior to surgery. Functionally, circCAMSAP1 promoted the malignant behavior of CRC. Mechanism study of upstream biogenesis of circCAMSAP1 indicated that circCAMSAP1 cyclization in CRC was mediated by splicing factor epithelial-splicing regulatory protein 1. Moreover, circCAMSAP1 acted as a sponge for miR-328-5p and abrogated its suppression on transcription factor E2F1. Taken together, our data indicated an essential role of the circCAMSAP1/miR-328-5p/E2F1 axis in the progression of CRC, which implied that circCAMSAP1 could serve as a diagnostic and prognostic biomarker as well as a potential therapeutic target for CRC.

An ortholog of the Vasa intronic gene is required for small RNA-mediated translation repression in Chlamydomonas reinhardtii

Small RNAs (sRNAs) associate with Argonaute (AGO) proteins in effector complexes, termed RNA-induced silencing complexes (RISCs), which regulate complementary transcripts by translation inhibition and/or RNA degradation. In the unicellular alga Chlamydomonas, several metazoans, and land plants, emerging evidence indicates that polyribosome-associated transcripts can be translationally repressed by RISCs without substantial messenger RNA (mRNA) destabilization. However, the mechanism of translation inhibition in a polyribosomal context is not understood. Here we show that Chlamydomonas VIG1, an ortholog of the Drosophila melanogaster Vasa intronic gene (VIG), is required for this process. VIG1 localizes predominantly in the cytosol and comigrates with monoribosomes and polyribosomes by sucrose density gradient sedimentation. A VIG1-deleted mutant shows hypersensitivity to the translation elongation inhibitor cycloheximide, suggesting that VIG1 may have a nonessential role in ribosome function/structure. Additionally, FLAG-tagged VIG1 copurifies with AGO3 and Dicer-like 3 (DCL3), consistent with it also being a component of the RISC. Indeed, VIG1 is necessary for the repression of sRNA-targeted transcripts at the translational level but is dispensable for cleavage-mediated RNA interference and for the association of the AGO3 effector with polyribosomes or target transcripts. Our results suggest that VIG1 is an ancillary ribosomal component and plays a role in sRNA-mediated translation repression of polyribosomal transcripts.

6mer Seed Toxicity in Viral microRNAs.

SummaryMicroRNAs (miRNAs) are short double-stranded noncoding RNAs (19-23 nucleotides) that regulate gene expression by suppressing mRNAs through RNA interference. Targeting is determined by the seed sequence (position 2-7/8) of the mature miRNA. A minimal G-rich seed of just six nucleotides is highly toxic to cells by targeting genes essential for cell survival. A screen of 215 miRNAs encoded by 17 human pathogenic viruses (v-miRNAs) now suggests that a number of v-miRNAs can kill cells through a G-rich 6mer sequence embedded in their seed. Specifically, we demonstrate that miR-K12-6-5p, an oncoviral mimic of the tumor suppressive miR-15/16 family encoded by human Kaposi sarcoma-associated herpes virus, harbors a noncanonical toxic 6mer seed (position 3-8) and that v-miRNAs are more likely than cellular miRNAs to utilize a noncanonical 6mer seed. Our data suggest that during evolution viruses evolved to use 6mer seed toxicity to kill cells.

Hit two birds with one stone: the multiple properties of (viral) RNA silencing suppressors

In any organism, gene expression regulation is provided by multiple factors to maintain a harmonious development of individuals. Discovered in the late 1990s, RNA interference (RNAi) completely remodelled the way in which gene expression regulations were initially apprehended. RNAi provides fine regulation at the cellular level and allows organisms to control their development, maintain their genomic integrity and fight against different stresses like viral infection. Exogenous or endogenous double-stranded RNAs initiate RNAi and are recognized and cleaved by Dicer protein in about 20nucleotide duplexes small RNAs (sRNAs). One strand of the duplex is loaded into a ribonucleoproteic complex, named RISC (RNA-induced silencing complex), composed of at least one ARGONAUTE protein and a sRNA. Therefore, the expression of any RNA possessing the complementary siRNA sequence of the small RNA will be specifically silenced either at the transcriptional or post-transcriptional level. RNAi plays a prominent role in the defence against viral infection and the last two decades of research have refined our knowledge of proteins involved in this pathway. Many viruses counteract the antiviral action of RNAi through the expression of factors (viral suppressor of RNA silencing [VSR]) that were first identified on virally infected plants. However, in mammals, the antiviral role of RNAi remains controversial. Indeed, viral infections are controlled by the interferon response and the antiviral action of RNAi has not been clearly demonstrated in vivo. In this review, the main modes of defence suppression used by VSR and endogenous RNAi suppressors will be presented. Finally, the role of viral non-coding RNAs (ncRNAs) acting as suppressors of RNAi will be discussed.

Hit two birds with one stone: the multiple properties of (viral) RNA silencing suppressors.

In any organism, gene expression regulation is provided by multiple factors to maintain a harmonious development of individuals. Discovered in the late 1990s, RNA interference (RNAi) completely remodelled the way in which gene expression regulations were initially apprehended. RNAi provides fine regulation at the cellular level and allows organisms to control their development, maintain their genomic integrity and fight against different stresses like viral infection. Exogenous or endogenous double-stranded RNAs initiate RNAi and are recognized and cleaved by Dicer protein in about twenty nucleotide duplexes small RNAs (sRNAs). One strand of the duplex is loaded into a ribonucleoproteic complex, named RISC (RNA induced silencing complex), composed of at least one ARGONAUTE protein and a sRNA. Therefore, the expression of any RNA possessing the complementary siRNA sequence will be specifically silenced either at the transcriptional or post-transcriptional level. RNAi plays a prominent role in the defence against viral infection and the last two decades of research have refined our knowledge of proteins involved in this pathway. Many viruses counteract the antiviral action of RNAi through the expression of factors (VSR, Viral suppressor of RNA silencing) that were first identified on virally infected plants. However, in mammals the antiviral role of RNAi remains controversial. Indeed, viral infections are controlled by the interferon response and the antiviral action of RNAi has not been clearly demonstrated in vivo. In this review, the main modes of defence suppression used by VSR and endogenous RNAi suppressors will be presented. Finally, the role of viral non-coding RNAs (ncRNAs) acting as suppressors of RNAi will be discussed.

Characterization and differential expression of microRNA in the freshwater shrimp (Neocaridina heteropoda) with different body length

ABSTRACTMicroRNAs (miRNAs) are a class of small non-coding RNAs which promote mRNA degradation and inhibit mRNA translation. Through post-transcriptional regulation, microRNAs (miRNAs) play a significant role in regulating growth, development, metabolic, and reproductive processes. However, current knowledge on miRNAs in crustaceans is limited. In this study, we explored the potential roles of miRNAs in abdominal muscle of Neocaridina heteropoda of different body length. We constructed two small RNA libraries from muscle samples, and 26 novel miRNAs were identified. Among these novel miRNAs, 13 were found significantly differentially expressed. Specifically, 10 miRNAs were significantly up-regulated and 3 miRNAs were down-regulated in small shrimps compared with large ones. Furthermore, bioinformatics analysis revealed that miR-26 was associated with a large number of differentially expressed genes, including glucose transporter 1 (Glut1), phosphoenolpyruvate carboxykinase (Pepck), and phosphofructokinase (Pfk), related to glycolysis and gluconeogenesis pathways. Besides, luciferase activity assay verified that Pepck was a direct target gene of the novel miR-26. The results imply potential growth regulatory roles for miRNAs and provide a valuable resource for further investigation of the miRNA mechanisms controlling muscle growth in N. heteropoda and other crustaceans.Abbreviations: Ago: argonaute; ANOVA: one-way analysis of variance; CRISPR: clustered regularly interspaced short palindromic repeats; DEGs: differentially expressed genes; DMEM: Dulbecco’s Modified Eagle Medium; Glut1: glucose transporter 1; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; miRNA: microRNA; Pepck: phosphoenolpyruvate carboxykinase; PPIs: predict protein-protein interactions; Pfk: phosphofructokinase; RISC: RNA-induced silencing complex; siRNAs: small interfering RNAs; sRNA: small RNA; dsDNA: double-stranded DNA; qPCR: quantitative PCR

The Non-Canonical Aspects of MicroRNAs: Many Roads to Gene Regulation.

MicroRNAs (miRNAs) are critical regulators of gene expression. As miRNAs are frequently deregulated in many human diseases, including cancer and immunological disorders, it is important to understand their biological functions. Typically, miRNA-encoding genes are transcribed by RNA Polymerase II and generate primary transcripts that are processed by RNase III-endonucleases DROSHA and DICER into small RNAs of approximately 21 nucleotides. All miRNAs are loaded into Argonaute proteins in the RNA-induced silencing complex (RISC) and act as post-transcriptional regulators by binding to the 3′- untranslated region (UTR) of mRNAs. This seed-dependent miRNA binding inhibits the translation and/or promotes the degradation of mRNA targets. Surprisingly, recent data presents evidence for a target-mediated decay mechanism that controls the level of specific miRNAs. In addition, several non-canonical miRNA-containing genes have been recently described and unexpected functions of miRNAs have been identified. For instance, several miRNAs are located in the nucleus, where they are involved in the transcriptional activation or silencing of target genes. These epigenetic modifiers are recruited by RISC and guided by miRNAs to specific loci in the genome. Here, we will review non-canonical aspects of miRNA biology, including novel regulators of miRNA expression and functions of miRNAs in the nucleus.

Click RNA for Rapid Capture and Identification of Intracellular MicroRNA Targets.

Rapid capture and identification of the intracellular target genes of microRNAs (miRNAs) are the key to understanding miRNA functions and development of RNA-based therapeutics. However, developing biochemical tools that can fish out the target genes of miRNAs in live cells is a significant technical challenge. Here, we report a remarkably simple yet powerful technology capable of loading virtually any miRNA into Ago2 of the RNA-induced silencing complexes (RISCs). This surprising discovery enables rapid capture and identification of target mRNAs and long noncoding RNAs. It is achieved by linking dibenzocyclooctyne (DBCO), a classical chemical moiety in copper-free click chemistry, to the 3' end of miRNAs. DBCO serves as a high-affinity tag to the Ago2 protein, thus boosting the formation of RISCs with miRNA target genes in living cells. Upon cell lysing, DBCO's routine function in click chemistry allows rapid enrichment of target genes for analysis without the need of additional molecular handles. A series of miR-21 and miR-27a target genes that were previously unknown were pulled down from various cell lines and identified with qRT-PCR, demonstrating the utility of this innovative technology in both transcriptomic research and RNA-based studies.

A DNA virus-encoded immune antagonist fully masks the potent antiviral activity of RNAi in Drosophila

Coevolution of viruses and their hosts may lead to viral strategies to avoid, evade, or suppress antiviral immunity. An example is antiviral RNA interference (RNAi) in insects: the host RNAi machinery processes viral double-stranded RNA into small interfering RNAs (siRNAs) to suppress viral replication, whereas insect viruses encode suppressors of RNAi, many of which inhibit viral small interfering RNA (vsiRNA) production. Yet, many studies have analyzed viral RNAi suppressors in heterologous systems, due to the lack of experimental systems to manipulate the viral genome of interest, raising questions about in vivo functions of RNAi suppressors. To address this caveat, we generated an RNAi suppressor-defective mutant of invertebrate iridescent virus 6 (IIV6), a large DNA virus in which we previously identified the 340R protein as a suppressor of RNAi. Loss of 340R did not affect vsiRNA production, indicating that 340R binds siRNA duplexes to prevent RNA-induced silencing complex assembly. Indeed, vsiRNAs were not efficiently loaded into Argonaute 2 during wild-type IIV6 infection. Moreover, IIV6 induced a limited set of mature microRNAs in a 340R-dependent manner, most notably miR-305-3p, which we attribute to stabilization of the miR-305-5p:3p duplex by 340R. The IIV6 340R deletion mutant did not have a replication defect in cells, but was strongly attenuated in adult Drosophila This in vivo replication defect was completely rescued in RNAi mutant flies, indicating that 340R is a bona fide RNAi suppressor, the absence of which uncovers a potent antiviral immune response that suppresses virus accumulation ∼100-fold. Together, our work indicates that viral RNAi suppressors may completely mask antiviral immunity.

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.

Leading RNA Interference Therapeutics Part 1: Silencing Hereditary Transthyretin Amyloidosis, with a Focus on Patisiran.

In 2018, patisiran was the first-ever RNA interference (RNAi)-based drug approved by the US Food and Drug Administration. Now pharmacology textbooks may include a new drug class thatresults in the effect firstdescribed by Fire and Mello 2 decades ago: post-transcriptional gene silencing by a small-interfering RNA (siRNA). Patients with hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) present withmutations in the transthyretin (TTR) gene that lead to the formation of amyloid deposits in peripheral nerves and heart. The disease may also affect the eye and central nervous system. The formulation of patisiran comprises the RNAi drug encapsulated into a nanoparticle especially developed to deliver the anti-TTR siRNA into the main TTR producer: the liver. Hepatic cells contain apolipoprotein E receptors that recognize ApoE proteins opsonized in the lipid carrier and internalize the drug by endocytosis. Lipid vesicles are disrupted in the cell cytoplasm, and siRNAs are free to trigger the RNAi-based TTR gene silencing. The silencing processinvolves the binding of siRNA guide strand to 3'-untranslated region sequence of both mutant and wild-type TTR messenger RNA, which culminates in the TTR mRNA cleavage by the RNA-induced silencing complex (RISC)asthe first biochemical drug effect. Patisiran 0.3mg/kg is administered intravenously every 3weeks. Patients require premedication with anti-inflammatory drugs and antagonists of histamine H1 and H2 receptors to prevent infusion-related reactions and may require vitamin A supplementation. Following patisiran treatment, TTR knockdown remained stable for at least 2years. Adverse effects were mild to moderate with unchanged hematological, renal, or hepatic parameters. No drug-related severe adverse effects occurred in a 24-month follow-up phase II open-label extension study.At the recommended dosage of patisiran, Cmax and AUC values (mean ± standard deviation) were 7.15±2.14μg/mL and 184±159μg·h/mL, respectively. The drug showed stability in circulation with > 95% encapsulated in lipid particles. Metabolization occurred by ribonuclease enzymes, with less than 1% excreted unchanged in the urine. Patisiran ameliorated neuropathy impairment according to the modified Neuropathy Impairment Score + 7 analysis of the phase III study. The Norfolk Quality of Life-Diabetic Neuropathy score and gait speed improved in 51% of the patisiran-treated group in 18months. Additionally,the modified body mass index showed positive outcomes. Altogether, the data across phase I-III clinical trials points to patisiran as an effective and safe drug for the treatment of hATTR amyloidosis. It is hoped that real-world data from a larger number of patients treated with patisiran will confirm the effectiveness of this first-approved siRNA-based drug.

New opportunities for designing effective small interfering RNAs

Small interfering RNAs (siRNAs) that silence genes of infectious diseases are potentially potent drugs. A continuing obstacle for siRNA-based drugs is how to improve their efficacy for adequate dosage. To overcome this obstacle, the interactions of antiviral siRNAs, tested in vivo, were computationally examined within the RNA-induced silencing complex (RISC). Thermodynamics data show that a persistent RISC cofactor is significantly more exothermic for effective antiviral siRNAs than their ineffective counterparts. Detailed inspection of viral RNA secondary structures reveals that effective antiviral siRNAs target hairpin or pseudoknot loops. These structures are critical for initial RISC interactions since they partially lack intramolecular complementary base pairing. Importing two temporary RISC cofactors from magnesium-rich hairpins and/or pseudoknots then kickstarts full RNA hybridization and hydrolysis. Current siRNA design guidelines are based on RNA primary sequence data. Herein, the thermodynamics of RISC cofactors and targeting magnesium-rich RNA secondary structures provide additional guidelines for improving siRNA design.

AEG-1/miR-221 Axis Cooperatively Regulates the Progression of Hepatocellular Carcinoma by Targeting PTEN/PI3K/AKT Signaling Pathway.

Hepatocellular carcinoma (HCC) is the third leading malignancy worldwide, causing mortality in children and adults. AEG-1 is functioned as a scaffold protein for the proper assembly of RNA-induced silencing complex (RISC) to optimize or increase its activity. The increased activity of oncogenic miRNAs leads to the degradation of target tumor suppressor genes. miR-221 is an oncogenic miRNA, that plays a seminal role in carcinogenesis regulation of HCC. However, the molecular mechanism and biological functions of the miR-221/AEG-1 axis have not been investigated extensively in HCC. Here, the expression of miR-221/AEG-1 and their target/associate genes was analyzed by qRT-PCR and Western blot. The role of the miR-221/AEG-1 axis in HCC was evaluated by proliferation assay, migration assay, invasion assay, and flow cytometry analysis. The expression level of miR-221 decreased in AEG-1 siRNA transfected HCC cells. On the other hand, there were no significant expression changes of AEG-1 in miR-221 mimic and miR-221 inhibitor transfected HCC cells and inhibition of miR-221/AEG-1 axis decreased cell proliferation, invasion, migration, and angiogenesis and induced apoptosis, cell cycle arrest by upregulating p57, p53, PTEN, and RB and downregulating LSF, MMP9, OPN, Bcl-2, PI3K, AKT, and LC3A in HCC cells. Furthermore, these findings suggest that the miR-221/AEG-1 axis plays a seminal oncogenic role by modulating PTEN/PI3K/AKT signaling pathway in HCC. In conclusion, the miR-221/AEG-1 axis may serve as a potential target for therapeutics, diagnostics, and prognostics of HCC.

Hepatitis C Virus Mediated Inhibition of miR-181c Activates ATM Signaling and Promotes Hepatocyte Growth.

Hepatitis C virus (HCV) infection promotes hepatocyte growth and progress to hepatocellular carcinoma. We previously observed that HCV infection of hepatocytes transcriptionally down-regulates miR-181c expression through CCAAT/enhancer binding protein β (C/EBP-β). Here, we examined the role of miR-181c in the regulation of cell cycle progression in relation to HCV infection. In silico analysis suggested that ataxia-telangiectasia mutated (ATM) protein, a protein kinase, is a direct target of miR-181c. ATM is a central mediator of response for cellular DNA double-strand break. Our results demonstrated that ATM expression is higher in HCV-infected hepatocytes and chronic HCV-infected liver biopsy specimens. We have shown a direct interaction of miR-181c with the 3' untranslated region of ATM, and the presence of ATM in miR-181c-associated RNA-induced silencing complex. Exogenous expression of miR-181c inhibited ATM expression and activation of its downstream molecules, Chk2 and Akt. On the other hand, introduction of anti-miR-181c restored ATM and phosphorylated Akt. Furthermore, introduction of miR-181c significantly inhibited phospho-cyclin-dependent kinase 2 (CDK2) and cyclin-A expression, arresting cell cycle progression, whereas overexpression of miR-181c promoted apoptosis of HCV-infected hepatocytes and can be inhibited by overexpression of ATM from a clone lacking miR-181c binding sites. In addition, miR-181c significantly regressed tumor growth in the xenograft human hepatocellular carcinoma mouse model. Together, our results suggest that HCV infection suppresses miR-181c in hepatocytes, resulting in ATM activation and apoptosis inhibition for promotion of cell cycle progression. The results provide mechanistic insight into understanding the role of miR-181c in HCV-associated hepatocyte growth promotion, and may have the potential for therapeutic intervention.

Association study of AGO1 and AGO2 genes polymorphisms with recurrent pregnancy loss

An Argonaute (AGO) protein within the RNA-induced silencing complex binds a microRNA, permitting the target mRNA to be silenced. We hypothesized that variations in AGO genes had the possibility including affected the miRNA function and associated with recurrent pregnancy loss (RPL) susceptibility. Especially, we were chosen the AGO1 (rs595961, rs636832) and AGO2 (rs2292779, rs4961280) polymorphisms because of those polymorphisms have already reported in other diseases excluding the RPL. Here, we conducted a case-control study (385 RPL patients and 246 controls) to evaluate the association of four polymorphisms with RPL. We found that the AGO1 rs595961 AA genotype, recessive model (P = 0.039; P = 0.043, respectively), the AGO1 rs636832 GG genotype, and recessive model (P = 0.037; P = 0.016, respectively) were associated with RPL in women who had had four or more consecutive pregnancy losses. The patients with the AGO1 rs636832 GG genotypes had greater platelet counts (P = 0.023), while the patients with the AGO2 rs4961280 CA genotypes had less homocysteine (P = 0.027). Based on these results, we propose that genetic variations with respect to the AGO1 and AGO2 genotypes are associated with risk for RPL, and might serve as useful biomarkers for the prognosis of RPL.

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.

Functional Characterization of Entamoeba histolytica Argonaute Proteins Reveals a Repetitive DR-Rich Motif Region That Controls Nuclear Localization.

The RNA interference (RNAi) pathway regulates gene expression in many eukaryotic organisms. Argonaute (Ago) proteins, together with bound small RNAs (sRNAs), are key effectors that mediate gene silencing function. However, there is limited knowledge of Ago proteins and their functions in nonmodel systems. In the protozoan parasite Entamoeba histolytica, RNAi is a robust means for stable gene silencing mediated via large populations of antisense sRNAs. Here, we report functional characterization of three Ago proteins in E. histolytica (EhAgo2-1, EhAgo2-2, and EhAgo2-3). Our data show that each EhAgo protein has a distinct subcellular localization and binds 27-nucleotide (nt) sRNAs and that the localization of EhAgo proteins is altered in response to stress conditions. Via mutagenesis analyses, we demonstrated that the Ago PAZ (Piwi/Argonaute/Zwille) domain in all three EhAgos is essential for sRNA binding. With mutation of the PAZ domain in EhAgo2-2, there was no effect on the nuclear localization of the protein but a strong phenotype and a growth defect. We further show that EhAgo2-2 contains an unusual repetitive DR-rich (aspartic acid, arginine-rich) motif region which functions as a nuclear localization signal (NLS) and is both necessary and sufficient to mediate nuclear localization. Overall, our data delineate the localization and sRNA binding features of the three E. histolytica Ago proteins and demonstrate that the PAZ domain is necessary for sRNA binding. The repetitive DR-rich motif region in EhAgo2-2 has not previously been defined in other systems, which adds to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems.IMPORTANCE The protozoan parasite Entamoeba histolytica, which causes amebiasis and affects over 50 million people worldwide, contains an important RNAi pathway for gene silencing. Gene silencing via the RNAi pathway is mediated by the Argonaute (Ago) proteins. However, we lack knowledge on Ago function(s) in this nonmodel system. In this paper, we discovered that three E. histolytica Ago proteins (EhAgo2-1, EhAgo2-2, and EhAgo2-3) all bind 27-nt small RNAs and have distinct subcellular localizations, which change in response to stress conditions. The EhAgos bind small RNA populations via their PAZ domains. An unusual repetitive DR-rich motif region is identified in EhAgo2-2 that functions as a nuclear localization signal. Our results show for the first time an active nuclear transport process of the EhAgo2-2 RNA-induced silencing complex (RISC) in this parasite. These data add to the novel observations that can be made when studies of the RNAi pathway are extended to nonmodel systems.

MiR-302: The Multifunctional MicroRNA

MicroRNAs (miRNAs) are short single-stranded noncoding RNAs (20- to 25-nucleotide (nt) long) representing a class of small regulatory RNAs. By inhibiting the translation of target mRNAs, miRNAs regulate gene expression posttranscriptionally and thus play an important role in a wide range of cellular processes. Currently, there are two known types of miRNAs: intergenic and intronic miRNAs. Biogenesis of an intergenic miRNA starts with the synthesis of a primary miRNA transcript (pri-miRNA) catalyzed by types-II or -III RNA polymerase (Pol-II/III). Pri-miRNAs are processed in the nucleus by the ribonuclease Drosha into a miRNA precursor (pre-miRNA) approximately 60-nt in length. After being transported into the cytoplasm, these pre-miRNAs are further processed into mature and functional miRNAs by the cytoplasmic ribonuclease Dicer. Mature miRNAs then associate with a number of proteins to form the RNA-induced silencing complex (RISC) that bind with target mRNAs having total or partial complementary sequences to the miRNAs and initiate the inhibition of subsequent protein translation via RNA interference (RNAi).