RNA-induced Silencing Complex Research Articles

Telomerase, an Accomplice of RNA-induced Silencing Complex in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most lethal and widespread cancers in the human race. Despite its fatal attributes, there is only a limited understanding of the factors contributing to its pathogenesis at the cellular and molecular levels. Consequently, unraveling new facets involved in HCC progression is elemental for establishing novel targets and biomarkers for this disease. Over the last few years, emerging evidence signifies the role of RNA-induced silencing complex in mediating gene silencing and contributing to HCC. Recent studies also highlight the importance of human telomerase holoenzyme and its complex accessory proteins in the development of HCC. The current review encompasses the multifaceted roles of RNA-induced silencing complex and telomerase activity as well as their synergistic function in HCC.

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.

Metabolically Stable Anomeric Linkages Containing GalNAc-siRNA Conjugates: An Interplay among ASGPR, Glycosidase, and RISC Pathways.

Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural β-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and β-anomeric linkages, N-glycosides with β-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.

A conserved SNP variation in the pre-miR396c flanking region in Oryza sativa indica landraces correlates with mature miRNA abundance

Plant precursor miRNAs (pre-miRNA) have conserved evolutionary footprints that correlate with mode of miRNA biogenesis. In plants, base to loop and loop to base modes of biogenesis have been reported. Conserved structural element(s) in pre-miRNA play a major role in turn over and abundance of mature miRNA. Pre-miR396c sequences and secondary structural characteristics across Oryza species are presented. Based on secondary structure, twelve Oryza pre-miR396c sequences are divided into three groups, with the precursor from halophytic Oryza coarctata forming a distinct group. The miRNA-miRNA* duplex region is completely conserved across eleven Oryza species as are other structural elements in the pre-miRNA, suggestive of an evolutionarily conserved base-to-loop mode of miRNA biogenesis. SNPs within O. coarctata mature miR396c sequence and miRNA* region have the potential to alter target specificity and association with the RNA-induced silencing complex. A conserved SNP variation, rs10234287911 (G/A), identified in O. sativa pre-miR396c sequences alters base pairing above the miRNA-miRNA* duplex. The more stable structure conferred by the ‘A10234287911’ allele may promote better processing vis-à-vis the structure conferred by ‘G10234287911’ allele. We also examine pri- and pre-miR396c expression in cultivated rice under heat and salinity and their correlation with miR396c expression.

MiRNA accessibility in cellular RISC revealed by single molecule kinetic fingerprinting.

MicroRNAs (miRNAs) in association with Argonaute (Ago) proteins in the RNA induced silencing complex (RISC) regulate the levels of translation of messenger RNAs (mRNAs). The major parameter thought to explain the selection of the target mRNA is the base pairing between the ‘5’ seed’ region of the miRNA and its counterpart mRNA. Ago2 crystalizes in a conformation where the 5’ seed region is exposed to the target mRNA. However, both 5’ seed and 3’ non-seed-based target recognition has been reported. In fact, thousands of miRNA target sites depend on a non-seed pairing interaction, highlighting a gap in our understanding of miRNA targeting rules. Here, we present a ‘single molecule kinetic fingerprinting’ approach using split miRNA complementary probes (i.e., target mimics) that trace the differential accessibility of the 5’ seed and 3’ non-seed segments of the miRNA in RISC immunoprecipitated from human HeLa cell extract. Two distinct populations are observed with either the 5’ seed or the 3’ non-seed segment accessible. Only a minor population (∼5-10%) shows simultaneous accessibility of both segments. Compared to the protein-free miRNA, the target interaction kinetics of the seed and non-seed segments of the Ago2-bound miRNA are differentially controlled by the surrounding protein. In particular, tight target binding to the 5’ seed region alters the kinetics of the 3’ non-seed interaction, resulting in kinetic heterogeneity in that interaction with the target mimic. This kinetic heterogeneity most likely arises from differential accessibility of the 3’ non-seed region of miRNA in Ago2, which adopts multiple conformations. In summary, our data indicate that both 5’ seed and 3’ non-seed-based target recognition is possible, but the accessibility and the kinetics of mRNA interactions is largely governed by the surrounding protein conformation as well as the guide-target complementarity.

Molecular modeling of PARP1 inhibition through miRNA gene silencing for the treatment of retinitis pigmentosa.

Retinitis Pigmentosa (RP) is a category of diseases that are characterized by the death of rod photoreceptors and subsequent death of cone receptors. Parthanatos, a form of regulated necrosis, has been reported as being the underlying mechanism of cell death in RP. Parthanatos in RP occurs with the downstream expression of poly-ADP-ribose-polymerase (PARP) and the subsequent nuclear translocation of the mitochondrial apoptosis-inducing factor (AIF), leading to condensing of chromatin and DNA fragmentation. Extracellular vesicles (EVs) are small-scale liposomes that carry miRNAs and the RNA-induced silencing complex (RISC) associated with gene regulation. miRNAs, loaded on the argonaute protein 2 (Ago-2) bind to the mature mRNA and initiate the recruitment of the RISC complex triggering the degradation of the mRNA, dramatically scaling down gene expression. In vitro validation of gene silencing is a time expensive process and computational resources provide faster and cheaper options. Here, we identified the miRNAs target sequences on the mature PARP1 mRNA construct from the TargetScan database and obtained their complementary miRNA sequences from the miRbase. Using the human Ago-2 crystal structure deposited to the PDB database (PDB ID: 4W5Q) as a template, mutational studies were conducted on the protein/mRNA/miRNA complex to reproduce PARP1 mRNA seed pairing to miRNAs. Upon initial geometry optimization, further conformational search studies were done for thorough exploration of the conformational space of the protein/RNA complex by implementing conformational flooding (CF) molecular dynamics (MD) simulations. CF MD introduces a flooding potential to the force field, thus lowering energy barriers and facilitating observations of macroscale conformational changes. Clustering of the CF trajectories reveal the stable conformations of the Ago-2/miRNA/mRNA complexes. Energetically favorable miRNAs discovered in silico will further be validated with in vitro silencing assays.

LncRNA MEG3 inhibits renal fibrinoid necrosis of diabetic nephropathy via the MEG3/miR-21/ORAI1 axis.

Diabetic nephropathy (DN) is one of the most common and lethal diabetic complications worldwide and is associated with a high risk of mortality. However, the exact mechanism behind its development is unknown. The mesangial cells (MCs) and non-coding RNAs are critical for DN, but it is unknown whether a MEG3/miR-21/ORAI1 regulatory axis exists in MCs. Hence, in this study, we aimed to understand whether the MEG3/miR-21/ORAI1 regulatory axis has a role in the pathophysiology of DN. We demonstrated that high-glucose stimuli downregulated MEG3 and ORAI1 expression while enhancing miR-21 expression. Exogenous miR-21 mimics inhibited ORAI1 expression, which was partially salvaged or reversed by MEG3 overexpression. Furthermore, RIP assay demonstrated that the beads labeled with AGO2 antibody could enrich more miR-21 and MEG3 than those labeled with control IgG antibody; both of them formed the RNA-induced silencing complex. Further, the biochemical indicators of db/db mice significantly improved, and renal fibrinoid necrosis was ameliorated using a miR-21 inhibitor. The MEG3/miR-21/ORAI1 axis regulates the manifestation of DN in diabetic mice and MCs, and the miR-21 inhibitor can be a potential therapeutic strategy to alleviate DN, once the presence of such an axis is found in humans.

Genome-Wide Identification and Posttranscriptional Regulation Analyses Elucidate Roles of Key Argonautes and Their miRNA Triggers in Regulating Complex Yield Traits in Rapeseed.

Argonautes (AGOs) interact with microRNAs (miRNAs) to form the RNA-induced silencing complex (RISC), which can posttranscriptionally regulate the expression of targeted genes. To date, however, the AGOs and their miRNA triggers remain elusive in rapeseed (Brassica napus). Here, we systematically performed a phylogenetic analysis and examined the collinear relationships of the AGOs among four Brassicaceae species. Their physicochemical properties, gene structures, and expression patterns among 81 tissues from multiple materials and developmental stages were further analyzed. Additionally, their posttranscriptional regulation was analyzed using psRNATarget prediction, miRNA-/mRNA-Seq analyses, and a qRT-PCR verification. We finally identified 10 AtAGOs, 13 BolAGOs, 11 BraAGOs, and 24 BnaAGOs. An expression analysis of the BnaAGOs in the B. napus cultivar ZS11, as well as genotypes with extreme phenotypes in various yield-related traits, revealed the conservation and diversity of these genes. Furthermore, we speculated the posttranscriptional regulation of the B. napus miR168a-AGO1s and miR403-AGO2s modules. Combining miRNA-Seq and mRNA-Seq analyses, we found that the B. napus miR168a-AGO1s module may play an essential role in negatively regulating yield traits, whereas the miR403-AGO2s module positively impacts yield. This is the first attempt to comprehensively analyze the AGOs and their miRNA triggers in B. napus and provides a theoretical basis for breeding high-yielding varieties through the manipulation of the miRNA-AGOs modules.

Suppression of Phytophthora capsici using double-stranded RNAs targeting NLP effector genes in Nicotiana benthamiana

RNA interference (RNAi) is a gene regulatory mechanism that involves the interaction of small interfering RNAs (siRNAs) and RNA-induced silencing complex (RISC). Dicer cleaves exogenous double-stranded RNA (dsRNA) into siRNAs, which get incorporated into RISC and bind to complementary sequences on the target mRNA to induce its degradation. In this study, we adopted RNAi technology using dsRNAs to suppress Phytophthora capsici, which causes diseases in solanaceous crops, including pepper. We designed and synthesized dsRNAs targeting the P. capsici effector genes PcNLP2 and PcNLP6, respectively. These genes encode necrosis and ethylene-inducing peptide 1-like proteins in P. capsici, which are known to promote oomycete infection. Nicotiana benthamiana leaves were first infiltrated with dsRNAs and inoculated with P. capsici 2 days later. We confirmed significant suppression of P. capsici and PcNLP2, PcNLP6 expression in dsRNA-treated leaves. In addition, we found that downregulation of PcNLP2 and PcNLP6 distinctly affected the expression of some defense-related genes. These results suggest that dsRNA mediated RNAi technology can be used to suppress various pathogens, and may contribute toward crop protection.

An OsPRMT5-OsAGO2/miR1875-OsHXK1 module regulates rice immunity to blast disease.

Rice ARGONAUTE2 (OsAGO2) is a core component of the rice RNA-induced silencing complex (RISC), which is repressed by Magnaporthe oryzae (M. oryzae) infection. Whether and how OsAGO2-mediated gene silencing plays a role in rice blast resistance and which sRNAs participate in this process are unknown. Our results indicate that OsAGO2 is a key immune player that manipulates rice defense responses against blast disease. OsAGO2 associates with the 24-nt miR1875 and binds to the promoter region of HEXOKINASE1 (OsHXK1), which causes DNA methylation and leads to gene silencing. Our multiple genetic evidence showed that, without M. oryzae infection, OsAGO2/miR1875 RISC promoted OsHXK1 promoter DNA methylation and OsHXK1 silencing; after M. oryzae infection, the reduced OsAGO2/miR1875 led to a relatively activated OsHXK1 expression. OsHXK1 acts as a positive regulator of blast disease resistance that OsHXK1-OE rice exhibited enhanced resistance, whereas Cas9-Oshxk1 rice showed reduced resistance against M. oryzae infection. OsHXK1 may function through its sugar sensor activity as glucose induced defense-related gene expression and reactive oxygen species (ROS) accumulation in Nipponbare and OsHXK1-OE but not in Cas9-Oshxk1 rice. OsAGO2 itself is delicately regulated by OsPRMT5, which senses M. oryzae infection and attenuates OsAGO2-mediated gene silencing through OsAGO2 arginine methylation. Our study reveals an OsPRMT5-OsAGO2/miR1875-OsHXK1 regulatory module that fine tunes the rice defense response to blast disease.

CD95/Fas ligand induced toxicity.

The role of CD95/Fas ligand (CD95L/FasL) in the induction of CD95-mediated extrinsic apoptosis is well characterized. Trimerized, membrane-bound CD95L ligates the CD95 receptor activating downstream signaling resulting in the execution of cells by caspase proteins. However, the expression of CD95L has been reported to induce cell death in contexts in which this pathway is unlikely to be activated, such as in cell autonomous activation induced cell death (AICD) and in CD95-resistant cancer cell lines. Recent data suggests that the CD95L mRNA exerts toxicity through death induced by survival gene elimination (DISE). DISE results from the targeting of networks of survival genes by toxic short RNA (sRNA)s in the RNA-induced silencing complex (RISC). CD95L mRNA contributes to this death directly, through the processing of its mRNA into toxic sRNAs that are loaded into the RISC, and indirectly, by promoting the loading of other toxic sRNAs. Interestingly, CD95L is not the only mRNA that is processed and loaded into the RISC. Protein-coding mRNAs involved in protein translation are also selectively loaded. We propose a model in which networks of mRNA-derived sRNAs modulate DISE, with networks of genes providing non-toxic RISC substrate sRNAs that protect against DISE, and opposing networks of stress-activated genes that produce toxic RISC substrate sRNAs that promote DISE.

Androgen receptor suppresses lung cancer invasion and increases cisplatin response via decreasing TPD52 expression.

Lung cancer, as the most commonly diagnosed malignancy, still accounts for the leading cause of cancer-related deaths worldwide. The high rate of mortality and tumor recurrence has prompted clinicians and scientists to urgently explore new targets for improved treatment. Previous studies have indicated a potential role of the androgen receptor (AR) in the progression of non-small cell lung cancer (NSCLC). However, the precise mechanisms underlying this association, particularly its relation to TPD52-mediated cell invasion and cisplatin (DDP) response, have not been fully elucidated. Therefore, further investigation is necessary to gain a better understanding of these mechanisms and their potential implications for lung cancer treatment. In this study, we discovered that AR can suppress NSCLC cell invasion and increase cisplatin response by downregulating the expression of circular RNA (circRNA), specifically circ-SLCO1B7. This suppression is achieved through the direct binding of AR to the 5' promoter region of the host gene SLCO1B7. The decreased expression of circ-SLCO1B7, mediated by AR, released miR-139-5p back to the RISC (RNA induced silencing complex), where it bonds to the 3' untranslated region (3'UTR) of Tumor Protein D52 (TPD52) messenger RNA, resulting in TPD52 reduction. The in vivo data also validated the functional contribution of AR/circ-SLCO1B7/miR-139-5p/TPD52 axis to lung cancer progression. Furthermore, analysis of human NSCLC databases and clinical specimens confirmed the association of the AR/circ-SLCO1B7/miR-139-5p/TPD52 signaling pathway with NSCLC progression. Collectively, the results from our study suggest that AR can suppress lung cancer cell invasion and increase DDP response by modulating the circ-SLCO1B7/miR-139-5p/TPD52 signaling pathway. Targeting this novel signaling pathway may be a new therapeutic strategy to effectively constrain NSCLC development.

Determination of mRNA Targets of miR-376c within the RNA-Induced Silencing Complex

Determination of mRNA Targets of miR-376c within the RNA-Induced Silencing Complex

Rational optimization of siRNA to ensure strand bias in the interaction with the RNA-induced silencing complex.

To ensure specificity of small interfering RNAs (siRNAs), the antisense strand must be selected by the RNA-induced silencing complex (RISC). We have previously demonstrated that a 5'-morpholino-modified nucleotide at the 5'-end of the sense strand inhibits its interaction with RISC ensuring selection of the desired antisense strand. To improve this antagonizing binding property even further, a new set of morpholino-based analogues, Mo2 and Mo3, and a piperidine analogue, Pip, were designed based on the known structure of Argonaute2, the slicer enzyme component of RISC. Sense strands of siRNAs were modified with these new analogues, and the siRNAs were evaluated in vitro and in mice for RNAi activity. Our data demonstrated that Mo2 is the best RISC inhibitor among the modifications tested and that it effectively mitigates sense strand-based off-target activity of siRNA.

AraC-induced neuron-like differentiation of human NTERA2/D1 cells and quantification of endogenous pre-mir-106b and 19b levels.

MicroRNAs (miRNAs) are approximately 22 nucleotide-long non-coding RNAs that are encoded in the genome. miRNAs form base pairs with target mRNAs in the RNA-induced silencing complex and repress their expression through a mechanism called RNA silencing. Expression profiles of miRNAs differ between cells and tissues. In this study, we performed cytosine β-D-arabinofuranoside (AraC)-induced neuron-like differentiation of human NTERA2/D1 (NT2) cells and quantified endogenous miRNA levels using quantitative RT-PCR. In conclusion, pre-mir-106b and pre-mir-19b levels were decreased after AraC-induced neuron-like differentiation of NT2 cells, indicating the functional relevance of miRNAs in the differentiation of mammalian cells.

Interaction Between Argonaute2 and RNA Molecules: AGO2 Molecular Structure and Different Regions in Nucleotide Chain

Argonaute2 (AGO2) is an important protein connecting the construction of RNA-induced silencing complex (RISC) and micro-RNA (miRNA) biogenesis. This paper explores the mechanism during the function through previous studies which reveal the crystal structure and affinity comparisons. It is concluded that the AGO2 have compartmentalized domains for certain functions, which have both individual and cooperative role in the entire process. The characters of AGO2 and miRNA suggest a model of regiondifferent nucleotide chains in miRNA, which means its epigenetic information is based on base sequence and its special information. According to these findings, further studies are advised to monitor the reactions in a dynamical method, which would be a new potential entry point for clinical utilization.

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

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

RNA interference: Promising approach for breast cancer diagnosis and treatment.

Today, cancer is one of the main health-related challenges, and in the meantime, breast cancer (BC) is one of the most common cancers among women, with an alarming number of incidences and deaths every year. For this reason, the discovery of novel and more effective approaches for the diagnosis, treatment, and monitoring of the disease are very important. In this regard, scientists are looking for diagnostic molecules to achieve the above-mentioned goals with higher accuracy and specificity. RNA interference(RNAi)is a posttranslational regulatory process mediated by microRNAintervention and small interfering RNAs. After transcription and edition, these two noncoding RNAs are integrated and activated with the RNA-induced silencing complex (RISC) and AGO2 to connect the target mRNA by their complementary sequence and suppress their translation, thus reducing the expression of their target genes. These two RNAi categories show different patterns in different BC types and stages compared to healthy cells, andhence, these molecules have high diagnostic, monitoring, and therapeutic potentials. This article aims to review the RNAi pathway and diagnostic and therapeutic potentials with a special focus on BC.

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.

RNA:RNA interaction in ternary complexes resolved by chemical probing.

RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.