miRNA Duplex Research Articles

Unveiling the intriguing role of nuclear microRNA-34a: A journey from discovery to functional exploration

MicroRNAs (miRNAs) are endogenous ~22 nt RNAs. miRNAs are traditionally considered primarily cytoplasmic molecules that regulate gene expression posttranscriptionally by targeting mRNAs for degradation or translational repression. In the canonical pathway of biogenesis, pri-miRNAs are transcribed from DNA sequences inside the cell nucleus. Pri-miRNAs are then processed by Drosha into pre-miRNAs and exported to the cytoplasm via the exportin 5 (XPO5)/RanGTP complex. Pre-miRNAs are then processed into mature miRNA duplexes by the RNase III endonuclease Dicer. In the intricate world of molecular biology, the discovery of mature microRNA-34a (miR-34a) inside the nucleus of atypical rat kidney epithelial cells (karyomegalic cells) has ignited scientific curiosity, revealing its multifaceted roles in cellular processes. This perspective embarks on a journey, beginning with the groundbreaking work and culminating in the recent breakthrough, shedding light on the association of miR-34a with cell proliferation and its exploration within the nuclear landscape. Other examples of mature miRNAs that have been reported to be present inside the nucleus include miR-21, miR-122, miR-223, and miR-29b.

Chemical modification patterns for microRNA therapeutic mimics: a structure-activity relationship (SAR) case-study on miR-200c.

microRNA (miRNA) mimics are an emerging class of oligonucleotide therapeutics, with a few compounds already in clinical stages. Synthetic miRNAs are able to restore downregulated levels of intrinsic miRNAs, allowing for parallel regulation of multiple genes involved in a particular disease. In this work, we examined the influence of chemical modifications patterns in miR-200c mimics, assessing the regulation of a selection of target messenger RNAs (mRNA) and, subsequently, of the whole transcriptome in A549 cells. We have probed 37 mimics and provided an initial set of instructions for designing miRNA mimics with potency and selectivity similar to an unmodified miRNA duplex. Additionally, we have examined the stability of selected mimics in serum. Finally, the selected two modification patterns were translated to two other miRNAs, miR-34a and miR-155. To differing degrees, these designs acted on targetmRNAs in a similar manner to the unmodified mimic. Here, for the first time, we describe a structured overview of 'miRNA mimics modification templates' that are chemically stabilised and optimised for use in an in vitro set up and highlight the need of further sequence specific optimization when mimics are to be used beyond in vitro tool experiments.

Rolling circle transcription: A new system to produce RNA microspheres for improving RNAi efficiency in an agriculturally important lepidopteran pest (Mythimna separate)

Rolling circle transcription: A new system to produce RNA microspheres for improving RNAi efficiency in an agriculturally important lepidopteran pest (Mythimna separate)

A combined molecular approach utilizing microbial DNA and microRNAs in a qPCR multiplex for the classification of five forensically relevant body fluids.

Body fluid identification is an essential step in the forensic biology workflow that can assist DNA analysts in determining where to collect DNA evidence. Current presumptive tests lack the specificity that molecular techniques can achieve; therefore, molecular methods, including microRNA (miRNA) and microbial signature characterization, have been extensively researched in the forensic community. Limitations of each method suggest combining molecular markers to increase the discrimination efficiency of multiple body fluids from a single assay. While microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have shown promise in fluids with low microbial abundance (blood and semen). This project synergized the benefits of microRNAs and microbial DNA to identify multiple body fluids using DNA extracts. A reverse transcription (RT)-qPCR duplex targeting miR-891a and let-7g was validated, and miR-891a differential expression was significantly different between blood and semen. The miRNA duplex was incorporated into a previously reported qPCR multiplex targeting 16S rRNA genes of Lactobacillus crispatus, Bacteroides uniformis, and Streptococcus salivarius to presumptively identify vaginal/menstrual secretions, feces, and saliva, respectively. The combined classification regression tree model resulted in the presumptive classification of five body fluids with 94.6% overall accuracy, now including blood and semen identification. These results provide proof of concept that microRNAs and microbial DNA can classify multiple body fluids simultaneously at the quantification step of the current forensic DNA workflow.

Development of Shortened miR-506-3p Mimics Exhibiting Strong Differentiation-Inducing Activity in Neuroblastoma Cells.

microRNA mimics are synthetic RNA molecules that imitate the mature miRNA duplexes and their functions. These mimics have shown promise in treating cancers. Nucleotide chemical modifications of microRNA mimics have been investigated and have improved the stability of miRNA mimics. However, the potential therapeutic benefit of mimic analogs based on sequence modifications has not been explored. miR-506-3p was identified as a differentiation-inducing microRNA in neuroblastoma cells, suggesting the potential of applying the miR-506-3p mimic in neuroblastoma differentiation therapy. In this study, we explored the possibility of developing shortened miR-506-3p analogs that can maintain differentiation-inducing activities comparable to the wild-type miR-506-3p mimic. We found that deleting up to two nucleotides at either the 3' end or within the middle region of the miR-506-3p sequence fully maintained the differentiation-inducing activity when compared to the wild-type mimic. Deleting up to four nucleotides from the 3' end or deleting three nucleotides in the middle positions diminished the differentiation-inducing activity, but the analogs still maintained differentiation-inducing activities that were significantly higher than the negative control oligo. The shortened analog designs potentially benefit patients from two perspectives: (1) the reduced cost of manufacturing shortened analogs, and (2) the reduced non-specific toxicity due to their smaller molecular sizes.

Specific Adsorption of Magnetic Fe3O4@hydroxyapatite Nanocomposites to a Hybridized Duplex for Immobilization and Label-Free Electrochemical Biosensing of MicroRNA

The unique discriminating ability of hydroxyapatite (HAP) to single- and double-stranded nucleic acid implies its potential as a biosensing material for nucleic acid analysis. Herein, a novel immobilization- and label-free electrochemical strategy has been developed for miRNA let-7a detection based on the specific adsorption of magnetic Fe3O4@hydroxyapatite (Fe3O4@HAP) to a duplex structure. Typically, the highly efficient homogeneous reaction between probe DNA (pDNA) and target miRNA let-7a produces the nucleic acid duplex, which is specifically adsorbed on the surface of Fe3O4@HAP. Thereafter, the electroactive intercalator Nile blue (NB) is introduced as the label-free signal molecule and inserts between the stacked base pairs of the pDNA–miRNA duplex. Thus, the Fe3O4@HAP bearing pDNA–miRNA and numerous intercalated NBs are adsorbed by the magnetic glassy carbon electrode, giving rise to an intense sensing signal without the need of nuclease-assisted cyclic amplification. With this advantage, the sensing system shows a wide linear range from 1.0 fM to 100 nM and a low detection limit of 0.051 fM for miRNA let-7a analysis. What is more, the synergy of the base-pairing-driven hybridization, the specific intercalation of NB to the duplex structure, and the selective adsorption of Fe3O4@HAP contribute to a high sensing specificity of the biosensing strategy. The sensing method provides a state-of-the-art strategy for the sensitive and facile bioanalysis and clinical diagnosis of miRNA.

The Human Pre-miRNA Distance Distribution for Exploring Disease Association.

MicroRNAs (miRNAs), playing an important role in cell differentiation, development, gene regulation, and apoptosis, have attracted much attention in recent years. miRNAs were shown to be involved in the mechanisms of various diseases, and certainly, they can be employed as useful disease biomarkers. The phylogenetic tree analysis of miRNA biomarkers is a useful tool to investigate the association between various diseases as well as the association between viruses and disease. In addition to the phylogenetic tree analysis, a more advanced study is to use the miRNA distance distribution to evaluate the similarity of the miRNA biomarkers. The mature miRNA distance distribution based on mature miRNA sequences has been derived. The averages of the pairwise distances of miRNA biomarkers for several associated diseases were shown to be smaller than the overall mean of all miRNAs, which indicates the high similarity of miRNA biomarkers for associated diseases. In addition to the mature miRNA, the precursor miRNA (pre-miRNA) may be more useful to explore the similarity of miRNAs because the mature miRNA duplex is released from the pre-miRNA. Therefore, in this study, the distance distributions based on human pre-miRNA stem-loop sequences were derived. The 1917 human miRNA stem-loop sequences in the miRBase dataset were used to derive the pre-miRNA distance distribution, and this is the first study to provide the distance distribution based on the human pre-miRNAs. The similarity of miRNA biomarkers for several associated diseases or vaccines was examined using the derived distribution, and the results show that the similarity of pre-miRNA biomarkers may be a feasible way to help explore the disease association.

Structural basis of microRNA biogenesis by Dicer-1 and its partner protein Loqs-PB

In animals and plants, Dicer enzymes collaborate with double-stranded RNA-binding domain (dsRBD) proteins to convert precursor-microRNAs (pre-miRNAs) into miRNA duplexes. We report six cryo-EM structures of Drosophila Dicer-1 that show how Dicer-1 and its partner Loqs‑PB cooperate (1) before binding pre-miRNA, (2) after binding and in a catalytically competent state, (3) after nicking one arm of the pre-miRNA, and (4) following complete dicing and initial product release. Our reconstructions suggest that pre-miRNA binds a rare, open conformation of the Dicer‑1⋅Loqs‑PB heterodimer. The Dicer-1 dsRBD and three Loqs‑PB dsRBDs form a tight belt around the pre-miRNA, distorting the RNA helix to place the scissile phosphodiester bonds in the RNase III active sites. Pre-miRNA cleavage shifts the dsRBDs and partially closes Dicer-1, which may promote product release. Our data suggest a model for how the Dicer‑1⋅Loqs‑PB complex affects a complete cycle of pre-miRNA recognition, stepwise endonuclease cleavage, and product release.

The molecular mechanism of microRNA duplex selectivity of Arabidopsis ARGONAUTE10.

Small RNAs (sRNAs), including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are essential gene regulators for plant and animal development. The loading of sRNA duplexes into the proper ARGONAUTE (AGO) protein is a key step to forming a functional silencing complex. In Arabidopsis thaliana, the specific loading of miR166/165 into AGO10 (AtAGO10) is critical for the maintenance of the shoot apical meristem, the source of all shoot organs, but the mechanism by which AtAGO10 distinguishes miR166/165 from other cellular miRNAs is not known. Here, we show purified AtAGO10 alone lacks loading selectivity towards miR166/165 duplexes. However, phosphate and HSP chaperone systems reshape the selectivity of AtAGO10 to its physiological substrates. A loop in the AtAGO10 central cleft is essential for recognizing specific mismatches opposite the guide strand 3′ region in miR166/165 duplexes. Replacing this loop with the equivalent loop from Homo sapiens AGO2 (HsAGO2) changes AtAGO10 miRNA loading behavior such that 3′ region mismatches are ignored and mismatches opposite the guide 5′ end instead drive loading, as in HsAGO2. Thus, this study uncovers the molecular mechanism underlying the miR166/165 selectivity of AtAGO10, essential for plant development, and provides new insights into how miRNA duplex structures are recognized for sRNA sorting.

Liquid Biopsy beyond Cancer: A miRNA Detection in Serum with Electrochemical Chip for Non‐Invasive Coeliac Disease Diagnosis

Coeliac disease is a very common autoimmune disease estimated to affect 1 in 100 people worldwide. It occurs in genetically predisposed people where the ingestion of gluten leads to damage in the small intestine, and it is accurately diagnosticated through duodenal biopsy, an invasive diagnostic method. The liquid biopsy, generally used for monitoring cancer, is an appealing alternative even for autoimmune pathology such as coeliac disease, allowing for detecting disease progression or resistance to treatment. For this reason, an electrochemical peptide nucleic acid (PNA) device combined with a smartphone‐assisted potentiostat for non‐invasive coeliac disease diagnosis is proposed, by measuring the selected overexpressed miRNA‐486‐5p in serum, enlarging the application of liquid biopsy in nontumor pathologies. For highly sensitive detection, the polyester‐based printed sensor is nanomodified with gold nanoparticles and a synthetic customized PNA probe. The designed sensor can detect the target analyte in the range of 10–100 nm with a limit of detection of 0.7 nm by measuring the variation of the response of the electrochemical mediator hexaammineruthenium in the presence of PNA–miRNA duplex on the nanostructured working electrode surface. The analyses testing serum samples are found in agreement with ones obtained by real‐time quantitative polymerase chain reaction (RT‐qPCR), demonstrating the reliability of this innovative electrochemical chip developed.

MiR-155-3p: processing by-product or rising star in immunity and cancer?

MicroRNAs (miRNAs) are key players in gene regulation that target specific mRNAs for degradation or translational repression. Each miRNA is synthesized as a miRNA duplex comprising two strands (5p and 3p). However, only one of the two strands becomes active and is selectively incorporated into the RNA-induced silencing complex in a process known as miRNA strand selection. Recently, significant progress has been made in understanding the factors and processes involved in strand selection. Here, we explore the selection and functionality of the miRNA star strand (either 5p or 3p), which is generally present in the cell at low levels compared to its partner strand and, historically, has been thought to possess no biological activity. We also highlight the concepts of miRNA arm switching and miRNA isomerism. Finally, we offer insights into the impact of aberrant strand selection on immunity and cancer. Leading us through this journey is miR-155, a well-established regulator of immunity and cancer, and the increasing evidence that its 3p strand plays a role in these arenas. Interestingly, the miR-155-5p/-3p ratio appears to vary dependent on the timing of the immune response, and the 3p strand seems to play a regulatory role upon its partner 5p strand.

RNA and Protein Determinants Mediate Differential Binding of miRNAs by a Viral Suppressor of RNA Silencing Thus Modulating Antiviral Immune Responses in Plants.

Many plant viruses express suppressor proteins (VSRs) that can inhibit RNA silencing, a central component of antiviral plant immunity. The most common activity of VSRs is the high-affinity binding of virus-derived siRNAs and thus their sequestration from the silencing process. Since siRNAs share large homologies with miRNAs, VSRs like the Tombusvirus p19 may also bind miRNAs and in this way modulate cellular gene expression at the post-transcriptional level. Interestingly, the binding affinity of p19 varies considerably between different miRNAs, and the molecular determinants affecting this property have not yet been adequately characterized. Addressing this, we analyzed the binding of p19 to the miRNAs 162 and 168, which regulate the expression of the important RNA silencing constituents Dicer-like 1 (DCL1) and Argonaute 1 (AGO1), respectively. p19 binds miRNA162 with similar high affinity as siRNA, whereas the affinity for miRNA168 is significantly lower. We show that specific molecular features, such as mismatches and ‘G–U wobbles’ on the RNA side and defined amino acid residues on the VSR side, mediate this property. Our observations highlight the remarkable adaptation of VSR binding affinities to achieve differential effects on host miRNA activities. Moreover, they show that even minimal changes, i.e., a single base pair in a miRNA duplex, can have significant effects on the efficiency of the plant antiviral immune response.

Artificial microRNA guide strand selection from duplexes with no mismatches shows a purine-rich preference for virus- and non-virus-based expression vectors in plants.

SummaryArtificial microRNA (amiRNA) technology has allowed researchers to direct efficient silencing of specific transcripts using as few as 21 nucleotides (nt). However, not all the artificially designed amiRNA constructs result in selection of the intended ~21‐nt guide strand amiRNA. Selection of the miRNA guide strand from the mature miRNA duplex has been studied in detail in human and insect systems, but not so much for plants. Here, we compared a nuclear‐replicating DNA viral vector (tomato mottle virus, ToMoV, based), a cytoplasmic‐replicating RNA viral vector (tobacco mosaic virus, TMV, based), and a non‐viral binary vector to express amiRNAs in plants. We then used deep sequencing and mutational analysis and show that when the structural factors caused by base mismatches in the mature amiRNA duplex were excluded, the nucleotide composition of the mature amiRNA region determined the guide strand selection. We found that the strand with excess purines was preferentially selected as the guide strand and the artificial miRNAs that had no mismatches in the amiRNA duplex were predominantly loaded into AGO2 instead of loading into AGO1 like the majority of the plant endogenous miRNAs. By performing assays for target effects, we also showed that only when the intended strand was selected as the guide strand and showed AGO loading, the amiRNA could provide the expected RNAi effects. Thus, by removing mismatches in the mature amiRNA duplex and designing the intended guide strand to contain excess purines provide better control of the guide strand selection of amiRNAs for functional RNAi effects.

Regulation of Oncogenic Targets by Tumor-Suppressive miR-150-3p in Lung Squamous Cell Carcinoma.

Several recent studies have shown that both strands of certain miRNAs derived from miRNA duplexes are involved in cancer pathogenesis. Our own recent studies revealed that both strands of the miR-150 duplex act as tumor-suppressive miRNAs in lung adenocarcinoma (LUAD) through the targeting of several oncogenes. The aim of the study here was to further investigate the tumor-suppressive roles of miR-150-3p (the passenger strand) in lung squamous cell carcinoma (LUSQ) and its control of cancer-promoting genes in LUSQ cells. The downregulation of miR-150-3p in LUSQ tissues was confirmed by data in The Cancer Genome Atlas (TCGA). The ectopic expression of miR-150-3p attenuated cancer cell aggressive features, e.g., cell cycle arrest, migration and invasive abilities. Our target search strategy successfully identified a total of 49 putative targets that were listed as subjects of miR-150-3p regulation in LUSQ cells. Interestingly, among these targets, 17 genes were categorized as related to the “cell cycle” based on Gene Ontology (GO) classification, namely CENPA, CIT, CCNE1, CCNE2, TIMELESS, BUB1, MCM4, HELLS, SKA3, CDCA2, FANCD2, NUF2, E2F2, SUV39H2, CASC5, ZWILCH and CKAP2). Moreover, we show that the expression of HELLS (helicase, lymphoid specific) is directly controlled by miR-150-3p, and its expression promotes the malignant phenotype of LUSQ cells.

Controlled RISC loading efficiency of miR168 defined by miRNA duplex structure adjusts ARGONAUTE1 homeostasis.

Micro RNAs (miRNAs) are processed from precursor RNA molecules with precisely defined secondary stem-loop structures. ARGONAUTE1 (AGO1) is the main executor component of miRNA pathway and its expression is controlled via the auto-regulatory feedback loop activity of miR168 in plants. Previously we have shown that AGO1 loading of miR168 is strongly restricted leading to abundant cytoplasmic accumulation of AGO-unbound miR168. Here, we report, that intrinsic RNA secondary structure of MIR168a precursor not only defines the processing of miR168, but also precisely adjusts AGO1 loading efficiency determining the biologically active subset of miR168 pool. Our results show, that modification of miRNA duplex structure of MIR168a precursor fragment or expression from artificial precursors can alter the finely adjusted loading efficiency of miR168. In dcl1-9 mutant where, except for miR168, production of most miRNAs is severely reduced this mechanism ensures the elimination of unloaded AGO1 proteins via enhanced AGO1 loading of miR168. Based on this data, we propose a new competitive loading mechanism model for miR168 action: the miR168 surplus functions as a molecular buffer for controlled AGO1 loading continuously adjusting the amount of AGO1 protein in accordance with the changing size of the cellular miRNA pool.

Vorinostat triggers miR-769-5p/3p-mediated suppression of proliferation and induces apoptosis via the STAT3-IGF1R-HDAC3 complex in human gastric cancer.

Previous reports have shown that histone deacetylase inhibitors (HDACi) can alter miRNA expression in a range of cancers. Both the 5p-arm and 3p-arm of mature miRNAs can be expressed from the same precursor and involved in cancer progress. Nevertheless, the detailed mechanism by which vorinostat (SAHA), a HDACi, triggers miR-769-5p/miR-769-3p-mediated suppression of proliferation and induces apoptosis in gastric cancer (GC) cells remains elusive. Here, we showed that the miRNA-seq analysis of GC cells treated with SAHA identified seven differentially expressed miRNAs with both strands of the miRNA duplex. miR-769-5p/miR-769-3p expression was downregulated in GC tissues compared with normal tissues. Functionally, high expression of miR-769-5p/miR-769-3p blocked the malignant abilities of GC cells. Mechanistically, miR-769-5p/miR-769-3p targeted IGF1R and IGF1R overexpression rescued the effects of miR-769-5p/miR-769-3p on GC cells growth and metastasis. Moreover, STAT3 bound to the promoter of miR-769. Furthermore, miR-769-5p/miR-769-3p expression was negatively regulated by the STAT3-IGF1R-HDAC3 complex. Besides, miR-769-5p/miR-769-3p synergized with SAHA to promote GC cells apoptosis. Our studies suggest that miR-769-5p/miR-769-3p acts as a tumor suppressor by the STAT3-IGF1R-HDAC3 complex. Moreover, SAHA triggers miR-769-5p/miR-769-3p-mediated inhibition of proliferation and induces apoptosis in GC cells.

Modulation of MicroRNA Processing by Dicer via Its Associated dsRNA Binding Proteins.

MicroRNAs (miRNAs) are small non-coding RNAs that are about 22 nucleotides in length. They regulate gene expression post-transcriptionally by guiding the effector protein Argonaute to its target mRNA in a sequence-dependent manner, causing the translational repression and destabilization of the target mRNAs. Both Drosha and Dicer, members of the RNase III family proteins, are essential components in the canonical miRNA biogenesis pathway. miRNA is transcribed into primary-miRNA (pri-miRNA) from genomic DNA. Drosha then cleaves the flanking regions of pri-miRNA into precursor-miRNA (pre-miRNA), while Dicer cleaves the loop region of the pre-miRNA to form a miRNA duplex. Although the role of Drosha and Dicer in miRNA maturation is well known, the modulation processes that are important for regulating the downstream gene network are not fully understood. In this review, we summarized and discussed current reports on miRNA biogenesis caused by Drosha and Dicer. We also discussed the modulation mechanisms regulated by double-stranded RNA binding proteins (dsRBPs) and the function and substrate specificity of dsRBPs, including the TAR RNA binding protein (TRBP) and the adenosine deaminase acting on RNA (ADAR).

Regulation of MicroRNAs.

MicroRNAs are RNAs of about 18-24 nucleotides in lengths, which are found in the small noncoding RNA class and have a crucial role in the posttranscriptional regulation of gene expression, cellular metabolic pathways, and developmental events. These small but essential molecules are first processed by Drosha and DGCR8 in the nucleus and then released into the cytoplasm, where they cleaved by Dicer to form the miRNA duplex. These duplexes are bound by the Argonaute (AGO) protein to form the RNA-induced silencing complex (RISC) in a process called RISC loading. Transcription of miRNAs, processing with Drosha and DGCR8 in the nucleus, cleavage by Dicer, binding to AGO proteins and forming RISC are the most critical steps in miRNA biogenesis. Additional molecules involved in biogenesis at these stages can enhance or inhibit these processes, which can radically change the fate of the cell. Biogenesis is regulated by many checkpoints at every step, primarily at the transcriptional level, in the nucleus, cytoplasm, with RNA regulation, RISC loading, miRNA strand selection, RNA methylation/uridylation, and turnover rate. Moreover, in recent years, different regulation mechanisms have been discovered in noncanonical Drosha or Dicer-independent pathways. This chapter seeks answers to how miRNA biogenesis and function are regulated through both canonical and non-canonical pathways.

Developing miR‐506‐3p Mimics with Enhanced Differentiation‐Inducing Activity

Neuroblastoma is one of the most prominent childhood cancers that results from the failure of neural crest cell differentiation. Inducing cell differentiation is a vital therapeutic strategy for neuroblastoma. MicroRNAs (miRNAs) are a class of endogenously expressed RNAs that regulate expression of target genes by complementary binding to the 3’ untranslated region (3’UTR) of mRNA. Previous studies have shown the miRNA-506-3p seed family to be a strong inducer of neuroblastoma cell differentiation. Differentiation-inducing miRNA mimics, synthetic RNA molecules that imitate the mature miRNA duplexes and their functions, are potential therapeutic agents for neuroblastoma differentiation therapy. In the effort to develop miR-506-3p-based differentiation agents, we identified mutant miRNA-506-3p mimics that potentially have enhanced differentiation-inducing activity based on the predicted interaction of the mutant miR-506-3p with its key differentiation-regulating targets, STAT3 and CDK4, suggesting these mutant mimics may have enhanced therapeutic potential compared to wild type (WT) miR-506-3p mimic. The objectives of my project are: 1) to compare the effect of these mutant mimics on neuroblastoma cell differentiation to that of WT mimic by measuring neurite outgrowth, a morphological differentiation marker, in neuroblastoma cells, and 2) to compare the effect of these mutants on cell growth arrest to that of WT mimic using MTT cell viability assay. This study will help to determine whether the mutant miR-506-3p mimics have higher therapeutic potential to treat neuroblastoma and widen efforts to develop more targeted therapies for high-risk patients.

MicroRNA strand selection: Unwinding the rules.

microRNAs (miRNAs) play a central role in the regulation of gene expression by targeting specific mRNAs for degradation or translational repression. Each miRNA is post‐transcriptionally processed into a duplex comprising two strands. One of the two miRNA strands is selectively loaded into an Argonaute protein to form the miRNA‐Induced Silencing Complex (miRISC) in a process referred to as miRNA strand selection. The other strand is ejected from the complex and is subject to degradation. The target gene specificity of miRISC is determined by sequence complementarity between the Argonaute‐loaded miRNA strand and target mRNA. Each strand of the miRNA duplex has the capacity to be loaded into miRISC and possesses a unique seed sequence. Therefore, miRNA strand selection plays a defining role in dictating the specificity of miRISC toward its targets and provides a mechanism to alter gene expression in a switch‐like fashion. Aberrant strand selection can lead to altered gene regulation by miRISC and is observed in several human diseases including cancer. Previous and emerging data shape the rules governing miRNA strand selection and shed light on how these rules can be circumvented in various physiological and pathological contexts.This article is categorized under:RNA Processing > Processing of Small RNAsRegulatory RNAs/RNAi/Riboswitches > Regulatory RNAsRegulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs