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MicroRNAs have recently emerged as key posttranscriptional regulators of eukaryotic gene expression, yet our understanding of how microRNA expression is itself controlled has remained rudimentary. This review describes recent insights into the mechanisms governing microRNA transcription and processing in vertebrates and their implications for understanding the regulation of microRNA biogenesis.
In this article, we show that a subset of conventional mammalian microRNAs is derived from LINE-2 transposable elements and other genome repeats. These repeat-derived microRNAs arise from conventional precursor hairpins and are distinct from the rasiRNAs, which appear to be processed from long double-stranded RNA precursors. The insertion of transposable elements into new genomic sites appears to be one of the driving-forces that create new microRNAs during mammalian evolution. Two of the LINE-2-derived microRNAs exhibit perfect complementarity to a large family of mRNA and EST transcripts that contain portions of MIR and other LINE-2 elements in their 3'-untranslated regions.
Recently, we reported that four microRNAs show perfect complementarity with MIR/LINE-2 elements within human mRNAs. This finding raises the question of whether microRNAs might also target other genomic repeats and transposable elements. Here, we demonstrate that almost 30 human microRNAs exhibit typical short-seed complementarity with a specific site within Alu elements that is highly conserved within 3' untranslated regions of human mRNAs. The results suggest that at least some Alu elements within human mRNAs serve as microRNA targets.
MicroRNAs are an abundant class of small RNAs that are thought to regulate the expression of protein-coding genes in plants and animals. Here we show that the target sequence of two microRNAs, known to regulate genes in the class-III homeodomain-leucine zipper (HD-Zip) gene family of the flowering plant Arabidopsis, is conserved in homologous sequences from all lineages of land plants, including bryophytes, lycopods, ferns and seed plants. We also find that the messenger RNAs from these genes are cleaved within the same microRNA-binding site in representatives of each land-plant group, as they are in Arabidopsis. Our results indicate not only that microRNAs mediate gene regulation in non-flowering as well as flowering plants, but also that the regulation of this class of plant genes dates back more than 400 million years.
To date, more than 200 microRNAs have been described in humans; however, the precise functions of these regulatory, non-coding RNAs remains largely obscure. One cluster of microRNAs, the mir-17-92 polycistron, is located in a region of DNA that is amplified in human B-cell lymphomas. Here we compared B-cell lymphoma samples and cell lines to normal tissues, and found that the levels of the primary or mature microRNAs derived from the mir-17-92 locus are often substantially increased in these cancers. Enforced expression of the mir-17-92 cluster acted with c-myc expression to accelerate tumour development in a mouse B-cell lymphoma model. Tumours derived from haematopoietic stem cells expressing a subset of the mir-17-92 cluster and c-myc could be distinguished by an absence of apoptosis that was otherwise prevalent in c-myc-induced lymphomas. Together, these studies indicate that non-coding RNAs, specifically microRNAs, can modulate tumour formation, and implicate the mir-17-92 cluster as a potential human oncogene.
MicroRNAs are small, non-coding RNAs that control the translation of target messenger RNAs, thereby regulating critical aspects of plant and animal development. In the mammalian nervous system, the spatiotemporal control of mRNA translation has an important role in synaptic development and plasticity. Although a number of microRNAs have been isolated from the mammalian brain, neither the specific microRNAs that regulate synapse function nor their target mRNAs have been identified. Here we show that a brain-specific microRNA, miR-134, is localized to the synapto-dendritic compartment of rat hippocampal neurons and negatively regulates the size of dendritic spines--postsynaptic sites of excitatory synaptic transmission. This effect is mediated by miR-134 inhibition of the translation of an mRNA encoding a protein kinase, Limk1, that controls spine development. Exposure of neurons to extracellular stimuli such as brain-derived neurotrophic factor relieves miR-134 inhibition of Limk1 translation and in this way may contribute to synaptic development, maturation and/or plasticity.
In animals, the majority of microRNAs regulate gene expression through the RNA interference (RNAi) machinery without inducing small-interfering RNA (siRNA)-directed mRNA cleavage. Thus, the mechanisms by which microRNAs repress their targets have remained elusive. Recently, Argonaute proteins, which are key RNAi effector components, and their target mRNAs were shown to localize to cytoplasmic foci known as P-bodies or GW-bodies. Here, we show that the Argonaute proteins physically interact with a key P-/GW-body subunit, GW182. Silencing of GW182 delocalizes resident P-/GW-body proteins and impairs the silencing of microRNA reporters. Moreover, mutations that prevent Argonaute proteins from localizing in P-/GW-bodies prevent translational repression of mRNAs even when Argonaute is tethered to its target in a siRNA-independent fashion. Thus, our results support a functional link between cytoplasmic P-bodies and the ability of a microRNA to repress expression of a target mRNA.
Chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults in the Western world, is characterized by predominantly nondividing malignant CD5+ B cells overexpressing the anti-apoptotic Bcl2 protein. Significant familial aggregation with largely unknown mode of inheritance has been demonstrated. Until recently little else was known regarding the events leading to CLL initiation and progression. New findings support the view that CLL is a genetic disease where the main alterations occur at the level of transcriptional/post-transcriptional regulation of the malignant cells genome because of deregulations of a new class of genes named microRNAs (miRNAs). miRNA genes miR-15a and miR-16-1, located at 13q14.3, are frequently deleted and/or downregulated in patients with B-cell CLL. Both microRNAs negatively regulate Bcl2 at a post-transcriptional level and this repression is enough to induce apoptosis. Therefore, miR-15 and miR-16 are natural antisense Bcl2 interactors that could be used for therapy of Bcl2-overexpressing tumors. Furthermore, microRNA expression profiles can distinguish normal B cells from malignant B cells in CLL. A unique microRNA signature is associated with prognostic factors such as mutations in the immunoglobulin heavy-chain variable-region gene (IgV(H)) or high expression of the 70-kd zeta-associated protein (ZAP-70+) and disease progression in CLL. Mutations in miRNA transcripts are frequent, some of them germ-line, and may have functional importance and may predispose to CLL and to a spectrum of associated malignancies.
Three muscle-specific microRNAs, miR-206, -1, and -133, are induced during differentiation of C2C12 myoblasts in vitro. Transfection of miR-206 promotes differentiation despite the presence of serum, whereas inhibition of the microRNA by antisense oligonucleotide inhibits cell cycle withdrawal and differentiation, which are normally induced by serum deprivation. Among the many mRNAs that are down-regulated by miR-206, the p180 subunit of DNA polymerase alpha and three other genes are shown to be direct targets. Down-regulation of the polymerase inhibits DNA synthesis, an important component of the differentiation program. The direct targets are decreased by mRNA cleavage that is dependent on predicted microRNA target sites. Unlike small interfering RNA-directed cleavage, however, the 5' ends of the cleavage fragments are distributed and not confined to the target sites, suggesting involvement of exonucleases in the degradation process. In addition, inhibitors of myogenic transcription factors, Id1-3 and MyoR, are decreased upon miR-206 introduction, suggesting the presence of additional mechanisms by which microRNAs enforce the differentiation program.
Here we describe an episomal, one-vector system which allows the generation of cell populations displaying homogenous, inducible gene inactivation by RNA interference in a one step procedure. A dual tet-repressor/activator system tightly controls a bi-directional promoter, which simultaneously drives expression of microRNAs and a fluorescent marker protein. We demonstrate the effectiveness of this vector by knockdown of p53 expression in a human cell line which resulted in the expected loss of G1-arrest after DNA damage. The generation of a cell pool homogenously expressing the ectopic microRNAs was achieved in 1 week without the need for viral infections. Induction of microRNA expression did not elicit an interferon response. Furthermore, the vector was adapted for convenient ligation-free transfer of microRNA cassettes from public libraries. This conditional knockdown-system should prove useful for many research and gene therapeutic applications.
In eukaryotes, 21- to 24-nucleotide-long RNAs engage in sequence-specific interactions that inhibit gene expression by RNA silencing. This process has regulatory roles involving microRNAs and, in plants and insects, it also forms the basis of a defense mechanism directed by small interfering RNAs that derive from replicative or integrated viral genomes. We show that a cellular microRNA effectively restricts the accumulation of the retrovirus primate foamy virus type 1 (PFV-1) in human cells. PFV-1 also encodes a protein, Tas, that suppresses microRNA-directed functions in mammalian cells and displays cross-kingdom antisilencing activities. Therefore, through fortuitous recognition of foreign nucleic acids, cellular microRNAs have direct antiviral effects in addition to their regulatory functions.
Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 11 distinct microRNAs, all of which are found clustered within the major latency-associated region of the KSHV genome in the same transcriptional orientation. Because the KSHV microRNAs are all expressed in latently infected cells and are largely unaffected by induction of lytic replication, it appeared probable that they would be processed out of KSHV transcripts that are derived from a latent promoter(s) present in this region. Here, we define three latent transcripts, derived from two distinct KSHV latent promoters, that function as both KSHV primary microRNA precursors and as kaposin pre-mRNAs. These activities require the readthrough of a leaky viral polyadenylation signal located at nucleotide 122070 in the KSHV genome. In contrast, recognition of this polyadenylation signal gives rise to previously identified mRNAs that encode the KSHV open reading frames (ORFs) 71, 72 and 73 proteins as well as a novel unspliced KSHV mRNA that encodes only ORF72 and ORF71. Thus, transcripts initiating at the two latent promoters present in the KSHV latency-associated region can undergo two entirely distinct fates, i.e., processing to give a kaposin mRNA and viral microRNAs on the one hand or expression as KSHV ORF71, ORF72, or ORF73 mRNAs on the other, depending on whether the viral polyadenylation site located at position 122070 is ignored or recognized, respectively.
In Caenorhabditis elegans the timing of many developmental events is regulated by heterochronic genes. Such genes orchestrate the timing of cell divisions and fates appropriate for the developmental stage of an organism. Analyses of heterochronic mutations in the nematode C. elegans have revealed a genetic pathway that controls the timing of post-embryonic cell divisions and fates. Two of the genes in this pathway encode small regulatory RNAs. The 22 nucleotide (nt) RNAs downregulate the expression of protein-coding mRNAs of target heterochronic genes. Analogous variations in the timing of appearance of particular features have been noted among closely related species, suggesting that such explicit control of developmental timing may not be exclusive to C. elegans. In fact, some of the genes that globally pattern the temporal progression of C. elegans development, including one of the tiny RNA genes, are conserved and temporally regulated across much of animal phylogeny, suggesting that the molecular mechanisms of temporal control are ancient and universal. A very large family of tiny RNA genes called microRNAs, which are similar in structure to the heterochronic regulatory RNAs, have been detected in diverse animal species and are likely to be present in most metazoans. Functions of the newly discovered microRNAs are not yet known. Other examples of temporal programs during growth include the exquisitely choreographed temporal sequences of developmental fates in neurogenesis in Drosophila and the sequential programs of epidermal coloration in insect wing patterning. An interesting possibility is that microRNAs mediate transitions on a variety of time scales to pattern the activities of particular target protein-coding genes and in turn generate sets of cells over a period of time. Plasticity in these microRNA genes or their targets may lead to changes in relative developmental timing between related species, or heterochronic change. Instead of inventing new gene functions, even subtle changes in temporal expression of pre-existing control genes can result in speciation by altering the time at which they function.
MicroRNAs (miRNAs) are a new class of 18-23 nucleotide long non-coding RNAs that play critical roles in a wide spectrum of biological processes. Recent reports also throw light into the role of microRNAs as critical effectors in the intricate host-pathogen interaction networks. Evidence suggests that both virus and hosts encode microRNAs. The exclusive dependence of viruses on the host cellular machinery for their propagation and survival also make them highly susceptible to the vagaries of the cellular environment like small RNA mediated interference. It also gives the virus an opportunity to fight and/or modulate the host to suite its needs. Thus the range of interactions possible through miRNA-mRNA cross-talk at the host-pathogen interface is large. These interactions can be further fine-tuned in the host by changes in gene expression, mutations and polymorphisms. In the pathogen, the high rate of mutations adds to the complexity of the interaction network. Though evidence regarding microRNA mediated cross-talk in viral infections is just emerging, it offers an immense opportunity not only to understand the intricacies of host-pathogen interactions, and possible explanations to viral tropism, latency and oncogenesis, but also to develop novel biomarkers and therapeutics.
We present a new microRNA target prediction algorithm called TargetBoost, and show that the algorithm is stable and identifies more true targets than do existing algorithms. TargetBoost uses machine learning on a set of validated microRNA targets in lower organisms to create weighted sequence motifs that capture the binding characteristics between microRNAs and their targets. Existing algorithms require candidates to have (1) near-perfect complementarity between microRNAs' 5' end and their targets; (2) relatively high thermodynamic duplex stability; (3) multiple target sites in the target's 3' UTR; and (4) evolutionary conservation of the target between species. Most algorithms use one of the two first requirements in a seeding step, and use the three others as filters to improve the method's specificity. The initial seeding step determines an algorithm's sensitivity and also influences its specificity. As all algorithms may add filters to increase the specificity, we propose that methods should be compared before such filtering. We show that TargetBoost's weighted sequence motif approach is favorable to using both the duplex stability and the sequence complementarity steps. (TargetBoost is available as a Web tool from http://www.interagon.com/demo/.).
AIM: Early cervical cancer diagnosis is a global challenge that needs to be addressed by the discovery of less invasive diagnostic and prognostic approaches. Circulating miRNAs are stable in plasma and their diagnostic potentials have been elucidated in some cancers. Therefore, in this cross-sectional study, we determined the patterns of expression of 7 selected circulating microRNAs that differ between patients with cervical cancer receiving therapy, patients with cervical not on therapy and healthy females. The goal was to investigate the diagnostic and prognostic potential of these selected miRNAs. METHODS: Total RNA was extracted from plasma samples collected from 53 participants recruited from Komfo Anokye Teaching Hospital and the Cape Coast Teaching Hospital, Ghana. Complementary DNA (cDNA) synthesis was performed, followed by quantitative polymerase chain reaction (qPCR) to amplify and quantify the expression levels of the target microRNAs. Expression levels of seven microRNAs-hsa-miR-146a, hsa-miR-29a, hsa-miR-29b, hsa-miR-34a, hsa-miR-233, hsa-miR-155, and hsa-miR-27a were compared among three groups: healthy controls (n = 27), patients with cervical cancer on therapy (n = 13), and those not on therapy (n = 13). RESULTS: miR-155 and miR-27a showed statistically significant differential expression between cancer patients and healthy controls. In addition, miR-29b expression levels differed significantly between stage 4b and stage 4a of patient with cervical cancer undergoing treatment. CONCLUSION: These findings suggest that circulating plasma miRNAs may serve as non-invasive biomarkers for the early detection of cervical cancer, monitoring disease progression, and evaluating treatment response.
Dengue infection is a mosquito-borne disease that can cause mild illness or severe haemorrhagic fever. Predictive biomarkers for severe cases, especially those with bleeding manifestations or plasma leakage, are currently lacking. This systematic review and meta-analysis synthesises findings from studies on circulating miRNAs in dengue infection, sourced from PubMed, Scopus, Science Direct, and Web of Science, with quality assessed by the Newcastle-Ottawa Scale (PROSPERO: CRD42024521685). Quantitative meta-analyses were conducted using RStudio to evaluate diagnostic Area Under the Curve (AUC) metrics across five eligible studies and Standardised Mean Differences (SMD) for differential miRNA expression. Key miRNAs, such as miR-146a, miR-150, miR-378, miR-21-5p, and miR-1246, were found to regulate immune responses, inflammation, and viral replication. Meta-analysis demonstrated high overall diagnostic accuracy for circulating miRNAs, with individual reported AUC values ranging from 0.79 to 1.00; however, substantial statistical heterogeneity was observed across pooled metrics (I-squared exceeding 89%). SMD analysis highlighted significant upregulation of acute inflammatory drivers, including miR-21-5p (SMD = 4.67) and miR-450b-5p (SMD = 6.76), alongside marked downregulation of endothelial-protective markers such as miR-126-3p (SMD = -1.31). Rather than viewing dysregulated microRNAs as a uniform class of clinical tools, this review highlights that candidate microRNAs span distinct translational tiers. Recognising these validation levels alongside pre-analytical and biological heterogeneity is essential for prioritising the most viable microRNA candidates for future prospective clinical translation and targeted therapies in dengue management.
MXene, a two-dimensional nanomaterial, has metal conductivity, high electronegativity, functionalized with surface groups, which make it widely applicable in catalysis and biosensing. However, studies on the principle of enhanced electrochemiluminescence (ECL) by MXene composites and the improvement of their performance in catalyzing the ECL reaction are still in their infancy. In this study, gold nanoparticles (AuNPs) are obtained by mild reductive reduction and loaded in situ on the Ti3C2Tx MXene surface to form the composites (AuNPs@MXene). In oxygenated PBS test buffer, AuNPs@MXene enhance the ECL emission of silver nanoclusters (AgNCs) with aggregation-induced electrochemiluminescence (AIECL) properties as luminophore. Approximately 7.5-fold enhancement of ECL signals is obtained by using two ECL enhancement strategies: an efficient AIECL emitter and a co-reaction accelerator. The special nucleic acid structure with "Three Way Junction (TWJ)" enables an ultra-sensitive detection of microRNA, providing an efficient and ultra-sensitive method for microRNA detection. The biosensor achieves a wide detection range of microRNA-21 from 100 aM to 1 nM, with a low detection limit of 31 aM, and exhibits excellent stability, selectivity and high reproducibility in real samples.