Published ahead of print on August 17, 2006, doi:10.1165/rcmb.2006-0227TR
© 2007 American Thoracic Society DOI: 10.1165/rcmb.2006-0227TR
Regulation of MicroRNA by AntagomirsA New Class of Pharmacological Antagonists for the Specific Regulation of Gene Function?School of Biomedical Sciences, Faculty of Health and Hunter Medical Research Institute, University of Newcastle; John Hunter Children's Hospital, Newcastle, New South Wales; and John Curtin School for Medical Research, Australian National University, Canberra, Australia Correspondence and requests for reprints should be addressed to Joerg Mattes and Paul Foster, School of Biomedical Sciences, Faculty of Health and Hunter Medical Research Institute, University of Newcastle, NSW 2300, Australia. E-mail: Joerg.mattes{at}newcastle.edu.au and Paul.foster{at}newcastle.edu.au
The discovery of small "noncoding" or "nonmessenger" RNA molecules that are repressors of translation (microRNAs) has provided the opportunity to specifically suppress a gene or clusters of genes. Moreover, the recent employment of synthetic analogs of these small RNA molecules termed "antagomirs" has shown that microRNAs of interest can be specifically targeted. Understanding the role of microRNAs in fundamental processes associated with complex diseases such as asthma, chronic obstructive pulmonary disease, cancer, chronic infections, and immune disorders may aid in disease diagnosis and prognosis and potentially identify new therapeutic targets.
Approaches to the suppression of gene function to attenuate inappropriate cellular signals that lead to disease have been a key focus of fundamental biology for a number of years. The main problems with this approach are accessibility of the cellular machinery that regulates gene function and target specificity. The recent discovery of small "noncoding" or "nonmessenger" RNA molecules that are critical regulators of translation of specific gene products has provided the opportunity to target specific suppression of a gene or clusters of genes. Moreover, the recent employment of synthetic analogs of these small RNA molecules termed "antagomirs" has shown that cells expressing genes of interest can be specifically targeted. Although in its infancy, the development of antagomirs that can target cell-specific genes or families of genes may pave the way forward for the generation of a new class of therapeutics to treat complex inflammatory diseases.
Small "noncoding" or "nonmessenger" RNA molecules are integral components of the genetic program that are found widely in the genome of most cells. Genes coding for small RNA molecules account for  1–5% of the predicted genes in plants, worms, and vertebrates and are localized in the introns of protein-coding genes or in the noncoding regions of the genome (1). Distinct classes of small "noncoding" or "nonmessenger" RNA molecules, microRNA (miRNA) and small interfering RNA (siRNA), have been identified as key regulators of gene activation and suppression (2). Since the discovery of the first miRNA, Lin 4, a decade ago (3), more than 3,500 unique miRNAs have been identified and are tabulated in an miRNA registry (http://microrna.sanger.ac.uk) (4). Among them, more than 300 miRNAs have been discovered in humans to date. Each miRNA regulates the expression of a large number of target genes by blocking the translation of the target protein, and experimental evidence suggests that as many as 200 target genes may be regulated by a single miRNA (5). Consequently, these molecules are proposed to regulate up to one-third of all human genes at the post-transcriptional level by degrading or repressing target messenger RNA (6). Post-transcriptional regulation of gene function by miRNA is elegantly guided by the base-pairing rules of Watson and Crick. Although the human genome contains hundreds of miRNAs (many of which may not be unique to humans), their role in physiological and pathological processes are only now emerging. The cellular miRNA milieu is likely to be unique in each cell type and associated with distinct and highly specific processes. For example, miRNA may regulate differentiation and maintenance of cell identity in the hematopoietic system (7, 8), contribute to establishing muscle phenotypes (9), control morphogenesis of epithelial tissues (10), and regulate aspects of organogenesis (11, 12) and metabolic processes (13, 14). These structures may also be important in innate immune responses that control viral infections by blocking the synthesis of viral proteins (15). Viral genomes also encode miRNAs and these seem to evolve rapidly, and regulate both the viral life-cycle and the interaction between viruses and their hosts (16). Thus, emerging data suggest that small RNAs are involved in a remarkable spectrum of biological pathways. Abnormal miRNA signatures may exist in diseased states and be valuable diagnostic and/or prognostic markers (17). They may also be used to identify individuals at risk and be indicative of the altered genetic programs that lead to susceptibility and disease expression. Furthermore, modulation of their activity may be of therapeutic benefit (14). The identification of specific miRNA signatures has been achieved in some forms of cancer (17–19), and we await characterization of respiratory diseases (Figure 1).
A complex set of proteins is required for the biogenesis of miRNAs and miRNA-mediated gene regulation (Figure 2). Mature miRNA molecules are generated from long primary miRNA molecules (pri-miRNA), which usually contain a few hundred base pairs. The pri-miRNAs are processed in the nucleus into "stem-loop precursors" (pre-miRNAs) of around 70 nucleotides by RNase II endonucleases, Drosha and its partner Pasha (20–22). The pre-miRNAs are actively transported into the cytoplasm by proteins involved in active nuclear transport such as exportin 5 and Ran-GTP (23, 24). In the cytoplasm they may be further processed into small RNA duplexes of 22 nucleotides by Dicer RNase III and its double-stranded RNA-binding protein partner, the tar-binding protein (TRBP) (25, 26). The functional miRNA strand dissociates from its complementary nonfunctional strand and locates within the RNA-induced-silencing-complex (RISC), which is composed of Dicer, TRBP, and Argonaute2 protein (27) (Figure 2). Alternatively, RISC can cleave target mRNA using pre-miRNA hairpin as the source of small RNA with a nearly 10-fold greater activity, suggesting that miRNA processing and target-RNA cleavage are coupled (28) (Figure 2).
It is this complex that plays a key role in binding messenger RNA via the attached (pre-) miRNA for inactivation. The specific miRNA–RISC complex binds to its cognate messenger RNA target through a small region in the 5' end (29, 30). Binding is usually incomplete and some bases of the messenger RNA remain free and not bound to the miRNA–RISC complex. The 3' region of the miRNA may subsequently zipper up with the free 5' regions of the target messenger RNA (31). In general, small RNAs can function by two different mechanisms: by cleavage of the target messenger RNA or by blocking the process of translation of the encoded protein. Complete binding of small RNA directs cleavage of a single phosphodiester bond in the target messenger RNA, directly across from nucleotide 10 and 11 (32). Even if binding is fully complementary, cleavage only occurs when the 5' end of the small RNA is bound to an Argonaute protein that bears an endonuclease in its Piwi domain (33, 34). This pathway is known as RNA interference and is the principal mechanism by which plant miRNAs regulate gene expression (35). In animals, however, mature miRNA is only partially complementary to the sequence of their target messenger RNAs, enabling them to bind to other messenger RNAs. Rather than inducing cleavage of the target messenger RNA, animal miRNAs block translation of the encoded protein (36, 37). One miRNA may control translation of hundreds of genes, underscoring the potential influence of miRNA on almost every genetic pathway and their possible contribution to disease. However, binding of one miRNA may not be sufficient to block translation, and several other target-specific miRNAs may be required to bind to the messenger RNA for combinatorial control of gene expression (31). Initially, it was thought that miRNA bound to the RISC inhibited protein synthesis by degrading the newly synthesized protein as it emerged from the ribosome, or by freezing ribosomes in place on the messenger RNA and thus blocking protein elongation (36). Recent evidence, however, suggests that miRNA may induce target instability. The miRNA–RISC complex when bound to its target blocks the initiation of translation (38) by promoting the movement of the messenger RNA from the cytosol to sites of RNA degradation termed "P-bodies" (39). This is achieved by Argonaute2 protein and associated enzymes through the removal of the 5'-7-methylguanosine cap, which is characteristic of messenger RNAs and is a prerequisite for their destruction in P-bodies (39). Thus, miRNAs potentially inhibit the initiation of mRNA translation into protein by promoting the localization of target mRNAs into sites of RNA destruction (40). This pathway is presumed to be distinct from that of the siRNA-directed cleavage pathway in plants, where Argonaute first cleaves the target messenger RNA into small fragments before degradation in exosomes.
Strategies for the identification of miRNAs and prediction of their respective targets include genome sequencing combined with computational approaches, isolation of mutant genes from organisms showing abnormal phenotypic characteristics, and miRNA knockout or overexpression technologies. Although the human genome is predicted to contain more than 1,000 miRNAs, recent efforts to discover and categorize all small RNA species and classes have identified only 53 miRNAs that are unique to primates (41). This suggests that miRNA may evolve rapidly, with new miRNA genes arising from mutations and duplication. However, given their predicted role in gene regulation, miRNAs are likely to be highly conserved and stable within a species and cell type. A prominent characteristic of miRNAs is that their genes are closely clustered on the genome, and are often organized in tandems (42, 43). Clustered miRNAs are probably processed from the same polycistronic precursor transcript. Clusters can contain miRNA of similar sequences and these molecules collectively (additively) contribute to post-transcriptional regulation of their target RNAs. Alternatively, a clustered arrangement of miRNAs with distinct sequences may be co-ordinately deployed towards their various target RNAs, and/or be expressed only in specific tissues. One of these miRNA gene clusters, which encodes six closely related genes (miR-290 to miR-295), is specifically expressed in embryonic stem cells and may contribute to the maintenance of stem cell potency (43). Another cluster of 46 miRNAs (comprising, among others, miR-127 and miR-136), is located on human chromosome 14q32, implying a key role for this chromosome in the regulation of gene function (44). While expressed exclusively on the maternal but not the paternal chromosome, the function of these imprinted small RNA genes are as yet unknown (45). A unique miRNA signature within the miRNA gene-clusters miR-15a to miR-16–1 and miR-24–1 to miR-23b has recently been linked with prognostic factors and disease progression in chronic lymphocytic leukemia (CLL) (19). Moreover, germ-line and somatic mutations were described in some of these miRNA genes in more than 10% of patients with CLL, while no such mutations were observed in individuals without cancer. Notably, the target of some of these miRNAs is the anti-apoptotic BCL-2 gene, which is overexpressed in most of the patients with CLL. Therefore, mutations in distinct miRNA clusters may be associated with specific diseased states through the modulation of complex cellular responses such as apoptosis. As miRNAs can function as tumor suppressors and oncogenes ("oncomirs"), they might prove useful in the treatment and diagnosis of cancer (46, 47). As expected, polymorphisms have also been identified in other human pre-miRNA genome regions (48). However, it has yet to be established whether complex diseases as diverse as cancer, allergy, and chronic infections (which share disease pathways that compromise cellular proliferation, differentiation, apoptosis, and metabolism) may be modulated, at least in part, by genetic variations within the non–protein-coding genes containing distinct miRNA clusters.
Efficient loss-of-function and/or overexpression strategies in vivo would be required to investigate the effects of miRNA-directed regulation of gene expression. A fundamental step toward this goal has recently been made by Krutzfeldt and coworkers (13). These investigators have identified a novel class of chemically engineered oligonucleotides they termed "antagomirs" as specific and effective silencers of miRNA expression in mice. These cholesterol-conjugated single-stranded RNA molecules are 21–23 nucleotides in length and complementary to the mature target miRNA. They specifically silenced miRNA expression (miR-122) in the liver, lung, intestine, heart, skin, and bone marrow for more than a week after one intravenous injection. This resulted in upregulated expression of hundreds of genes predicted to be repressed by miR-122 because these genes had a miR-122 recognition motif in the 3' untranslated region. Paradoxically, antagomir treatment also revealed a significant number of downregulated genes that may be activated (as opposed to repressed) by miR-122. Although the mechanism by which miRNAs may activate gene expression in vivo is unknown, it may involve an indirect effect—namely, the suppression of a transcriptional repressor (13). Alternatively, miRNA may have a direct effect on gene activation (e.g., chromatin remodelling) (13). Esau and colleagues extended these studies and highlighted that the inhibition of miRNAs may be a possible therapeutic approach to the treatment of disease (14). Specifically, they inhibited miR-122 expression, which resulted in reduced plasma cholesterol levels and a decrease in hepatic fatty-acid and cholesterol synthesis rates in normal mice and in mice with diet-induced obesity. Thus miR-122 has been identified as a key regulator of cholesterol and fatty-acid metabolism. Recently gene-manipulated mice have been generated overexpressing miR-155, which has been reported to accumulate in human B cell lymphomas. Notably, miR-155 transgenic mice exhibited a spontaneous B cell malignancy, indicating that the role of miR155 is to induce polyclonal expansion (49). These studies demonstrate that powerful technologies are now available to characterize the in vivo role of individual miRNAs in gene regulation and in disease models. As this field rapidly develops, we anticipate that significant improvements in our understanding of fundamental processes associated with complex diseases such as asthma, chronic obstructive pulmonary disease, cancer, chronic infections, immune disorders, and metabolic diseases will eventuate. Moreover, understanding the miRNA signature in susceptible individuals may facilitate the partitioning of patients into distinct subpopulations for targeted therapy, aid in disease diagnosis and prognosis, and potentially identify new therapeutic targets. The employment of this technology to understand origins and mechanisms of respiratory disease is eagerly awaited.
This study was supported by the NHMRC, Australia; the Cooperative Research Centre for Asthma and Airways, Australia; the Hunter Medical Research Institute, Australia; and the Landesstiftung Baden-Wuerttemberg, Germany. Originally Published in Press as DOI: 10.1165/rcmb.2006-0227TR on August 17, 2006 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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Abstract
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