Sensational MicroRNAs: Neurosensory Roles of the MicroRNA-183 Family.

MicroRNAs (miRNAs, miRs) are short noncoding RNAs that act to repress expression of proteins from target mRNA transcripts. miRNAs influence many cellular processes including stemness, proliferation, differentiation, maintenance, and survival, and miRNA mutations or misexpression are associated with a variety of disease states. The miR-183 family gene cluster including miR-183, miR-96, and miR-182 is highly conserved among vertebrate and invertebrate organisms, and the miRNAs are coordinately expressed with marked specificity in sensory neurons and sensory epithelial cells. The crucial functions of these miRNAs in normal cellular processes are not yet fully understood, but expectedly dependent upon the transcriptomes of specific cell types at different developmental stages or in various maintenance circumstances. This article provides an overview of evidence supporting roles for miR-183 family members in normal biology of the nervous system, including mechanoreception for auditory and vestibular function, electroreception, chemoreception, photoreception, circadian rhythms, sensory ganglia and pain, and memory formation.

A mouse model of miR-96, miR-182 and miR-183 misexpression implicates miRNAs in cochlear cell fate and homeostasis.

Germline mutations in Mir96, one of three co-expressed polycistronic miRNA genes (Mir96, Mir182, Mir183), cause hereditary hearing loss in humans and mice. Transgenic FVB/NCrl- Tg(GFAP-Mir183,Mir96,Mir182)MDW1 mice (Tg1MDW), which overexpress this neurosensory-specific miRNA cluster in the inner ear, were developed as a model system to identify, in the aggregate, target genes and biologic processes regulated by the miR-183 cluster. Histological assessments demonstrate Tg1MDW/1MDW homozygotes have a modest increase in cochlear inner hair cells (IHCs). Affymetrix mRNA microarray data analysis revealed that downregulated genes in P5 Tg1MDW/1MDW cochlea are statistically enriched for evolutionarily conserved predicted miR-96, miR-182 or miR-183 target sites. ABR and DPOAE tests from 18 days to 3 months of age revealed that Tg1MDW/1MDW homozygotes develop progressive neurosensory hearing loss that correlates with histologic assessments showing massive losses of both IHCs and outer hair cells (OHCs). This mammalian miRNA misexpression model demonstrates a potency and specificity of cochlear homeostasis for one of the dozens of endogenously co-expressed, evolutionally conserved, small non-protein coding miRNA families. It should be a valuable tool to predict and elucidate miRNA-regulated genes and integrated functional gene expression networks that significantly influence neurosensory cell differentiation, maturation and homeostasis.

Transcriptome-wide comparison of the impact of Atoh1 and miR-183 family on pluripotent stem cells and multipotent otic progenitor cells.

Over 5% of the global population suffers from disabling hearing loss caused by multiple factors including aging, noise exposure, genetic predisposition, or use of ototoxic drugs. Sensorineural hearing loss is often caused by the loss of sensory hair cells (HCs) of the inner ear. A barrier to hearing restoration after HC loss is the limited ability of mammalian auditory HCs to spontaneously regenerate. Understanding the molecular mechanisms orchestrating HC development is expected to facilitate cell replacement therapies. Multiple events are known to be essential for proper HC development including the expression of Atoh1 transcription factor and the miR-183 family. We have developed a series of vectors expressing the miR-183 family and/or Atoh1 that was used to transfect two different developmental cell models: pluripotent mouse embryonic stem cells (mESCs) and immortalized multipotent otic progenitor (iMOP) cells representing an advanced developmental stage. Transcriptome profiling of transfected cells show that the impact of Atoh1 is contextually dependent with more HC-specific effects on iMOP cells. miR-183 family expression in combination with Atoh1 not only appears to fine tune gene expression in favor of HC fate, but is also required for the expression of some HC-specific genes. Overall, the work provides novel insight into the combined role of Atoh1 and the miR-183 family during HC development that may ultimately inform strategies to promote HC regeneration or maintenance.

Identifying microRNAs involved in aging of the lateral wall of the cochlear duct.

Age-related hearing loss is a progressive sensorineural hearing loss that occurs during aging. Degeneration of the organ of Corti and atrophy of the lateral wall of the cochlear duct (or scala media) in the inner ear are the two primary causes. MicroRNAs (miRNAs), a class of short non-coding RNAs that regulate the expression of mRNA/protein targets, are important regulators of cellular senescence and aging. We examined miRNA gene expression profiles in the lateral wall of two mouse strains, along with exploration of the potential targets of those miRNAs that showed dynamic expression during aging. We show that 95 and 60 miRNAs exhibited differential expression in C57 and CBA mice during aging, respectively. A majority of downregulated miRNAs are known to regulate pathways of cell proliferation and differentiation, while all upregulated miRNAs are known regulators in the pro-apoptotic pathways. By using apoptosis-related gene array and bioinformatic approaches to predict miRNA targets, we identify candidate miRNA-regulated genes that regulate apoptosis pathways in the lateral wall of C57 and CBA mice during aging.

Conditional and target-specific transgene induction through RNA replacement using an allosteric trans-splicing ribozyme.

Gene therapeutic approaches are needed that can simultaneously induce the well-controlled expression of therapeutic genes and suppress the expression of disease-causing genes for maximization of their efficacy. To address this challenge, we designed an allosteric ribozyme that comprises a Tetrahymena group I-based trans-splicing ribozyme as an active domain for RNA replacement, a small molecule-specific RNA aptamer as a sensor domain, and a communication module as an active transfer domain. The effectiveness of this approach was assessed by constructing various ribozymes in combination with a theophylline-binding aptamer to identify an allosteric ribozyme, which is controlled by theophylline both in vitro and in cells. Moreover, we constructed adenoviral vectors encoding the ribozymes and validated allosteric regulation of trans-gene expression via theophylline-dependent RNA replacement in target RNA-expressing cells. Results demonstrate that an allosteric trans-splicing ribozyme is an applicable RNA-based framework for engineering external ligand-controlled gene expression regulatory systems that exhibit adjustable regulation, design modularity, and target specificity.

Characterization of transcriptomes of cochlear inner and outer hair cells.

Inner hair cells (IHCs) and outer hair cells (OHCs) are the two types of sensory receptor cells that are critical for hearing in the mammalian cochlea. IHCs and OHCs have different morphology and function. The genetic mechanisms that define their morphological and functional specializations are essentially unknown. The transcriptome reflects the genes that are being actively expressed in a cell and holds the key to understanding the molecular mechanisms of the biological properties of the cell. Using DNA microarray, we examined the transcriptome of 2000 individually collected IHCs and OHCs from adult mouse cochleae. We show that 16,647 and 17,711 transcripts are expressed in IHCs and OHCs, respectively. Of those genes, ∼73% are known genes, 22% are uncharacterized sequences, and 5.0% are noncoding RNAs in both populations. A total of 16,117 transcripts are expressed in both populations. Uniquely and differentially expressed genes account for <15% of all genes in either cell type. The top 10 differentially expressed genes include Slc17a8, Dnajc5b, Slc1a3, Atp2a3, Osbpl6, Slc7a14, Bcl2, Bin1, Prkd1, and Map4k4 in IHCs and Slc26a5, C1ql1, Strc, Dnm3, Plbd1, Lbh, Olfm1, Plce1, Tectb, and Ankrd22 in OHCs. We analyzed commonly and differentially expressed genes with the focus on genes related to hair cell specializations in the apical, basolateral, and synaptic membranes. Eighty-three percent of the known deafness-related genes are expressed in hair cells. We also analyzed genes involved in cell-cycle regulation. Our dataset holds an extraordinary trove of information about the molecular mechanisms underlying hair cell morphology, function, pathology, and cell-cycle control.

Identifying microRNAs involved in degeneration of the organ of corti during age-related hearing loss.

MicroRNAs (miRNAs), a class of short non-coding RNAs that regulate the expression of mRNA targets, are important regulators of cellular senescence and aging. We questioned which miRNAs are involved in age-related degeneration of the organ of Corti (OC), the auditory sensory epithelium that transduces mechanical stimuli to electrical activity in the inner ear. Degeneration of the OC is generally accepted as the main cause of age-related hearing loss (ARHL), a progressive loss of hearing in individuals as they grow older. To determine which miRNAs are involved in the onset and progression of ARHL, miRNA gene expression in the OC of two mouse strains, C57BL/6J and CBA/J, was compared at three different ages using GeneChip miRNA microarray and was validated by real-time PCR. We showed that 111 and 71 miRNAs exhibited differential expression in the C57 and CBA mice, respectively, and that downregulated miRNAs substantially outnumbered upregulated miRNAs during aging. miRNAs that had approximately 2-fold upregulation included members of miR-29 family and miR-34 family, which are known regulators of pro-apoptotic pathways. In contrast, miRNAs that were downregulated by about 2-fold were members of the miR-181 family and miR-183 family, which are known to be important for proliferation and differentiation, respectively. The shift of miRNA expression favoring apoptosis occurred earlier than detectable hearing threshold elevation and hair cell loss. Our study suggests that changes in miRNA expression precede morphological and functional changes, and that upregulation of pro-apoptotic miRNAs and downregulation of miRNAs promoting proliferation and differentiation are both involved in age-related degeneration of the OC.

Conditional deletion of the human ortholog gene Dicer1 in Pax2-Cre expression domain impairs orofacial development.

BACKGROUND: Orofacial clefts are common worldwide and result from insufficient growth and/or fusion during the genesis of the derivatives of the first pharyngeal arch and the frontonasal prominence. Recent studies in mice carrying conditional and tissue-specific deletions of the human ortholog Dicer1, an RNAse III family member, have highlighted its importance in cell survival, differentiation, proliferation, and morphogenesis. Nevertheless, information regarding Dicer1 and its dependent microRNAs (miRNAs) in mammalian palatogenesis and orofacial development is limited. AIMS: To describe the craniofacial phenotype, gain insight into potential mechanisms underlying the orofacial defects in the Pax2-Cre/Dicer1 CKO mouse, and shed light on the role of Dicer1 in mammalian palatogenesis. MATERIALS AND METHODS: Histological and molecular assays of wild type (WT) and Pax2-Cre/Dicer1(loxP/loxP) (Dicer1 CKO) mice dissected tissues have been performed to characterize and analyze the orofacial dysmorphism in Pax2-Cre/Dicer1(loxP/loxP) mouse. RESULTS: Dicer1 CKO mice exhibit late embryonic lethality and severe craniofacial dysmorphism, including a secondary palatal cleft. Further analysis suggest that Dicer1 deletion neither impacts primary palatal development nor the initial stages of secondary palatal formation. Instead, Dicer1 is implicated in growth, differentiation, mineralization, and survival of cells in the lateral palatal shelves. Histological and molecular analysis demonstrates that secondary palatal development becomes morphologically arrested prior to mineralization around E13.5 with a significant increase in the expression levels of apoptotic markers (P < 0.01). CONCLUSIONS: Pax2-Cre-mediated Dicer1 deletion disrupts lateral palatal outgrowth and bone mineralization during palatal shelf development, therefore providing a mammalian model for investigating the role of miRNA-mediated signaling pathways during palatogenesis.

MicroRNA-183 family expression in hair cell development and requirement of microRNAs for hair cell maintenance and survival.

MicroRNAs (miRNAs) post-transcriptionally repress complementary target gene expression and can contribute to cell differentiation. The coordinate expression of miRNA-183 family members (miR-183, miR-96, and miR-182) has been demonstrated in sensory cells of the mouse inner ear and other vertebrate sensory organs. To further examine hair cell miRNA expression in the mouse inner ear, we have analyzed miR-183 family expression in wild type animals and various mutants with defects in neurosensory development. miR-183 family member expression follows neurosensory cell specification, exhibits longitudinal (basal-apical) gradients in maturating cochlear hair cells, and is maintained in sensory neurons and most hair cells into adulthood. Depletion of hair cell miRNAs resulting from Dicer1 conditional knockout (CKO) in Atoh1-Cre transgenic mice leads to more disparate basal-apical gene expression profiles and eventual hair cell loss. Results suggest that hair cell miRNAs subdue cochlear gradient gene expression and are required for hair cell maintenance and survival.

The role of sensory organs and the forebrain for the development of the craniofacial shape as revealed by Foxg1-cre-mediated microRNA loss.

Cranial development is critically influenced by the relative growth of distinct elements. Previous studies have shown that the transcription factor Foxg1 is essential the for development of the telencephalon, olfactory epithelium, parts of the eye and the ear. Here we investigate the effects of a Foxg1-cre-mediated conditional deletion of Dicer1 and microRNA (miRNA) depletion on mouse embryos. We report the rapid and complete loss of the telencephalon and cerebellum as well as the severe reduction in the ears and loss of the anterior half of the eyes. These losses result in unexpectedly limited malformations of anterodorsal aspects of the skull. We investigated the progressive disappearance of these initially developing structures and found a specific miRNA of nervous tissue, miR-124, to disappear before reduction in growth of the specific neurosensory areas. Correlated with the absence of miR-124, these areas showed numerous apoptotic cells that stained positive for anticleaved caspase 3 and the phosphatidylserine stain PSVue® before the near or complete loss of those brain and sensory areas (forebrain, cerebellum, anterior retina, and ear). We conclude that Foxg1-cre-mediated conditional deletion of Dicer1 leads to the absence of functional miRNA followed by complete or nearly complete loss of neurons. Embryonic neurosensory development therefore depends critically on miRNA. Our data further suggest that loss of a given neuronal compartment can be triggered using early deletion of Dicer1 and thus provides a novel means to genetically remove specific neurosensory areas to investigate loss of their function on morphology (this study) or signal processing within the brain.

MicroRNA-221 controls expression of intercellular adhesion molecule-1 in epithelial cells in response to Cryptosporidium parvum infection.

Cryptosporidium parvum is a protozoan parasite that infects gastrointestinal epithelial cells and causes diarrhoeal disease in humans and animals globally. Pathological changes following C. parvum infection include crypt hyperplasia and a modest inflammatory reaction with increased infiltration of lymphocytes into intestinal mucosa. Expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), on infected epithelial cell surfaces may facilitate adhesion and recognition of lymphocytes at infection sites. MicroRNAs (miRNAs) are small RNA molecules of 23 nucleotides that negatively regulate protein-coding gene expression via translational suppression or mRNA degradation. We recently reported that microRNA-221 (miR-221) regulates ICAM-1 translation through targeting the ICAM-1 3'-untranslated region (UTR). In this study, we tested the role of miR-221 in regulating ICAM-1 expression in epithelial cells in response to C. parvum infection using an in vitro model of human biliary cryptosporidiosis. Up-regulation of ICAM-1 at both message and protein levels was detected in epithelial cells following C. parvum infection. Inhibition of ICAM-1 transcription with actinomycin D could only partially block C. parvum-induced ICAM-1 expression at the protein level. Cryptosporidium parvum infection decreased miR-221 expression in infected epithelial cells. When cells were transfected with a luciferase reporter construct covering the miR-221 binding site in the ICAM-1 3'-UTR and then exposed to C. parvum, an enhanced luciferase activity was detected. Transfection of miR-221 precursor abolished C. parvum-stimulated ICAM-1 protein expression. In addition, expression of ICAM-1 on infected epithelial cells facilitated epithelial adherence of co-cultured Jurkat cells. These results indicate that miR-221-mediated translational suppression controls ICAM-1 expression in epithelial cells in response to C. parvum infection.

Analysis of metal ion dependence in glmS ribozyme self-cleavage and coenzyme binding.

The bacterial glmS ribozyme is mechanistically unique among both riboswitches and RNA catalysts. Its self-cleavage activity is the basis of riboswitch regulation of glucosamine-6-phosphate (GlcN6P) production, and catalysis requires GlcN6P as a coenzyme. Previous work has shown that the coenzyme amine of GlcN6P is essential for glmS ribozyme self-cleavage, as is its protonation state. Metal ions are also essential within the glmS ribozyme core for both structure and function of the ribozyme. Although metal ions do not directly promote catalysis, we show that metal ion identity and the varying physicochemical properties of metal ions have an impact on the rate of glmS ribozyme self-cleavage. Specifically, these studies demonstrate that metal ion identity influences the overall apparent pK(a) of ribozyme self-cleavage, and metal ion binding largely reflects phosphate oxygen affinity. Results suggest that metal ions take alternative roles in supporting the mechanism of catalysis.

MicroRNAs sound off.

The message is loud and clear. MicroRNA-96, one in a cluster of three related neurosensory microRNAs, is crucial to the development and maintenance of inner ear hair cells and hearing in mice and humans. Two recent studies show that mutations in the critical seed region of the microRNA underlie the cause of hair cell degeneration and progressive hearing loss. Other recent reports reveal the general requirement of microRNAs for sensory epithelial development and maintenance in Dicer knockout mouse ear. The challenge begins to determine whether microRNAs will resonate as therapeutic agents or target molecules to preserve or restore hearing.

Identification of metabolite-riboswitch interactions using nucleotide analog interference mapping and suppression.

Riboswitches are RNA elements capable of modulating gene expression through interaction with cellular metabolites. One member of the riboswitch family, the glmS riboswitch, is unique among riboswitches in that it modulates gene expression by undergoing self-cleavage in the presence of its metabolite glucosamine-6-phosphate (GlcN6P). In order to investigate the interactions between the glmS RNA and GlcN6P we performed nucleotide analog interference mapping (NAIM) and suppression (NAIS). These techniques have been previously used to identify important functional groups in and tertiary contacts necessary for self-splicing and self-cleaving by catalytic RNAs, RNA-protein complexes, RNA folding, and RNA-metal ion interactions. Described here are the details of NAIM and NAIS experiments we have utilized to investigate RNA-ligand interactions between the glmS riboswitch and GlcN6P. These techniques can be employed to study a wide variety of RNA-small molecule interactions.

Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice.

Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morphohistogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.

Little but loud: small RNAs have a resounding affect on ear development.

The impact of small RNA function has resonated throughout nearly every aspect of eukaryotic biology and captured the varied interests of researchers, whether they are endeavoring to understand the basis of development and disease or seeking novel therapeutic targets and tools. The genetic regulatory roles of microRNAs (miRNAs) are particularly interesting given that these often highly conserved factors post-transcriptionally silence many complementary target genes by inhibiting messenger RNA translation. In this regard, miRNAs can be considered as counterparts to transcription factors, the ensemble of which establishes the set of expressed genes that define the characteristics of a specific cell type. In this review, evidence supporting a resounding role for small RNAs in development and maturation of sensory epithelia in the mouse inner ear will be considered with an emphasis on the contribution of one hair cell miRNA family (miR-183, miR-96, and miR-182). Although there is much yet to be explored in this fledgling aspect of ear biology, the breadth of miRNA expression and functional requirement for ear development are already sounding off.

MicroRNA-513 regulates B7-H1 translation and is involved in IFN-gamma-induced B7-H1 expression in cholangiocytes.

Biliary epithelial cells (cholangiocytes) respond to proinflammatory cytokines such as IFN-gamma and actively participate in the regulation of biliary inflammatory response in the liver. B7-H1 (also known as CD274 or PD-L1) is a member of the B7 costimulatory molecules and plays a critical immunoregulatory role in cell-mediated immune responses. In this study, we show that resting human cholangiocytes in culture express B7-H1 mRNA, but not B7-H1 protein. IFN-gamma induces B7-H1 protein expression and alters the microRNA (miRNA) expression profile in cholangiocytes. Of those IFN-gamma-down-regulated miRNAs, we identified microRNA-513 (miR-513) with complementarity to the 3'-untranslated region of B7-H1 mRNA. Targeting of the B7-H1 3'-untranslated region by miR-513 results in translational repression. Transfection of cholangiocytes with an antisense oligonucleotide to miR-513 induces B7-H1 protein expression. Additionally, transfection of miR-513 precursor decreases IFN-gamma-induced B7-H1 protein expression and consequently influences B7-H1-associated apoptotic cell death in cocultured Jurkat cells. Thus, miR-513 regulates B7-H1 translation and is involved in IFN-gamma-induced B7-H1 expression in human cholangiocytes, suggesting a role for miRNA-mediated gene silencing in the regulation of cholangiocyte response to IFN-gamma.

MicroRNA-183 family conservation and ciliated neurosensory organ expression.

MicroRNAs (miRNAs) are an integral component of the metazoan genome and affect posttranscriptional repression of target messenger RNAs. The extreme phylogenetic conservation of certain miRNAs suggests their ancient origin and crucial function in conserved developmental processes. We demonstrate that highly conserved miRNA-183 orthologs exist in both deuterostomes and protostomes and their expression is predominant in ciliated ectodermal cells and organs. The miRNA-183 family members are expressed in vertebrate sensory hair cells, in innervated regions of invertebrate deuterostomes, and in sensilla of Drosophila and C. elegans. Thus, miRNA-183 family member expression is conserved in possibly homologous but morphologically distinct sensory cells and organs. The results suggest that miR-183 family members contribute specifically to neurosensory development or function, and that extant metazoan sensory organs are derived from cells that share genetic programs of common evolutionary origin.

MicroRNA gene expression in the mouse inner ear.

MicroRNAs (miRNAs) are small non-coding RNAs that function through the RNA interference (RNAi) pathway and post-transcriptionally regulate gene expression in eukaryotic organisms. While miRNAs are known to affect cellular proliferation, differentiation, and morphological development, neither their expression nor roles in mammalian inner ear development have been characterized. We have investigated the extent of miRNA expression at various time points throughout maturation of the postnatal mouse inner ear by microarray analysis. Approximately one third of known miRNAs are detected in the inner ear, and their expression persists to adulthood. Expression of such miRNAs is validated by quantitative PCR and northern blot analysis. Further analysis by in situ hybridization demonstrates that certain miRNAs exhibit cell-specific expression patterns in the mouse inner ear. Notably, we demonstrate that miRNAs previously associated with mechanosensory cells in zebrafish are also expressed in hair cells of the auditory and vestibular endorgans. Our results demonstrate that miRNA expression is abundant in the mammalian inner ear and that certain miRNAs are evolutionarily associated with mechanosensory cell development and/or function. The data suggest that miRNAs contribute substantially to genetic programs intrinsic to development and function of the mammalian inner ear and that specific miRNAs might influence formation of sensory epithelia from the primitive otic neuroepithelium.