microRNA Pathway Research Articles

Possible role of plant MAP kinases in the biogenesis and transcription regulation of rice microRNA pathway factors

Signalling pathways play vital roles as determinants of almost all the molecular processes inside a eukaryotic cell. They are more often considered to be the link between extracellular and intracellular environmental cues. Gene silencing pathways have emerged to be involved in regulation of stress responses and developmental processes. However, very little is known about the crosstalk between signalling and silencing pathways and their influence on each other. The present work describes the effects of general protein kinase inhibitors and specific mitogen activated protein kinase (MAPK) pathway inhibitors on the components of microRNA pathway in rice. The kinase inhibitors significantly reduced the activities of miRNA biogenesis complex and changed the transcript expression of miRNA pathway factors. This study suggests a possible regulation of microRNA machinery by plant kinases and MAP kinases in particular.

NMDA receptor–dependent dephosphorylation of serine 387 in Argonaute 2 increases its degradation and affects dendritic spine density and maturation

Argonaute (AGO) proteins are essential components of the microRNA (miRNA) pathway. AGO proteins are loaded with miRNAs to target mRNAs and thereby regulate mRNA stability and protein translation. As such, AGO proteins are important actors in controlling local protein synthesis, for instance, at dendritic spines and synapses. Although miRNA-mediated regulation of dendritic mRNAs has become a focus of intense interest over the past years, the mechanisms regulating neuronal AGO proteins remain largely unknown. Here, using rat hippocampal neurons, we report that dendritic Ago2 is down-regulated by the proteasome upon NMDA receptor activation. We found that Ser-387 in Ago2 is dephosphorylated upon NMDA treatment and that this dephosphorylation precedes Ago2 degradation. Expressing Ser-387 phosphorylation-deficient or phosphomimetic Ago2 in neurons, we observed that this phosphorylation site is involved in modulating dendritic spine morphology and postsynaptic density protein 95 (PSD-95) expression in spines. Collectively, our results point toward a signaling pathway linking NMDA receptor-dependent Ago2 dephosphorylation and turnover to postsynaptic structural changes. They support a model in which NMDA receptor-mediated dephosphorylation of Ago2 and Ago2 turnover contributes to the de-repression of mRNAs involved in spine growth and maturation.

Protective paraspeckle hyper-assembly downstream of TDP-43 loss of function in amyotrophic lateral sclerosis

BackgroundParaspeckles are subnuclear bodies assembled on a long non-coding RNA (lncRNA) NEAT1. Their enhanced formation in spinal neurons of sporadic amyotrophic lateral sclerosis (ALS) patients has been reported but underlying mechanisms are unknown. The majority of ALS cases are characterized by TDP-43 proteinopathy. In current study we aimed to establish whether and how TDP-43 pathology may augment paraspeckle assembly.MethodsParaspeckle formation in human samples was analysed by RNA-FISH and laser capture microdissection followed by qRT-PCR. Mechanistic studies were performed in stable cell lines, mouse primary neurons and human embryonic stem cell-derived neurons. Loss and gain of function for TDP-43 and other microRNA pathway factors were modelled by siRNA-mediated knockdown and protein overexpression.ResultsWe show that de novo paraspeckle assembly in spinal neurons and glial cells is a hallmark of both sporadic and familial ALS with TDP-43 pathology. Mechanistically, loss of TDP-43 but not its cytoplasmic accumulation or aggregation augments paraspeckle assembly in cultured cells. TDP-43 is a component of the microRNA machinery, and recently, paraspeckles have been shown to regulate pri-miRNA processing. Consistently, downregulation of core protein components of the miRNA pathway also promotes paraspeckle assembly. In addition, depletion of these proteins or TDP-43 results in accumulation of endogenous dsRNA and activation of type I interferon response which also stimulates paraspeckle formation. We demonstrate that human or mouse neurons in vitro lack paraspeckles, but a synthetic dsRNA is able to trigger their de novo formation. Finally, paraspeckles are protective in cells with compromised microRNA/dsRNA metabolism, and their assembly can be promoted by a small-molecule microRNA enhancer.ConclusionsOur study establishes possible mechanisms behind paraspeckle hyper-assembly in ALS and suggests their utility as therapeutic targets in ALS and other diseases with abnormal metabolism of microRNA and dsRNA.

Crosstalk Between the Unfolded Protein Response, MicroRNAs, and Insulin Signaling Pathways: In Search of Biomarkers for the Diagnosis and Treatment of Type 2 Diabetes

Type 2 diabetes mellitus (T2DM) is a metabolic disorder that is characterized by functional defects in glucose metabolism and insulin secretion. Its complex etiology and multifaceted nature have made it difficult to design effective therapies for early diagnosis and treatment. Several lines of evidence indicate that aberrant activation of the unfolded protein response (UPR) in response to endoplasmic reticulum (ER) stress impairs the β cell’s ability to respond to glucose and promotes apoptosis. Elucidating the molecular mechanisms that govern β cell dysfunction and cell death can help investigators design therapies to halt or prevent the development of T2DM. Early diagnosis of T2DM, however, warrants additionally the identification of potential biomarkers. MicroRNAs (miRNAs) are key regulators of transcriptional processes that modulate various features of insulin signaling, such as insulin sensitivity, glucose tolerance, and insulin secretion. A deeper understanding of how changes in patterns of expression of miRNAs correlate with altered glucose metabolism can enable investigators to develop methods for the early diagnosis and treatment of T2DM. The first part of this review examines how altered expression of specific UPR pathway proteins disrupts ER function and causes β cell dysfunction, while the second part discusses the potential role of miRNAs in the diagnostic and treatment of T2DM.

Knockdown of RNA interference pathway genes in western corn rootworm, Diabrotica virgifera virgifera, identifies no fitness costs associated with Argonaute 2 or Dicer-2

The use of transgenic crops that induce silencing of essential genes using double-stranded RNA (dsRNA) through RNA interference (RNAi) in western corn rootworm, Diabrotica virgifera virgifera, is likely to be an important component of new technologies for the control of this important corn pest. Previous studies have demonstrated that the dsRNA response in D. v. virgifera depends on the presence of RNAi pathway genes including Dicer-2 and Argonaute 2, and that downregulation of these genes limits the lethality of environmental dsRNA. A potential resistance mechanism to lethal dsRNA may involve loss of function of RNAi pathway genes. Howver, the potential for resistance to evolve may depend on whether these pathway genes have essential functions such that the loss of function of core proteins in the RNAi pathway will have fitness costs in D. v. virgifera. Fitness costs associated with potential resistance mechanisms have a central role in determining how resistance can evolve to RNAi technologies in western corn rootworm. We evaluated the effect of dsRNA and microRNA pathway gene knockdown on the development of D. v. virgifera larvae through short-term and long-term exposures to dsRNA for Dicer and Argonaute genes. Downregulation of Argonaute 2, Dicer-2, Dicer-1 did not significantly affect larval survivorship or development through short and long-term exposure to dsRNA. However, downregulation of Argonaute 1 reduced larval survivorship and delayed development. The implications of these results as they relate to D. v. virgifera resistance to lethal dsRNA are discussed.

Nc886 is induced by TGF-\u03b2 and suppresses the microRNA pathway in ovarian cancer

Transforming growth factor-β (TGF-β) signaling and microRNAs (miRNAs) are important gene regulatory components in cancer. Usually in advanced malignant stages, TGF-β signaling is elevated but global miRNA expression is suppressed. Such a gene expression signature is well illustrated in a fibrosis (or mesenchymal) subtype of ovarian cancer (OC) that is of poor prognosis. However, the interplay between the two pathways in the OC subtype has not yet been elucidated. nc886 is a recently identified non-coding RNA implicated in several malignancies. The high expression of nc886 is associated with poor prognosis in 285 OC patients. Herein, we find that in OC nc886 expression is induced by TGF-β and that nc886 binds to Dicer to inhibit miRNA maturation. By preventing the miRNA pathway, nc886 emulates TGF-β in gene expression patterns and potentiates cell adhesion, migration, invasion, and drug resistance. Here we report nc886 to be a molecular link between the TGF-β and miRNA pathways.

FUS Regulates Activity of MicroRNA-Mediated Gene Silencing

MicroRNA-mediated gene silencing is a fundamental mechanism in the regulation of gene expression. It remains unclear how the efficiency of RNA silencing could be influenced by RNA-binding proteins associated with the microRNA-induced silencing complex (miRISC). Here we report that fused in sarcoma (FUS), an RNA-binding protein linked to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), interacts with the core miRISC component AGO2 and is required for optimal microRNA-mediated gene silencing. FUS promotes gene silencing by binding to microRNA and mRNA targets, as illustrated by its action on miR-200c and its target ZEB1. A truncated mutant form of FUS that leads its carriers to an aggressive form of ALS, R495X, impairs microRNA-mediated gene silencing. The C.elegans homolog fust-1 also shares a conserved role in regulating the microRNA pathway. Collectively, our results suggest a role for FUS in regulating the activity of microRNA-mediated silencing.

Cell-Based Reporter System for High-Throughput Screening of MicroRNA Pathway Inhibitors and Its Limitations.

MicroRNAs (miRNAs) are small RNAs repressing gene expression. They contribute to many physiological processes and pathologies. Consequently, strategies for manipulation of the miRNA pathway are of interest as they could provide tools for experimental or therapeutic interventions. One of such tools could be small chemical compounds identified through high-throughput screening (HTS) with reporter assays. While a number of chemical compounds have been identified in such high-throughput screens, their application potential remains elusive. Here, we report our experience with cell-based HTS of a library of 12,816 chemical compounds to identify miRNA pathway modulators. We used human HeLa and mouse NIH 3T3 cell lines with stably integrated or transiently expressed luciferase reporters repressed by endogenous miR-30 and let-7 miRNAs and identified 163 putative miRNA inhibitors. We report that compounds relieving miRNA-mediated repression via stress induction are infrequent; we have found only two compounds that reproducibly induced stress granules and relieved miRNA-targeted reporter repression. However, we have found that this assay type readily yields non-specific (miRNA-independent) stimulators of luciferase reporter activity. Furthermore, our data provide partial support for previously published miRNA pathway modulators; the most notable intersections were found among anthracyclines, dopamine derivatives, flavones, and stilbenes. Altogether, our results underscore the importance of appropriate negative controls in development of small compound inhibitors of the miRNA pathway. This particularly concerns validation strategies, which would greatly profit from assays that fundamentally differ from the routinely employed miRNA-targeted reporter assays.

Novel mechanisms of chemoresistance by Fusobacterium nucleatum involve not so novel pathways of microRNAs and autophagy

Colon cancer is the second leading cause of cancer death among both men and women in the United States. Even with available chemotherapies, a significant number of patients have recurrent disease, leading to the high incidence of morbidity and mortality. Understanding the mechanisms by which chemoresistance can occur is paramount to developing novel therapeutic approaches. Multiple past studies have highlighted the modes of resistance in cancer cells. The primary focus has been on molecular pathways and signaling cascades involving growth factors, tyrosine kinases and transcription factors that lead to tumorigenesis as well as chemoresistance (1,2). In the past few years, microRNAs (miRNAs) and small interfering-RNA (siRNA) have gained a signi cant role in cancer biology.

Investigation of mRNA Translation Regulation by FMRP via the Microrna Pathway

Investigation of mRNA Translation Regulation by FMRP via the Microrna Pathway

Biogenesis of small non-coding RNAs in animals

In recent years, the discovery of small non-coding RNAs has opened an all new field in molecular biology. Indeed, these non-coding sequences give rise to powerful regulators of gene expression. Nowadays, different types of small non-coding RNAs have been described. Of these, the best-characterized types are microRNAs, piRNAs (Piwi-interacting RNAs) and siRNAs (small interfering RNAs). Because of their fine-tuning important function in the regulation of gene and genome expression, an aberrant expression level of those small non-coding RNAs are associated to several pathologies. While this new research field is attracting attention, many aspects to be discovered. This review focuses on the biogenesis pathways of microRNAs, piRNAs and siRNAs in animals.

RNA-binding protein immunoprecipitation as a tool to investigate plant miRNA processing interference by regulatory proteins of diverse origin

BackgroundDue to the nature of viral RNA genomes, RNA viruses depend on many RNA-binding proteins (RBP) of viral and host origin for replication, dissemination and evasion of host RNA degradation pathways. Some viruses interfere with the microRNA (miRNA) pathway to generate better fitness. The development of an adjusted, reliable and sensitive ribonucleoprotein immunoprecipitation (RIP) assay is needed to study the interaction between RBP of different origin (including viral origin) and miRNA precursors. The method could be further applied to transiently expressed heterologous proteins in different plant species.ResultsHere we describe a modified RIP assay applied to nuclear epitope-tagged proteins of heterologous origin and transiently expressed in Nicotiana benthamiana. The assay includes a combination of optimized steps as well as the careful selection of control samples and rigorous data analysis. It has proven efficient to detect and quantify miRNA processing intermediates associated with regulatory proteins.ConclusionsThe RIP method described here provides a reliable tool to study the interaction of RBPs, such as transiently expressed regulatory proteins with lowly represented host RNA, as is the case of miRNA precursors. This modified method was efficiently adjusted to recover nuclear proteins and reduce unspecific background. The purification scheme optimized here for GFP-tagged proteins can be applied to a wide array of RBPs. The subsequent application of next-generation sequencing technologies will permit to sequence and characterize all RNA species bound in vivo by a given RBP.

CaSR signaling down-regulates AQP2 expression via a novel microRNA pathway in pendrin and NaCl cotransporter knockout mice.

High concentrations of urinary calcium counteract vasopressin action via the activation of the calcium-sensing receptor (CaSR) that is expressed in the luminal membrane of collecting duct cells, which impairs the trafficking of aquaporin-2 (AQP2). Pendrin/NaCl cotransporter double-knockout (dKO) mice display significant calcium wasting and develop severe volume depletion, despite increased circulating vasopressin levels. We hypothesized that the CaSR-mediated impairment of AQP2 expression/trafficking underlies vasopressin resistance in dKO mice. Compared with wild-type mice, in renal inner medulla, dKO mice had reduced total AQP2 sensitive to proteasome inhibitors, higher levels of AQP2-pS261, ubiquitinated AQP2, and p38-MAPK, an enzyme that is activated by CaSR signaling and known to phosphorylate AQP2 at Ser261. CaSR inhibition with the calcilytic NPS2143 reversed these effects, which indicates that CaSR mediates the up-regulation of AQP2-pS261, ubiquitination, and degradation. Of note, dKO mice demonstrated significantly higher AQP2-targeting miRNA-137 that was reduced upon CaSR inhibition, supporting a critical role for CaSR in the down-regulation of AQP2 expression. Our data indicate that CaSR signaling reduces AQP2 abundance both via AQP2-targeting miRNA-137 and the p38-MAPK/AQP2-pS261/ubiquitination/proteasomal axis. These effects may contribute to the reduced renal concentrating ability that has been observed in dKO mice and underscore a physiologic mechanism of the CaSR-dependent regulation of AQP2 abundance via a novel microRNA pathway.-Ranieri, M., Zahedi, K., Tamma, G., Centrone, M., Di Mise, A., Soleimani, M., Valenti, G. CaSR signaling down-regulates AQP2 expression via a novel microRNA pathway in pendrin and NaCl cotransporter knockout mice.

The Role of HPV Oncogenes in the Regulation of microRNA pathway in Cervical Cancer

The Role of HPV Oncogenes in the Regulation of microRNA pathway in Cervical Cancer

The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway.

In Arabidopsis, the F-box HAWAIIAN SKIRT (HWS) protein is important for organ growth. Loss of function of HWS exhibits pleiotropic phenotypes including sepal fusion. To dissect the HWS role, we EMS-mutagenized hws-1 seeds and screened for mutations that suppress hws-1 associated phenotypes. We identified shs-2 and shs-3 (suppressor of hws-2 and 3) mutants in which the sepal fusion phenotype of hws-1 was suppressed. shs-2 and shs-3 (renamed hst-23/hws-1 and hst-24/hws-1) carry transition mutations that result in premature terminations in the plant homolog of Exportin-5 HASTY (HST), known to be important in miRNA biogenesis, function and transport. Genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway also suppress the phenotypes associated with HWS loss of function, corroborating epistatic relations between the miRNA pathway genes and HWS. In agreement with these data, accumulation of miRNA is modified in HWS loss or gain of function mutants. Our data propose HWS as a new player in the miRNA pathway, important for plant growth.

Exploitation of microRNAs by Japanese Encephalitis virus in human microglial cells.

JEV infection in CNS leads to the JE neuroinflammation. Children and old age individual have been reported to be more prone to JEV infection. MicroRNAs are endogenous, small non-coding RNAs, which regulate the gene expression. These are ∼22 nucleotide long, conserved RNA sequence that binds at the 3'UTR of a target mRNA and regulate the post-transcriptional gene expression. The role of microRNAs has been reported in several diseases like cancer, viral infection, neuro-degeneration, diabetes etc. In the present study, the human microglial cells were infected with JEV (JaOArS982). The control and infected samples were subject to microarray profiling for microRNA expression. The microarray profile yielded differentially expressed microRNAs from JEV infected samples. The microRNA gene targets, gene ontology, annotations, and pathways were identified through various bioinformatics tools. Additionally, the pathways were mostly found common to "ubiquitin mediated proteolysis," "cytokine signaling," "maintenance of barrier function/cell junctions," JAK/STAT pathway" "Toll-like receptor signaling," "Wnt-signaling," "adhesion molecules," "apoptosis," "endocytosis," "vesicle mediated transport" etc.

Age-associated microRNA expression in human peripheral blood is associated with all-cause mortality and age-related traits.

SummaryRecent studies provide evidence of correlations of DNA methylation and expression of protein‐coding genes with human aging. The relations of microRNA expression with age and age‐related clinical outcomes have not been characterized thoroughly. We explored associations of age with whole‐blood microRNA expression in 5221 adults and identified 127 microRNAs that were differentially expressed by age at P < 3.3 × 10−4 (Bonferroni‐corrected). Most microRNAs were underexpressed in older individuals. Integrative analysis of microRNA and mRNA expression revealed changes in age‐associated mRNA expression possibly driven by age‐associated microRNAs in pathways that involve RNA processing, translation, and immune function. We fitted a linear model to predict ‘microRNA age’ that incorporated expression levels of 80 microRNAs. MicroRNA age correlated modestly with predicted age from DNA methylation (r = 0.3) and mRNA expression (r = 0.2), suggesting that microRNA age may complement mRNA and epigenetic age prediction models. We used the difference between microRNA age and chronological age as a biomarker of accelerated aging (Δage) and found that Δage was associated with all‐cause mortality (hazards ratio 1.1 per year difference, P = 4.2 × 10−5 adjusted for sex and chronological age). Additionally, Δage was associated with coronary heart disease, hypertension, blood pressure, and glucose levels. In conclusion, we constructed a microRNA age prediction model based on whole‐blood microRNA expression profiling. Age‐associated microRNAs and their targets have potential utility to detect accelerated aging and to predict risks for age‐related diseases.

MiR-21 silencing ameliorates experimental autoimmune encephalomyelitis by promoting the differentiation of IL-10-producing B cells.

IL-10-producing regulatory B (IL-10+ Breg) cells promote tolerance in autoimmune diseases and transplantation. However, it remains unclear whether microRNAs are involved in the development of IL-10+ Breg cells. Here, we found that microRNA-21 (miR-21) acts as an upstream regulator of IL-10 by targeting the 3' untranslated region of IL-10 mRNA. We also demonstrated that IL-10+ Breg cells exhibit lower miR-21 expression than non-Breg cells and that miR-21 acts as a potent negative regulator of the differentiation of IL-10+ Breg cells. Accordingly, specific inhibition of miR-21 using antisense oligonucleotides markedly promoted B cell IL-10 expression. Thus, IL-10 is a direct target of miR-21. Moreover, silencing of miR-21 significantly alleviated the severity of experimental autoimmune encephalomyelitis (EAE), and this change was associated with an increase in the number of IL-10+ Breg cells. Finally, we demonstrated that miR-21-silenced B cells exert their suppressive activity through effector T cells in an IL-10-dependent manner.Thus, we characterized a B cell-intrinsic microRNA pathway that inhibits the differentiation of IL-10+ Breg cells and promotes autoimmunity. miR-21 silencing therefore represents a new therapeutic strategy for the treatment of autoimmune diseases.

FUS Mutant Human Motoneurons Display Altered Transcriptome and microRNA Pathways with Implications for ALS Pathogenesis.

SummaryThe FUS gene has been linked to amyotrophic lateral sclerosis (ALS). FUS is a ubiquitous RNA-binding protein, and the mechanisms leading to selective motoneuron loss downstream of ALS-linked mutations are largely unknown. We report the transcriptome analysis of human purified motoneurons, obtained from FUS wild-type or mutant isogenic induced pluripotent stem cells (iPSCs). Gene ontology analysis of differentially expressed genes identified significant enrichment of pathways previously associated to sporadic ALS and other neurological diseases. Several microRNAs (miRNAs) were also deregulated in FUS mutant motoneurons, including miR-375, involved in motoneuron survival. We report that relevant targets of miR-375, including the neural RNA-binding protein ELAVL4 and apoptotic factors, are aberrantly increased in FUS mutant motoneurons. Characterization of transcriptome changes in the cell type primarily affected by the disease contributes to the definition of the pathogenic mechanisms of FUS-linked ALS.

Pathological processes and therapeutic advances in radioiodide refractory thyroid cancer.

While in most patients with non-medullary thyroid cancer (TC), disease remission is achieved by thyroidectomy and ablation of tumor remnants by radioactive iodide (RAI), a substantial subgroup of patients with metastatic disease present tumor lesions that have acquired RAI resistance as a result of dedifferentiation. Although oncogenic mutations in BRAF, TERT promoter and TP53 are associated with an increased propensity for induction of dedifferentiation, the role of genetic and epigenetic aberrations and their effects on important intracellular signaling pathways is not yet fully elucidated. Also immune, metabolic, stemness and microRNA pathways have emerged as important determinants of TC dedifferentiation and RAI resistance. These signaling pathways have major clinical implications since their targeting could inhibit TC progression and could enable redifferentiation to restore RAI sensitivity. In this review, we discuss the current insights into the pathological processes conferring dedifferentiation and RAI resistance in TC and elaborate on novel advances in diagnostics and therapy to improve the clinical outcome of RAI-refractory TC patients.