microRNA-mediated Gene Silencing Research Articles

Genome-Wide Analysis of C/S1-bZIP Subfamilies in Populus tomentosa and Unraveling the Role of PtobZIP55/21 in Response to Low Energy.

C/S1 basic leucine zipper (bZIP) transcription factors are essential for plant survival under energy deficiency. However, studies on the responses of C/S1-bZIPs to low energy in woody plants have not yet been reported. In this study, members of C/S1-bZIP subfamilies in Populus tomentosa were systematically analyzed using bioinformatic approaches. Four C-bZIPs and 10 S1-bZIPs were identified, and their protein properties, phylogenetic relationships, gene structures, conserved motifs, and uORFs were systematically investigated. In yeast two-hybrid assays, direct physical interactions between C-bZIP and S1-bZIP members were observed, highlighting their potential functional synergy. Moreover, expression profile analyses revealed that low energy induced transcription levels of most C/S1-bZIP members, with bZIP55 and bZIP21 (a homolog of bZIP55) exhibiting particularly significant upregulation. When the expression of bZIP55 and bZIP21 was co-suppressed using artificial microRNA mediated gene silencing in transgenic poplars, root growth was promoted. Further analyses revealed that bZIP55/21 negatively regulated the root development of P. tomentosa in response to low energy. These findings provide insights into the molecular mechanisms by which C/S1-bZIPs regulate poplar growth and development in response to energy deprivation.

Mature microRNA-binding protein QKI promotes microRNA-mediated gene silencing

ABSTRACT Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.

MicroRNA processing, role of GW182 Scaffolding proteins in RNA interference and Potential roles of let-7, miR-15 and miR-29 families as tumor suppressors

MicroRNAs are class of non-coding RNAs which are involved in many fundamental cellular and physiological process such as cell differentiation, proliferation and regulation of cell cycle and apoptosis. The vast majority of microRNAs are transcribed from non-coding regions (introns) of genes into primary miRNAs, which are then processed into precursor miRNAs and, lastly, microRNAs. Humans have at least 277 genes that are making microRNAs and these are conserved in mammals. MicroRNAs control gene expression by the sequence-specific targeting of mRNAs, which results in translational repression or mRNA destruction. Recent research has revealed that miRNAs have a role in tumor development, progression, and metastasis. MiRNA function dysregulation is linked to a growing variety of human disorders, including cancer, with miRNAs serving as tumor suppressors or oncogenes (oncomiRs). This review attempts to offer a snapshot of aspects of miRNA biogenesis, processing, role of GW182 scaffold proteins in microRNA mediated gene silencing and potential role of some microRNA families as tumor suppressors.

Epigenetic changes induced by pathogenic Chlamydia spp.

Chlamydia trachomatis, C. pneumoniae, and C. psittaci, the three Chlamydia species known to cause human disease, have been collectively linked to several pathologies, including conjunctivitis, trachoma, respiratory disease, acute and chronic urogenital infections and their complications, and psittacosis. In vitro, animal, and human studies also established additional correlations, such as between C. pneumoniae and atherosclerosis and between C. trachomatis and ovarian cancer. As part of their survival and pathogenesis strategies as obligate intracellular bacteria, Chlamydia spp. modulate all three major types of epigenetic changes, which include deoxyribonucleic acid (DNA) methylation, histone post-translational modifications, and microRNA-mediated gene silencing. Some of these epigenetic changes may be implicated in key aspects of pathogenesis, such as the ability of the Chlamydia spp. to induce epithelial-to-mesenchymal transition, interfere with DNA damage repair, suppress cholesterol efflux from infected macrophages, act as a co-factor in human papillomavirus (HPV)-mediated cervical cancer, prevent apoptosis, and preserve the integrity of mitochondrial networks in infected host cells. A better understanding of the individual and collective contribution of epigenetic changes to pathogenesis will enhance our knowledge about the biology of Chlamydia spp. and facilitate the development of novel therapies and biomarkers. Pathogenic Chlamydia spp. contribute to epigenetically-mediated gene expression changes in host cells by multiple mechanisms.

A specific type of Argonaute phosphorylation regulates binding to microRNAs during C. elegans development

Argonaute proteins are at the core of the microRNA-mediated gene silencing pathway essential for animals. In C. elegans, the microRNA-specific Argonautes ALG-1 and ALG-2 regulate multiple processes required for proper animal developmental timing and viability. Here we identified a phosphorylation site on ALG-1 that modulates microRNA association. Mutating ALG-1 serine 642 into a phospho-mimicking residue impairs microRNA binding and causes embryonic lethality and post-embryonic phenotypes that are consistent with alteration of microRNA functions. Monitoring microRNA levels in alg-1 phosphorylation mutant animals shows that microRNA passenger strands increase in abundance but are not preferentially loaded into ALG-1, indicating that the miRNA binding defects could lead to microRNA duplex accumulation. Our genetic and biochemical experiments support protein kinase A (PKA) KIN-1 as the putative kinase that phosphorylates ALG-1 serine 642. Our data indicate that PKA triggers ALG-1 phosphorylation to regulate its microRNA association during C. elegans development.

AGO4 suppresses tumor growth by modulating autophagy and apoptosis via enhancing TRIM21-mediated ubiquitination of GRP78 in a p53-independent manner.

Argonaute proteins, which consist of AGO1, AGO2, AGO3 and AGO4, are key players in microRNA-mediated gene silencing. So far, few non-microRNA related biological roles of AGO4 have been reported. Here, we first found that AGO4 had low expression in non-small cell lung cancer (NSCLC) patient tumor tissues and could suppress NSCLC cell proliferation and metastasis. Subsequent studies on the mechanism showed that AGO4 could interact with the tripartite motif-containing protein 21 (TRIM21) and the glucose-regulated protein 78 (GRP78). AGO4 promoted ubiquitination of GRP78 by stabilizing TRIM21, a new specific ubiquitin E3 ligase for promoting K48-linked polyubiquitination of GRP78 confirmed in this paper, which resulted in induced cell apoptosis and inhibited autophagy by activating mTOR signal pathway. Further studies showed that p53 had dominant effects on TRIM21-GRP78 axis by directly increasing the expression of TRIM21 in p53 wild-type cells and AGO4 may alternatively regulate TRIM21-GRP78 axis in p53-deficient cells. We also found that overexpression of AGO4 results in suppression of multiple p53-deficient cell growth both in vivo and vitro. Together, we showed for the first time that the AGO4-TRIM21-GRP78 axis, as a new regulatory pathway, may be a novel potential therapeutic target for p53-deficient tumor treatment.

Eukaryotic mRNA decapping factors: molecular mechanisms and activity.

Decapping is the enzymatic removal of 5' cap structures from mRNAs in eukaryotic cells. Cap structures normally enhance mRNA translation and stability, and their excision commits an mRNA to complete 5'-3' exoribonucleolytic digestion and generally ends the physical and functional cellular presence of the mRNA. Decapping plays a pivotal role in eukaryotic cytoplasmic mRNA turnover and is a critical and highly regulated event in multiple 5'-3' mRNA decay pathways, including general 5'-3' decay, nonsense-mediated mRNA decay (NMD), AU-rich element-mediated mRNA decay, microRNA-mediated gene silencing, and targeted transcript-specific mRNA decay. In the yeast Saccharomyces cerevisiae, mRNA decapping is carried out by a single Dcp1-Dcp2 decapping enzyme in concert with the accessory activities of specific regulators commonly known as decapping activators or enhancers. These regulatory proteins include the general decapping activators Edc1, 2, and 3, Dhh1, Scd6, Pat1, and the Lsm1-7 complex, as well as the NMD-specific factors, Upf1, 2, and 3. Here, we focus on in vivo mRNA decapping regulation in yeast. We summarize recently uncovered molecular mechanisms that control selective targeting of the yeast decapping enzyme and discuss new roles for specific decapping activators in controlling decapping enzyme targeting, assembly of target-specific decapping complexes, and the monitoring of mRNA translation. Further, we discuss the kinetic contribution of mRNA decapping for overall decay of different substrate mRNAs and highlight experimental evidence pointing to the functional coordination and physical coupling between events in mRNA deadenylation, decapping, and 5'-3' exoribonucleolytic decay.

Protein tyrosine phosphatase 1B regulates miR-208b-argonaute 2 association and thyroid hormone responsiveness in cardiac hypertrophy.

Increased production of reactive oxygen species plays an essential role in the pathogenesis of several diseases, including cardiac hypertrophy. In our search to identify redox-sensitive targets that contribute to redox signaling, we found that protein tyrosine phosphatase 1B (PTP1B) was reversibly oxidized and inactivated in hearts undergoing hypertrophy. Cardiomyocyte-specific deletion of PTP1B in mice (PTP1B cKO mice) caused a hypertrophic phenotype that was exacerbated by pressure overload. Furthermore, we showed that argonaute 2 (AGO2), a key component of the RNA-induced silencing complex, was a substrate of PTP1B in cardiomyocytes and in the heart. Our results revealed that phosphorylation at Tyr393 and inactivation of AGO2 in PTP1B cKO mice prevented miR-208b-mediated repression of thyroid hormone receptor-associated protein 1 (THRAP1; also known as MED13) and contributed to thyroid hormone-mediated cardiac hypertrophy. In support of this conclusion, inhibiting the synthesis of triiodothyronine (T3) with propylthiouracil rescued pressure overload-induced hypertrophy and improved myocardial contractility and systolic function in PTP1B cKO mice. Together, our data illustrate that PTP1B activity is cardioprotective and that redox signaling is linked to thyroid hormone responsiveness and microRNA-mediated gene silencing in pathological hypertrophy.

Single-molecule imaging of microRNA-mediated gene silencing in cells

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate the expression of thousands of genes; miRNAs silence gene expression from complementary mRNAs through translational repression and mRNA decay. For decades, the function of miRNAs has been studied primarily by ensemble methods, where a bulk collection of molecules is measured outside cells. Thus, the behavior of individual molecules during miRNA-mediated gene silencing, as well as their spatiotemporal regulation inside cells, remains mostly unknown. Here we report single-molecule methods to visualize each step of miRNA-mediated gene silencing in situ inside cells. Simultaneous visualization of single mRNAs, translation, and miRNA-binding revealed that miRNAs preferentially bind to translated mRNAs rather than untranslated mRNAs. Spatiotemporal analysis based on our methods uncovered that miRNAs bind to mRNAs immediately after nuclear export. Subsequently, miRNAs induced translational repression and mRNA decay within 30 and 60 min, respectively, after the binding to mRNAs. This methodology provides a framework for studying miRNA function at the single-molecule level with spatiotemporal information inside cells.

Recent Advances in Plant Gene Silencing Methods.

With the increasing understanding of fundamentals of gene silencing pathways in plants, various tools and techniques for downregulating the expression of a target gene have been developed across multiple plant species. This chapter provides an insight into the molecular mechanisms of gene silencing and highlights the advancements in various gene silencing approaches. The prominent aspects of different gene silencing methods, their advantages and disadvantages have been discussed. A succinct discussion on the newly emerged microRNA-based technologies like microRNA-induced gene silencing (MIGS) and microRNA-mediated virus-induced gene silencing (MIR-VIGS) are also presented. We have also discussed the gene-editing system like CRISPR-Cas. The prominent bottlenecks in gene silencing methods are the off-target effects and lack of universal applicability. However, the tremendous growth in understanding of this field reflects the potentials for improvements in the currently available approaches and the development of new widely applicable methods for easy, fast, and efficient functional characterization of plant genes.

EIF4E-homologous protein (4EHP): a multifarious cap-binding protein.

The cap-binding protein 4EHP/eIF4E2 has been a recent object of interest in the field of post-transcriptional gene regulation and translational control. From ribosome-associated quality control, to RNA decay and microRNA-mediated gene silencing, this member of the eIF4E protein family regulates gene expression through numerous pathways. Low in abundance but ubiquitously expressed, 4EHP interacts with different binding partners to form multiple protein complexes that regulate translation in a variety of biological contexts. Documented functions of 4EHP primarily relate to its role as a translational repressor, but recent findings indicate that it might also participate in the activation of translation in specific settings. In this review, we discuss the known functions, properties and mechanisms that involve 4EHP in the control of gene expression. We also discuss our current understanding of how 4EHP processes are regulated in eukaryotic cells, and the diseases implicated with dysregulation of 4EHP-mediated translational control.

CPSF4 regulates circRNA formation and microRNA mediated gene silencing in hepatocellular carcinoma.

CircRNAs play essential roles in various physiological processes and involves in many diseases, in particular cancer. Global downregulation of circRNA expression has been observed in hepatocellular carcinoma (HCC) in many studies. Previous studies revealed that the pre-mRNA 3' end processing complex participates in circRNA cyclization and plays an important role in HCC tumorigenesis. Therefore, we explored the role of CPSF4, for 3' end formation and cleavage, in circRNA formation. Clinical research has shown that CPSF4 expression is upregulated in HCC and that high expression of CPSF4 is associated with poor prognosis in HCC patients. Mechanistic studies have demonstrated that CPSF4 reduces the levels of circRNAs, which possess a polyadenylation signal sequence and this decrease in circRNAs reduces the accumulation of miRNA and disrupts the miRNA-mediated gene silencing in HCC. Experiments in cell culture and xenograft mouse models showed that CPSF4 promotes the proliferation of HCC cells and enhances tumorigenicity. Moreover, CPSF4 antagonizes the tumor suppressor effect of its downstream circRNA in HCC. In summary, CPSF4 acts as an oncogene in HCC through circRNA inhibition and disruption of miRNA-mediated gene silencing.

The RabGAP TBC-11 controls Argonaute localization for proper microRNA function in C. elegans.

Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3’UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo.

Abstract PO-048: MicroRNA-10b is a regulator of cellular viability and proliferation in fibrolamellar carcinoma

Abstract Fibrolamellar carcinoma (FLC) is a rare form of liver cancer that occurs mostly in adolescents without preexisting liver conditions. A genomic identifier of FLC is a ~400kb deletion on chromosome 19 which results in a fusion of two genes, DNAJB1 and PRKACA (referred to as “DP”). The DP chimera is a functional kinase, the over-expression of which is tumorigenic in mice. Targeting DP pharmacologically has proven challenging; therefore, it is of critical importance to define downstream mediators of tumor phenotypes in order to identify candidate druggable targets. Genome-scale analysis of FLC tumors shows reproducible changes in gene expression. DP is likely to mediate gene expression through both transcriptional and post-transcriptional mechanisms, the latter of which includes microRNA mediated gene silencing. In order to define aberrant microRNAs in FLC tumors, we performed small RNA-sequencing (small RNA-seq) in >25 tumors and 3 non-malignant liver samples. We identified multiple miRNAs significantly upregulated in FLC, including miR-182, miR-10b, miR-21, miR-449a, and miR-92b. We then compared the expression levels of these miRNAs to the levels reported in other cancers and identified the upregulation of miR-10b in FLC to be greater than any other tumor type. We also found that only miR-10b expression is further elevated in metastatic samples relative to primary tumor tissue. Finally, in a cell culture model of FLC, we found that only miR-10b expression is induced (greater than 10-fold) by DP activity. We hypothesized that miR-10b may function as a pro-survival, oncogenic factor in FLC. To test this hypothesis, we performed loss-of-function experiments using miR-10b locked nucleic acid (LNA) inhibitors in a cell culture model generated from a patient-derived xenograft tumor. We found that suppression of miR-10b significantly lowered FLC cell viability and proliferation. We then performed an integrative analysis of chromatin run-on sequencing (ChRO-seq) and RNA-sequencing (RNA-seq) data to identify genes that are likely regulated by miR-10b in the FLC tumor tissue. This analysis led to the discovery of TRIM35, or tripartite motif-containing protein family member 35, which we validated in the FLC cell line. TRIM35 is known to regulate the function of pyruvate kinase, which may explain the reduction of cellular viability when miR-10b is inhibited. Our future directions include resolving the direct relationship between miR-10b and TRIM35, performing cellular migration and invasion assays after miR-10b inhibition, and developing and analyzing a miR-10b null FLC cell line. Additionally, we are identifying other oncogenic miRNAs with which miR-10b coordinates and directs FLC cell survival. Citation Format: Adam Francisco, Andrew Massa, Matt Kanke, Praveen Sethupathy. MicroRNA-10b is a regulator of cellular viability and proliferation in fibrolamellar carcinoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-048.

Trim71 Links an Ancient MicroRNA Pathway to Neural Stem Cell Development and Human Congenital Hydrocephalus

Abstract INTRODUCTION Congenital hydrocephalus (CH), the most common brain malformation that affects 1:1000 live births, remains poorly treated owing to incomplete understanding of disease pathogenesis. Identification of novel CH disease genes is a powerful approach to understanding the molecular genetics of CH. Whole exome sequencing in humans has identified TRIM71 as a bona fide CH disease gene. Trim71 is the major target of the ancient let-7 pathway that famously regulates cell differentiation and organismal development via microRNA-mediated gene silencing in Caenorhabditis elegans. Previous works have implicated Trim71 in the regulation of embryonic neural stem cell (NSC) fate, yet its cellular and molecular function in NSCs remain poorly understood. METHODS To gain a better understanding of Trim71 as a paradigm of NSC involvement in CH, we have generated a mouse model that harbors the point mutation in Trim71 that results in human CH (R595H) using CRISPR-Cas9. RESULTS We found that a subset of mice heterozygous for the Trim71 R595H mutant allele developed severe communicating hydrocephalus with accompanying macrocephaly by 4 wk of age. CONCLUSION Investigation into the function of Trim71 and its CH-causing mutations using our novel mouse models presents outstanding opportunities to increase understanding of human NSC regulation, brain development, and CH pathogenesis.

Epigenetics of autoimmune liver diseases: current progress and future directions

Abstract Autoimmune liver disease, including autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis, is a chronic autoimmune liver condition characterized by abnormal accumulation of proinflammatory immune cells. As more and more genetic studies are conducted, it is becoming increasingly clear that genetic risk cannot fully explain disease pathogenesis. Epigenetic modifications, including CpG island DNA methylation, histone modification, and microRNA-mediated gene silencing, are gradually being acknowledged to play a role in immune cellular identity and disease heterogeneity. Given the heterogeneity and environmental exposure bias observed in patients, it is essential to uncover the epigenetic mechanisms of autoimmune liver disease, which could integrate environmental skewing, cell lineage commitment, and genetics to provide a fuller mechanistic understanding of the disease. Epigenetic profiling holds promise for identifying diagnostic and prognostic biomarkers, and epigenetic manipulations could result in novel therapies. Cutting-edge breakthroughs are frequently being made in epigenetics, which might guide future explorations into autoimmune liver disease.

MiRISC Composition Determines Target Fates in Time and Space

MicroRNA-mediated gene silencing can occur by either target mRNA degradation or translational repression. In this issue of Developmental Cell, Dallaire etal. (2018) show in C. elegans that tissue-specific composition of the silencing complex, miRISC, plays a major role in determining the fate of target mRNAs.

Obesity-induced upregulation of miR-361-5p promotes hepatosteatosis through targeting Sirt1

ObjectiveObesity is associated with an increased risk of many metabolic disorders, including non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanisms remain poorly understood. Recent studies have demonstrated that MicroRNA-mediated gene silencing plays an important role in hepatic triglyceride (TG) metabolism. In the present study, we aimed to investigate the pathological function of miR-361-5p in the development of NAFLD. MethodsExpression levels of miR-361-5p was determined by quantitative real-time PCR in livers of obese mice and NAFLD patients. Liver tissues from mice with miR-361-5p overexpression or inhibition were collected and analyzed by TG contents, gene expression profile. ResultsExpression of miR-361-5p was increased in the livers of two obese mouse models and NAFLD subjects. Overexpression of miR-361-5p in C57BL/6 mice led to hepatosteatosis, whereas inhibition of miR-361-5p expression in db/db mice improved TG accumulation and insulin sensitivity. Mechanistically, we identified Sirt1 as a direct target gene of miR-361-5p and re-introduction of Sirt1 largely abolished the metabolic action of miR-361-5p. ConclusionsOur results demonstrated the role of miR-361-5p in the regulation of hepatic TG homeostasis, which may provide potential therapeutic target for hepatosteatosis.

Mechanistic Insights into MicroRNA-Mediated Gene Silencing.

MicroRNAs (miRNAs) posttranscriptionally regulate gene expression by repressing protein synthesis and exert a broad influence over development, physiology, adaptation, and disease. Over the past two decades, great strides have been made toward elucidating how miRNAs go about shutting down messenger RNA (mRNA) translation and promoting mRNA decay.

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.