pri-miRNA Transcripts Research Articles

A systematical strategy for quality markers screening of different methods processing Platycodonis radix based on phytochemical analysis and the impact on Chronic Obstructive Pulmonary Disease

Ethnopharmacological relevanceBaihezhijiegeng is a processed product of Platycodonis radix, and it's effective in the treatment of Chronic Obstructive Pulmonary Disease (COPD). However, the specific mechanism of action has not been reported in the literature. Aim of the studyWe attempted to evaluate the phytochemical composition and pharmaco-dynamics of Platycodon grandiflorum (PG) and BJ to clarify the mechanism behind the expectorant effect of BJ. Materials and methodsWe integrated the ultra-high-performance liquid chromatography-linear trap quadrupole orbitrap velos mass spectrometry (UPLC-LTQ Orbitrap MS/MS) and the ultra-performance liquid chromatography quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) methods to identify the chemical constituents of PG and BJ. Moreover, correlation and multivariate statistical analyses were utilized to seek the candidate quality markers of PG and BJ. Analysis of effective herbal chemical components using UPLC-Q-TOF-MS/MS and retrieval of COPD disease targets from OMIM, TTD, GeneCard databases. Protein-protein interaction (PPI) and topology analyses were performed using the String database and Cytoscape 3.7.2 software; gene ontology (GO) functional enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed using the Metescape platform on common targets. Moreover, we used molecular docking to predict the potential mechanism of quality markers for developing anti-COPD activity. Simultaneously, the model of COPD was established by exposing the animals to cigarette smoke combined with a tracheal drip injection of lipopolysaccharide (LPS). Using the ELISA method, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB) to determine tumor-necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and matrix metalloproteinase (MMP)9 levels in serum and IL-4, IL-10, IFN-γ levels, epidermal growth factor receptor (EGFR) and MUC5AC expression in lung tissue of COPD rats to explore the therapeutic effects of PG and BJ on the COPD rat model. ResultsThe chemical identification of JG and PG extracts using UPLC-Q-TOF-MS/MS and UPLC-LTQ Orbitrap MS/MS showed 71 compounds, including 47 saponins, 16 phenolic acids, four flavonoids, and four other components. The multivariate statistical analysis showed that seven quality markers were screened. Network pharmacology results showed a role in biological processes such as cellular response to hydrogen peroxide, positive regulation of pri-miRNA transcription from RNA polymerase II promoter, molecular functions such as oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor, bile acid binding and other molecular functions. In COPD rats, histopathological findings depicted that BJ administration could effectively inhibit inflammatory cell infiltration and mucus hypersecretion, and improve the lung pathological status in rats with COPD. Moreover, BJ could significantly decrease TNF-α, IL-1β, IL-6, and matrix metalloproteinase (MMP)9 levels in the serum and interferon (IFN)-γ levels in lung tissues of rats with COPD (p < 0.01), and significantly increase IL-4 and IL-10 levels in their lung tissues (p < 0.01), suggesting its inhibition of the inflammatory response in vivo. Additionally, EGFR and MUC5AC were reduced in the lung tissues of rats with COPD and airway mucus hypersecretion in rats with COPD. ConclusionThis study revealed the material basis of PG and BJ for anti-COPD activity and discovered the quality markers of PG and BJ which could affect the anti-COPD activity. The therapeutic effects of BJ may be attributed to the regulation of the inflammatory mediators and mediation of the EGFR/MUC5AC pathway in rats with COPD.

Quercetin's Dual Mode of Action to Counteract the Sp1-miR-27a Axis in Colorectal Cancer Cells.

Quercetin (Qc) inhibits cell proliferation and induces apoptosis in a variety of cancer cells. The molecular mechanism of action has not been fully elucidated; however, interplay with some miRNAs has been reported, specifically with miR-27a, an onco-miRNA overexpressed in several malignancies. Here, we show that Qc reduces cell viability and induces apoptosis in HCT116 and HT-29 colon cancer cells, by upregulating negative modulators of proliferation pathways such as Sprouty2, PTEN and SFRP1. These are targets of miR-27a whose high expression is reduced by Qc. Moreover, miR-23a, and miR-24-2, the two other components of the unique gene cluster, and the pri-miRNA transcript are reduced, evoking a transcriptional regulation of the entire cluster by Sp1. Mechanistically, we show that Qc is rapidly internalized and localizes in the nucleus, where it likely interacts with Sp1, inducing its proteasomal degradation. Sp1 is further repressed by ZBTB10, an Sp1 competitor for DNA binding that is an miR-27a target and whose levels increase following Qc. SP1 mRNA is also reduced, supporting the regulation of its own gene transcription. Finally, Sp1 knockdown elicits the impaired transcription of the entire cluster and the upregulation of the miR-27a targets, phenocopying the effects of Qc. Through this dual mode of action, Qc counteracts the protumoral Sp1-miR-27a axis, opening the way for novel therapies based on its association as neoadjuvant with known anticancer treatments.

The HOS15-HDA9 complex associates with HYL1 to modulate miRNA expression in response to ABA signaling.

The regulation of microRNA (miRNA) biogenesis is crucial for maintaining plant homeostasis under biotic and abiotic stress. The crosstalk between the RNA polymerase II (Pol-II) complex and the miRNA processing machinery has emerged as a central hub modulating transcription and co-transcriptional processing of primary miRNA transcripts (pri-miRNAs). However, it remains unclear how miRNA-specific transcriptional regulators recognize MIRNA loci. Here, we show that the Arabidopsis (Arabidopsis thaliana) HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE15 (HOS15)-HISTONE DEACETYLASE9 (HDA9) complex is a conditional suppressor of miRNA biogenesis, particularly in response to ABA. When treated with ABA, hos15/hda9 mutants show enhanced transcription of pri-miRNAs that is accompanied by increased processing, leading to over-accumulation of a set of mature miRNAs. Moreover, upon recognition of the nascent pri-miRNAs, the ABA-induced recruitment of the HOS15-HDA9 complex to MIRNA loci is guided by HYPONASTIC LEAVES 1 (HYL1). The HYL1-dependent recruitment of the HOS15-HDA9 complex to MIRNA loci suppresses expression of MIRNAs and processing of pri-miRNA. Most importantly, our findings indicate that nascent pri-miRNAs serve as scaffolds for recruiting transcriptional regulators, specifically to MIRNA loci. This indicates that RNA molecules can act as regulators of their own expression by causing a negative feedback loop that turns off their transcription, providing a self-buffering system.

MiProBERT: identification of microRNA promoters based on the pre-trained model BERT.

Accurate prediction of promoter regions driving miRNA gene expression has become a major challenge due to the lack of annotation information for pri-miRNA transcripts. This defect hinders our understanding of miRNA-mediated regulatory networks. Some algorithms have been designed during the past decade to detect miRNA promoters. However, these methods rely on biosignal data such as CpG islands and still need to be improved. Here, we propose miProBERT, a BERT-based model for predicting promoters directly from gene sequences without using any structural or biological signals. According to our information, it is the first time a BERT-based model has been employed to identify miRNA promoters. We use the pre-trained model DNABERT, fine-tune the pre-trained model on the gene promoter dataset so that the model includes information about the richer biological properties of promoter sequences in its representation, and then systematically scan the upstream regions of each intergenic miRNA using the fine-tuned model. About, 665 miRNA promoters are found. The innovative use of a random substitution strategy to construct a negative dataset improves the discriminative ability of the model and further reduces the false positive rate (FPR) to as low as 0.0421. On independent datasets, miProBERT outperformed other gene promoter prediction methods. With comparison on 33 experimentally validated miRNA promoter datasets, miProBERT significantly outperformed previously developed miRNA promoter prediction programs with 78.13% precision and 75.76% recall. We further verify the predicted promoter regions by analyzing conservation, CpG content and histone marks. The effectiveness and robustness of miProBERT are highlighted.

Network Regulation of microRNA Biogenesis and Target Interaction.

MicroRNAs (miRNAs) are versatile, post-transcriptional regulators of gene expression. Canonical miRNAs are generated through the two-step DROSHA- and DICER-mediated processing of primary miRNA (pri-miRNA) transcripts with optimal or suboptimal features for DROSHA and DICER cleavage and loading into Argonaute (AGO) proteins, whereas multiple hairpin-structured RNAs are encoded in the genome and could be a source of non-canonical miRNAs. Recent advances in miRNA biogenesis research have revealed details of the structural basis of miRNA processing and cluster assistance mechanisms that facilitate the processing of suboptimal hairpins encoded together with optimal hairpins in polycistronic pri-miRNAs. In addition, a deeper investigation of miRNA-target interaction has provided insights into the complexity of target recognition with distinct outcomes, including target-mediated miRNA degradation (TDMD) and cooperation in target regulation by multiple miRNAs. Therefore, the coordinated or network regulation of both miRNA biogenesis and miRNA-target interaction is prevalent in miRNA biology. Alongside recent advances in the mechanistic investigation of miRNA functions, this review summarizes recent findings regarding the ordered regulation of miRNA biogenesis and miRNA-target interaction.

PrimiReference: a reference for analysis of primary-microRNA expression in single-nucleus sequencing data

Single-nucleus RNA-sequencing technology has revolutionized understanding of nuanced changes in gene expression between cell types within tissues. Unfortunately, our understanding of regulatory RNAs, such as microRNAs (miRNAs), is limited through both single-cell and single-nucleus techniques due to the short length of miRNAs in the cytoplasm and the incomplete reference of longer primary miRNA (pri-miRNA) transcripts in the nucleus. We build a custom reference to align and count pri-miRNA sequences in single-nucleus data. Using young and aged subventricular zone (SVZ) nuclei, we show differential expression of pri-miRNAs targeting genes involved in neural stem cells (NSC) differentiation in the aged SVZ. Furthermore, using wild-type and 5XFAD mouse model cortex nuclei, to validate the use of primiReference, we find cell-type-specific expression of pri-miRNAs known to be involved in Alzheimer's disease (AD). pri-miRNAs likely contribute to NSC dysregulation with age and AD pathology. primiReference is paramount in capturing a global profile of gene expression and regulation in single-nucleus data and can provide key insights into cell-type-specific expression of pri-miRNAs, paving the way for future studies of regulation and pathway dysregulation. By looking at pri-miRNA abundance and transcriptional differences, regulation of gene expression by miRNAs in disease and aging can be further explored.

PPMS: A framework to Profile Primary MicroRNAs from Single-cell RNA-sequencing datasets.

Single-cell/nuclei RNA-sequencing (scRNA-seq) technologies can simultaneously quantify gene expression in thousands of cells across the genome. However, the majority of the noncoding RNAs, such as microRNAs (miRNAs), cannot currently be profiled at the same scale. MiRNAs are a class of small noncoding RNAs and play an important role in gene regulation. MiRNAs originate from the processing of primary transcripts, known as primary-microRNAs (pri-miRNAs). The pri-miRNA transcripts, independent of their cognate miRNAs, can also function as long noncoding RNAs, code for micropeptides or even interact with DNA, acting like enhancers. Therefore, it is apparent that the significance of scRNA-seq pri-miRNA profiling expands beyond using pri-miRNA as proxies of mature miRNAs. However, there are no computational methods that allow profiling and quantification of pri-miRNAs at the single-cell-type resolution. We have developed a simple yet effective computational framework to profile pri-MiRNAs from single-cell RNA-sequencing datasets (PPMS). Based on user input, PPMS can profile pri-miRNAs at cell-type resolution. PPMS can be applied to both newly produced and publicly available datasets obtained via single cell or single-nuclei RNA-seq. It allows users to (i) investigate the distribution of pri-miRNAs across cell types and cell states and (ii) establish a relationship between the number of cells/reads sequenced and the detection of pri-miRNAs. Here, to demonstrate its efficacy, we have applied PPMS to publicly available scRNA-seq data generated from (i) individual chambers (ventricles and atria) of the human heart, (ii) human pluripotent stem cells during their differentiation into cardiomyocytes (the heart beating cells) and (iii) hiPSCs-derived cardiomyocytes infected with severe acute respiratory syndromecoronavirus 2.

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells.

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

R-loops at microRNA encoding loci promote co-transcriptional processing of pri-miRNAs in plants

In most organisms, the maturation of nascent RNAs is coupled to transcription. Unlike in animals, the RNA polymerase II (RNAPII) transcribes microRNA genes (MIRNAs) as long and structurally variable pri-miRNAs in plants. Current evidence suggests that the miRNA biogenesis complex assembly initiates early during the transcription of pri-miRNAs in plants. However, it is unknown whether miRNA processing occurs co-transcriptionally. Here, we used native elongating transcript sequencing data and imaging techniques to demonstrate that plant miRNA biogenesis occurs coupled to transcription. We found that the entire biogenesis occurs co-transcriptionally for pri-miRNAs processed from the loop of the hairpin but requires a second nucleoplasmic step for those processed from the base. Furthermore, we found that co- and post-transcriptional miRNA processing mechanisms co-exist for most miRNAs in a dynamic balance. Notably, we discovered that R-loops, formed near the transcription start site region of MIRNAs, promote co-transcriptional pri-miRNA processing. Furthermore, our results suggest the neofunctionalization of co-transcriptionally processed miRNAs, boosting countless regulatory scenarios.

Bioinformatics Analysis Reveals Hub Genes That May Reduce Inflammation and Complications After Cardiopulmonary Bypass.

Cardiopulmonary bypass (CPB), though indispensable in many cardiac surgery procedures, has several undesirable consequences. The aim of this study was to identify potential genes that may reduce the inflammatory response and complications after CPB. The GSE132176 dataset was selected from the Gene Expression Omnibus (GEO) database and included 10 patients with tetralogy of Fallot and 10 patients with an atrial septal defect who underwent CPB surgery. TSV files were downloaded after GEO2R processing. Protein-protein interaction analysis of common differentially expressed genes (DEGs) was performed using the Search Tool for the Retrieval of Interacting Genes. Gene modules and hub genes were visualized in the protein-protein interaction network using Cytoscape. Enrichment analysis was performed for all important DEGs, modular genes, and hub genes. A total of 72 DEGs were screened, including two functional and one hub gene module. FOS modular genes were primarily enriched in NGF-stimulated transcription, spinal cord injury, and PID AP1 pathway. The ATF3 modular gene was mainly enriched in cytomegalovirus infection and transcriptional misregulation in cancer. Hub gene modules were primarily enriched in the PID AP1 pathway, positive regulation of pri-miRNA transcription by RNA polymerase II, and the PID ATF2 pathway. FOS, JUN, ATF3, and EGR1 were the four most important hub genes; the top three hub genes were involved in the formation of AP-1 and enriched in the AP-1 pathway. Finally, we measured the expression levels of these four genes in patients undergoing CPB via qRT-PCR, and the results were consistent with those obtained in bioinformatic analysis. FOS, JUN, ATF3, and EGR1 and the AP-1 pathway may play key roles in inflammation and complications caused by CPB.

Serrate-Associated Protein 1, a splicing-related protein, promotes miRNA biogenesis in Arabidopsis.

MicroRNAs (miRNAs) are essential regulators of gene expression in metazoans and plants. In plants, most miRNAs are generated from primary miRNA transcripts (pri-miRNAs), which are processed by the Dicer-like 1 (DCL1) complex along with accessory proteins. Serrate-Associated Protein 1 (SEAP1), a conserved splicing-related protein, has been studied in human and yeast. However, the functions of SEAP1 in plants remain elusive. Lack of SEAP1 results in embryo lethality and knockdown of SEAP1 by an artificial miRNA (amiRSEAP1 ) causes pleiotropic developmental defects and reduction in miRNA accumulation. SEAP1 associates with the DCL1 complex, and may promote the interaction of the DCL1 complexes with pri-miRNAs. SEAP1 also enhances pri-miRNA accumulation, but does not affect pri-miRNA transcription, suggesting it may indirectly or directly stabilize pri-miRNAs. In addition, SEAP1 affects the splicing of some pri-miRNAs and intron retention of messenger RNAs at global levels. Our findings uncover both conserved and novel functions of SEAP1 in plants. Besides the role as a splicing factor, SEPA1 may promote miRNA biogenesis by positively modulating pri-miRNA splicing, processing and/or stability.

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Study on mechanism of Bushen Culuan Formula in treatment of polycystic ovary syndrome based on network pharmacology and molecular docking

This study used network pharmacology and molecular docking to study the mechanism of Bushen Culuan Formula in the treatment of infertility caused by polycystic ovary syndrome(PCOS). The active ingredients and potential drug targets of Bushen Cu-luan Decoction were obtained by searching the Traditional Chinese Medicine System Pharmacology(TCMSP) database, and the targets of PCOS by searching GeneCards. After the drug targets and disease targets were corrected by Uniprot, the intersection genes were obtained. STRING database and Cytoscape 3.7.2 were used for protein-protein interaction(PPI) analysis of the intersection genes. The ClueGO plug-in of Cytoscape 3.7.2 was employed to perform gene ontology(GO) enrichment and KEGG pathway enrichment for the intersection genes. Finally, molecular docking of the key active ingredients with the targets of Bushen Culuan Formula was performed using AutoDockVina and MGLtools. A total of 136 active ingredients and 314 drug targets of the decoction were obtained from TCMSP, and 136 disease targets from GeneCards. Finally, 49 drug-disease intersection genes were obtained. GO enrichment found that the genes were mainly involved in the regulation of muscle cell apoptosis, positive regulation of small molecule metabolism, core promoter binding, RNA polymerase Ⅱ regulation of pri-miRNA transcription, negative regulation of transmembrane transport and other biological functions. The enriched KEGG pathways mainly included MAPK, PI3 K-Akt, p53, and HIF-1 signaling pathways. The results of molecular docking showed that quercetin and PTGS2 can bind stably and interact through amino acid residues THR206, TRP387, ASN382, etc. This study preliminarily reveals the multi-component, multi-target, and multi-pathway mechanism of Bushen Culuan Formula in the treatment of PCOS-related infertility, which provides a basis for further research.

Construction of mRNA Regulatory Networks Reveals the Key Genes in Atrial Fibrillation

Atrial fibrillation (AF), the most familiar heart rhythm disorder, is a major cause of stroke in the world, whereas the mechanism behind AF remains largely unclear. In the current study, we used the RNA-seq method to identify 275 positively regulated mRNAs and 117 negatively regulated mRNAs in AF compared to healthy controls. Through bioinformatic analysis, it indicated that these distinctively expressed genes took part in regulating multiple AF-related biological processes and pathways, such as platelet aggregation, platelet activation, pri-miRNA transcription, and transforming growth factor-beta (TGF-β) receptor signaling pathway. Protein-protein interaction (PPI) network analysis identified ITGB5, SRC, ACTG1, ILK, ITGA2B, ITGB3, TUBB4B, CDK11A, PAFAH1B1, CDK11B, and TUBG1 as hub regulators in AF. Moreover, the quantitative real-time PCR (qRT-PCR) assay was conducted and revealed that these hub genes were remarkably overexpressed in AF samples compared to normal samples. We believed that this study would enrich the understanding of the pathogenesis of AF and enable further research on the pathogenesis of AF.

Identification of Key Genes and Pathways for Childhood Obesity Using System Biology Approach Based on Comprehensive Gene Information

Introduction: Childhood obesity is one of the most important public health issues of the 21st century. Epidemiological studies have suggested that obesity during childhood increases the risk of developing comorbidities, such as type 2 diabetes, later in life. Childhood obesity is a complex disease whose molecular mechanisms are not completely elucidated. In this context, a system biology approach could contribute to the scientific knowledge regarding genetic factors related to childhood obesity onset. Aim: To identify molecular mechanisms involved in childhood obesity by implementing a system biology approach. Methods: Experimentally validated and computationally predicted genes related to Pediatric Obesity (C2362324) were downloaded from the DisGeNET v7.0 database. The protein-protein interaction (PPI) network was constructed using the STRING v11.0 database and analyzed using NetworkAnalyst v3.0 and Cytoscape v3.8.1. The relevance of each node for the network structure and functionality was assessed using the degree method to define hub genes. Functional and pathway enrichment analyses were performed based on Gene Ontology (GO) terms and KEGG Pathways. Results: The search on the DisGeNET database retrieved 191 childhood obesity-related genes. The PPI network of these genes showed 19 hub genes (STAT3, SIRT1, BCL2, IRS1, PPARG, SOCS3, TGFB1, HDAC4, DNMT1, ADCY3, PPARA, NEDD4L, ACACB, NR0B2, VEGFA, APOA1, GHR, CALR, and MKKS). These hub genes were involved in biological processes of lipid storage / kinase activity, regulation of fatty-acid metabolic processes, regulation of pri-miRNA transcription by RNA polymerase II, and negative regulation of small molecules and carbohydrate metabolic processes. In terms of molecular functions, repressing of transcription factors biding was found enriched. Regarding KEGG Pathways, the hub genes are involved with adipocytokine signaling, insulin resistance, longevity regulation, and cytokine signaling pathways. Conclusion: Our approach identified 19 hub genes, which are highly connected and probably have a key role in childhood obesity. Moreover, functional enrichment analyses demonstrated they are enriched in several biological processes and pathways related to the underlying molecular mechanisms of obesity. These findings provide a more comprehensive information regarding genetic and molecular factors behind childhood obesity pathogenesis. Further experimental investigation of our findings may shed light on the pathophysiology of this disease and contribute to the identification of new therapeutic targets.

METTL3 regulates skeletal muscle specific miRNAs at both transcriptional and post-transcriptional levels

METTL3 increasing the mature miRNA levels via N6-Methyladenosine (m6A) modification of primary miRNA (pri-miRNA) transcripts has emerged as an important post-transcriptional regulation of miRNA biogenesis. Our previous studies and others have showed that muscle specific miRNAs are essential for skeletal muscle differentiation. Whether these miRNAs are also regulated by METTL3 is still unclear. Here, we found that m6A motifs were present around most of these miRNAs, which were indeed m6A modified as confirmed by m6A-modified RNA immunoprecipitation (m6A RIP). However, we surprisingly found that these muscle specific miRNAs were repressed instead of increased by METTL3 in C2C12 in vitro differentiation and mouse skeletal muscle regeneration after injury in vivo model. To elucidate the underlined mechanism, we performed reporter assays in 293T cells and validated METTL3 increasing these miRNAs at post-transcriptional level as expected. Furthermore, in myogenic C2C12 cells, we found that METTL3 not only repressed the expression of myogenic transcription factors (TFs) which can enhance the muscle specific miRNAs, but also increased the expression of epigenetic regulators which can repress these miRNAs. Thus, METTL3 could repress the muscle specific miRNAs at transcriptional level indirectly. Taken together, our results demonstrated that skeletal muscle specific miRNAs were repressed by METTL3 and such repression is likely synthesized transcriptional and post-transcriptional regulations.

Molecular characterization of miRNA genes and their expression in Dimocarpus longan Lour.

A genome-wide analysis of longan miRNA genes was conducted, and full-length pri-miRNA transcripts were cloned. Bioinformatics and expression analyses contributed to the functional characterization of longan miRNA genes. MicroRNAs are important for the post-transcriptional regulation of target genes. However, little is known about the transcription and regulation of miRNA genes in longan (Dimocarpus longan Lour.). In this study, 80 miRNA precursors (pre-miRNA) were predicted, and their secondary structure, size, conservation, and diversity were analyzed. Furthermore, the full-length cDNA sequences of 13 longan primary miRNAs (pri-miRNAs) were amplified by RLM-RACE and SMART-RACE and analyzed, which revealed that longan pri-miRNA transcripts have multiple transcription start sites (TSSs) and the downstream pre-miRNAs are polymorphic. Accordingly, the longan pri-miRNAs and protein-encoding genes may have similar transcriptional specificities. An analysis of the longan miRNA gene promoter elements indicated that the three most abundant cis-acting elements were light-responsive, stress-responsive, and hormone-responsive elements. A quantitative real-time PCR assay elucidated the potential spatial and temporal expression patterns of longan pre-miRNAs during the early stages of somatic embryogenesis (SE) and in different longan organs/tissues. This is the first report regarding the molecular characterization of miRNA genes and their expression profiles in longan. The generated data may serve as a foundation for future research aimed at clarifying the longan miRNA gene functions.

PRMT1 Modulates Processing of Asthma-Related Primary MicroRNAs (Pri-miRNAs) into Mature miRNAs in Lung Epithelial Cells.

Protein arginine methyltransferase-1 (PRMT1) is an important epigenetic regulator of cell function and contributes to inflammation and remodeling in asthma in a cell type-specific manner. Disease-specific expression patterns of microRNAs (miRNA) are associated with chronic inflammatory lung diseases, including asthma. The de novo synthesis of miRNA depends on the transcription of primary miRNA (pri-miRNA) transcript. This study assessed the role of PRMT1 on pri-miRNA to mature miRNA process in lung epithelial cells. Human airway epithelial cells, BEAS-2B, were transfected with the PRMT1 expression plasmid pcDNA3.1-PRMT1 for 48 h. Expression profiles of miRNA were determined by small RNA deep sequencing. Comparing these miRNAs with datasets of microarrays from five asthma patients (Gene Expression Omnibus dataset), 12 miRNAs were identified that related to PRMT1 overexpression and to asthma. The overexpression or knockdown of PRMT1 modulated the expression of the asthma-related miRNAs and their pri-miRNAs. Coimmunoprecipitation showed that PRMT1 formed a complex with STAT1 or RUNX1 and thus acted as a coactivator, stimulating the transcription of pri-miRNAs. Stimulation with TGF-β1 promoted the interaction of PRMT1 with STAT1 or RUNX1, thereby upregulating the transcription of two miRNAs: let-7i and miR-423. Subsequent chromatin immunoprecipitation assays revealed that the binding of the PRMT1/STAT1 or PRMT1/RUNX1 coactivators to primary let-7i (pri-let-7i) and primary miR (pri-miR) 423 promoter was critical for pri-let-7i and pri-miR-423 transcription. This study describes a novel role of PRMT1 as a coactivator for STAT1 or RUNX1, which is essential for the transcription of pri-let-7i and pri-miR-423 in epithelial cells and might be relevant to epithelium dysfunction in asthma.

HASTY modulates miRNA biogenesis by linking pri-miRNA transcription and processing

Post-transcriptional gene silencing mediated by microRNAs (miRNAs) modulates numerous developmental and stress response pathways. For the last two decades, HASTY (HST), the ortholog of human EXPORTIN 5, was considered to be a candidate protein that exports plant miRNAs from the nucleus to the cytoplasm. Here, we report that HST functions in the miRNA pathway independent of its cargo-exporting activity in Arabidopsis. We found that Arabidopsis mutants with impaired HST shuttling exhibit normal subcellular distribution of miRNAs. Interestingly, protein-protein interaction and microscopy assays showed that HST directly interacts with the microprocessor core component DCL1 through its N-terminal domain. Moreover, mass spectrometry analysis revealed that HST also interacts independently of its N-terminal domain with the mediator complex subunit MED37. Further experiments revealed that HST could act as a scaffold to facilitate the recruitment of DCL1 to genomic MIRNA loci by stabilizing the DCL1-MED37 complex, which in turn promotes the transcription and proper processing of primary miRNA transcripts (pri-miRNAs). Taken together, these results suggest that HST is likely associated with the formation of the miRNA biogenesis complex at MIRNA genes, promoting the transcription and processing of pri-miRNAs rather than the direct export of processed miRNAs from the nucleus.

Abstract 54: An miRNA transcriptome-wide association study of prostate cancer risk

Abstract Background: Large genome-wide association studies (GWAS) have identified hundreds of single-nucleotide polymorphisms (SNPs) associated with increased risk of prostate cancer (PRCA). The majority of these risk loci are intergenic and the underlying causal variants are presumed to confer regulatory effects on gene expression. While follow-up eQTL studies have begun to explore putative regulatory targets of PRCA risk SNPs, these have largely focused on protein-coding genes. Recent evidence demonstrates that pri-miRNA transcripts are susceptible to the same cis-acting regulatory effects as other long RNAs, and PRCA risk SNPs may confer systemic impacts on the prostate transcriptome through miRNA expression dysregulation. However, no miRNA transcriptome-wide association studies (TWAS) of PRCA risk have been conducted to date, and the role of miRNA dysregulation in PRCA risk is poorly understood. Methods: We performed a comprehensive miRNA TWAS of PRCA risk using small RNA sequencing and genome-wide genotyping data from N = 441 normal prostate tissue samples from Mayo Clinic along with N = 411 prostate adenocarcinoma (PRAD) tumor samples from the Cancer Genome Atlas (TCGA). Both miRNA expression datasets were uniformly processed separately using the CAP-miRSeq miRNA workflow and trimmed mean of M (TMM) normalization. Expressed mature miRNAs were identified as ≥ 5 reads in &amp;gt;25% of samples. PEER was used to remove known and latent factors on miRNA expression levels, and subsequent analyses were performed on residual values. Genetically regulated expression (GReX) prediction models were trained for all expressed miRNAs with evidence of cis-heritability (P&amp;lt;0.01) in the respective normal and tumor datasets using the FUSION TWAS software. TWAS for PRCA risk was performed with both sets of GReX models using single-SNP summary statistics from the recent PRACTICAL consortium PRCA case-control OncoArray GWAS meta-analysis. Significant TWAS associations were declared at a false discovery rate of 0.05. Results: A total of 615 and 571 distinct expressed miRNAs were identified in the normal and tumor tissue datasets, respectively (overlap: 480). Among these, 98 (16%) normal tissue miRNAs demonstrated significant cis-heritability (median cis-h2 = 0.09, range: 0.04-0.79) for GReX model training. Similar results were obtained from TCGA tumor samples, with 63 (11%) miRNA GReX models successfully trained (median cis-h2 = 0.11, range: 0.05-0.60). Using normal tissue GReX models, we identified three significant TWAS miRNA associations with PRCA risk: over-expression of mir-941 family miRNAs (PTWAS = 3.1E-04) and reduced expression of miR-3617-5p (PTWAS = 1.7E-04) and hsa-miR-16-2-3p (PTWAS = 9.1E-04). The TCGA tumor TWAS also identified a significant association with miR-941 overexpression (PTWAS = 9.7E-04). All associated miRNAs have previously reported oncogenic or tumor-suppressive associations in other cancer types. Conclusions: We identified significant associations with PRCA genetic risk and miRNA expression dysregulation using tumor and normal prostate tissue data. Future work will examine downstream regulatory effects of miRNA dysregulation as well as miRNA-mediated risk mechanisms via competing endogenous RNA relationships. Citation Format: Nicholas B. Larson, Shannon K. McDonnell, Zachary Fogarty, Stephen N. Thibodeau. An miRNA transcriptome-wide association study of prostate cancer risk [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 54.