Characteristics of microRNAs and Target Genes in Maize Root under Drought Stress

Maize (Zea mays) is an important multi-functional crop. The growth and yield of maize are severely affected by drought stress. Previous studies have shown that microRNAs (miRNAs) in maize play important roles in response to abiotic stress; however, their roles in response to drought stress in maize roots is unclear. In our study, we found 375 miRNAs in the roots of 16 inbred lines. Of the 16 lines, zma-MIR168, zma-MIR156, and zma-MIR166 were highly expressed, whereas zma-MIR399, zma-MIR2218, and zma-MIR2275 exhibited low expression levels. The expression patterns of miRNA in parental lines and their derived RILs are different. Over 50% of miRNAs exhibited a lower expression in recombinant inbred lines than in parents. The expression of 50 miRNAs was significantly altered under water stress (WS) in at least three inbred lines, and the expression of miRNAs in drought-tolerant lines changed markedly. To better understand the reasons for miRNA response to drought, the degree of histone modifications for miRNA genes was estimated. The methylation level of H3K4 and H3K9 in miRNA precursor regions changed more noticeably after WS, but no such phenomenon was seen for DNA methylation and m6A modification. After the prediction of miRNA targets using psRNATarget and psRobot, we used correlation analysis and qRT-PCR to further investigate the relationship between miRNAs and target genes. We found that 87 miRNA–target pairs were significantly negatively correlated. In addition, a weighted gene co-expression network analysis using miRNAs, as well as their predicted targets, was conducted to reveal that miR159, miR394, and miR319 may be related to maize root growth. The results demonstrated that miRNAs might play essential roles in the response to drought stress.     

ADAR Enzyme and miRNA Story: A Nucleotide that Can Make the Difference

Adenosine deaminase acting on RNA (ADAR) enzymes convert adenosine (A) to inosine (I) in double-stranded (ds) RNAs. Since Inosine is read as Guanosine, the biological consequence of ADAR enzyme activity is an A/G conversion within RNA molecules. A-to-I editing events can occur on both coding and non-coding RNAs, including microRNAs (miRNAs), which are small regulatory RNAs of ~20–23 nucleotides that regulate several cell processes by annealing to target mRNAs and inhibiting their translation. Both miRNA precursors and mature miRNAs undergo A-to-I RNA editing, affecting the miRNA maturation process and activity. ADARs can also edit 3′ UTR of mRNAs, further increasing the interplay between mRNA targets and miRNAs. In this review, we provide a general overview of the ADAR enzymes and their mechanisms of action as well as miRNA processing and function. We then review the more recent findings about the impact of ADAR-mediated activity on the miRNA pathway in terms of biogenesis, target recognition, and gene expression regulation.     

miRNA Profiling and Its Role in Multi-Omics Regulatory Networks Connected with Somaclonal Variation in Cucumber (Cucumis sativus L.)

The role of miRNAs in connection with the phenomenon of somaclonal variation, which occurs during plant in vitro culture, remains uncertain. This study aims to investigate the possible role of miRNAs in multi-omics regulatory pathways in cucumber somaclonal lines. For this purpose, we performed sRNA sequencing (sRNA-seq) from cucumber fruit samples identified 8, 10 and 44 miRNAs that are differentially expressed between somaclones (S1, S2, S3 lines) and the reference B10 line of Cucumis sativus. For miRNA identification, we use ShortStack software designed to filter miRNAs from sRNAs according to specific program criteria. The identification of predicted in-silico targets revealed 2,886 mRNAs encoded by 644 genes. The functional annotation of miRNA’s target genes and gene ontology classification revealed their association with metabolic processes, response to stress, multicellular organism development, biosynthetic process and catalytic activity. We checked with bioinformatic analyses for possible interactions at the level of target proteins, differentially expressed genes (DEGs) and genes affected by genomic polymorphisms. We assume that miRNAs can indirectly influence molecular networks and play a role in many different regulatory pathways, leading to somaclonal variation. This regulation is supposed to occur through the process of the target gene cleavage or translation inhibition, which in turn affects the proteome, as we have shown in the example of molecular networks. This is a new approach combining levels from DNA-seq through mRNA-seq, sRNA-seq and in silico PPI in the area of plants’ somaclonal variation.     

microRNA-mRNA Profile of Skeletal Muscle Differentiation and Relevance to Congenital Myotonic Dystrophy

microRNAs (miRNAs) regulate messenger RNA (mRNA) abundance and translation during key developmental processes including muscle differentiation. Assessment of miRNA targets can provide insight into muscle biology and gene expression profiles altered by disease. mRNA and miRNA libraries were generated from C2C12 myoblasts during differentiation, and predicted miRNA targets were identified based on presence of miRNA binding sites and reciprocal expression. Seventeen miRNAs were differentially expressed at all time intervals (comparing days 0, 2, and 5) of differentiation. mRNA targets of differentially expressed miRNAs were enriched for functions related to calcium signaling and sarcomere formation. To evaluate this relationship in a disease state, we evaluated the miRNAs differentially expressed in human congenital myotonic dystrophy (CMD) myoblasts and compared with normal control. Seventy-four miRNAs were differentially expressed during healthy human myocyte maturation, of which only 12 were also up- or downregulated in CMD patient cells. The 62 miRNAs that were only differentially expressed in healthy cells were compared with differentiating C2C12 cells. Eighteen of the 62 were conserved in mouse and up- or down-regulated during mouse myoblast differentiation, and their C2C12 targets were enriched for functions related to muscle differentiation and contraction.     

Identification of Novel miRNAs and Their Target Genes in the Response to Abscisic Acid in Arabidopsis

miRNAs are involved in various biological processes, including adaptive responses to abiotic stress. To understand the role of miRNAs in the response to ABA, ABA-responsive miRNAs were identified by small RNA sequencing in wild-type Arabidopsis, as well as in abi1td, mkkk17, and mkkk18 mutants. We identified 10 novel miRNAs in WT after ABA treatment, while in abi1td, mkkk17, and mkkk18 mutants, three, seven, and nine known miRNAs, respectively, were differentially expressed after ABA treatment. One novel miRNA (miRn-8) was differentially expressed in the mkkk17 mutant. Potential target genes of the miRNA panel were identified using psRNATarget. Sequencing results were validated by quantitative RT-PCR of several known and novel miRNAs in all genotypes. Of the predicted targets of novel miRNAs, seven target genes of six novel miRNAs were further validated by 5′ RLM-RACE. Gene ontology analyses showed the potential target genes of ABA-responsive known and novel miRNAs to be involved in diverse cellular processes in plants, including development and stomatal movement. These outcomes suggest that a number of the identified miRNAs have crucial roles in plant responses to environmental stress, as well as in plant development, and might have common regulatory roles in the core ABA signaling pathway.     

MicroRNA Expression Profile in TSC Cell Lines and the Impact of mTOR Inhibitor

The aim of this study was to assess the potential implication of microRNA on tuberous sclerosis (TSC) pathogenesis by performing microRNA profiling on cell lines silencing TSC1 or TSC2 genes using qPCR panels, before and after incubation with rapamycin. Significant differences in expression were observed between samples before and after rapamycin treatment in nineteen miRNAs in TSC1, five miRNAs in TSC2 and seven miRNAs in controls. Of miRNAs dysregulated before rapamycin treatment, three normalized after treatment in the TSC1 group (miR-21-3p, miR-433-3p, let-7g-3p) and one normalized in the TSC2 group (miR-1224-3p). Of the miRNAs dysregulated before rapamycin treatment in the TSC1 and TSC2 groups, two did not normalize after treatment (miR-33a-3p, miR-29a-3p). The results of the possible targets indicated that there are four common genes with seed regions susceptible to regulation by those miRNAs: ZBTB20, PHACTR2, PLXNC1 and ATP1B4. Our data show no changes in mRNA expression of these targets after rapamycin treatment. In conclusion, results of our study indicate the involvement of miRNA dysregulation in the pathogenesis of TSC. Some of the miRNA might be used as markers of treatment efficacy and autonomic miRNA as a target for future therapy.     

From miRNA Target Gene Network to miRNA Function: miR-375 Might Regulate Apoptosis and Actin Dynamics in the Heart Muscle via Rho-GTPases-Dependent Pathways

MicroRNAs (miRNAs) are short, single-stranded, non-coding ribonucleic acid (RNA) molecules, which are involved in the regulation of main biological processes, such as apoptosis or cell proliferation and differentiation, through sequence-specific interaction with target mRNAs. In this study, we propose a workflow for predicting miRNAs function by analyzing the structure of the network of their target genes. This workflow was applied to study the functional role of miR-375 in the heart muscle (myocardium), since this miRNA was previously shown to be associated with heart diseases, and data on its function in the myocardium are mostly unclear. We identified PIK3CA, RHOA, MAPK3, PAFAH1B1, CTNNB1, MYC, PRKCA, ERBB2, and CDC42 as key genes in the miR-375 regulated network and predicted the possible function of miR-375 in the heart muscle, consisting mainly in the regulation of the Rho-GTPases-dependent signaling pathways. We implemented our algorithm for miRNA function prediction into a Python module, which is available at GitHub.     

High-Throughput Sequencing Reveals Novel microRNAs Involved in the Continuous Flowering Trait of Longan (Dimocarpus longan Lour.)

A major determinant of fruit production in longan (Dimocarpus longan Lour.) is the difficulty of blossoming. In this study, high-throughput microRNA sequencing (miRNA-Seq) was carried out to compare differentially expressed miRNAs (DEmiRNAs) and their target genes between a continuous flowering cultivar ‘Sijimi’ (SJ), and a unique cultivar ‘Lidongben’ (LD), which blossoms only once in the season. Over the course of our study, 1662 known miRNAs and 235 novel miRNAs were identified and 13,334 genes were predicted to be the target of 1868 miRNAs. One conserved miRNA and 29 new novel miRNAs were identified as differently expressed; among them, 16 were upregulated and 14 were downregulated. Through the KEGG pathway and cluster analysis of DEmiRNA target genes, three critical regulatory pathways, plant–pathogen interaction, plant hormone signal transduction, and photosynthesis-antenna protein, were discovered to be strongly associated with the continuous flowering trait of the SJ. The integrated correlation analysis of DEmiRNAs and their target mRNAs revealed fourteen important flowering-related genes, including COP1-like, Casein kinase II, and TCP20. These fourteen flowering-related genes were targeted by five miRNAs, which were novel-miR137, novel-miR76, novel-miR101, novel-miR37, and csi-miR3954, suggesting these miRNAs might play vital regulatory roles in flower regulation in longan. Furthermore, novel-miR137 was cloned based on small RNA sequencing data analysis. The pSAK277-miR137 transgenic Arabidopsis plants showed delayed flowering phenotypes. This study provides new insight into molecular regulation mechanisms of longan flowering.