Identification of the highly accumulated microRNA*s in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa)

Plant microRNAs (miRNAs) are crucial for the regulation of gene expression, which is involved in almost all the important biological processes. In the cytoplasm, the miRNA strand is selectively incorporated into a specific Argonaute (AGO)-associated gene silencing complex, while the miRNA* is degraded rapidly. Thus, most miRNA*s were thought to be biologically meaningless. Interestingly, several recent reports in both plants and animals have shaken this notion. Many miRNA*s were demonstrated to possess regulatory roles in gene expression. However, the low accumulation levels of most miRNA*s raise the question whether the activities of this small RNA (sRNA) species are widespread in plants. Here, by using publicly available sRNA high-throughput sequencing data, we found that the accumulation levels of several miRNA*s could be much higher than those of their miRNA partners in certain organs, mutants and/or AGO-associated silencing complexes of both Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Based on target prediction and degradome sequencing data-based validation, some of these highly accumulated miRNA*s were indicated to possess cleavage-based potential regulatory role on certain targets. Besides, some interesting biological interpretations were obtained based on the accumulation patterns of the miRNA*s, the annotations of the target genes, and literature mining. Taken together, the expanded list of the highly accumulated miRNA*s along with their potential target genes discovered in this study further strengthened the current notion that certain members of the miRNA* species are biologically relevant, which needs further inspection.     

The role of microRNA-26b in human adipocyte differentiation and proliferation

Recent findings indicate that microRNAs (miRNAs) are involved in the regulatory network of adipogenesis and obesity. Thus far, only a few human miRNAs are known to function as adipogenic regulators, fanning interest in studies on the functional role of miRNAs during adipogenesis in humans. In a previous study, we used a microarray to assess miRNA expression during human preadipocyte differentiation. We found that expression of the miR-26b was increased in mature adipocytes. MiR-26b is an intronic miRNA located in the intron of CTDSP1 (carboxy terminal domain, RNA polymerase II, polypeptide A, small phosphatase 1). Target prediction and Renilla luciferase analyses revealed the phosphatase and tensin homolog gene (PTEN) as a putative target gene. In this study, we found that miR-26b was gradually upregulated during adipocyte differentiation. To understand the roles of miR-26b in adipogenesis, we adopted a loss-of-function approach to silence miR-26b stably in human preadipocytes. We found that miR-26b inhibition effectively suppressed adipocyte differentiation, as evidenced by decreased lipid droplets and the ability of miR-26b to decrease mRNA levels of adipocyte-specific molecular markers and triglyceride accumulation. Furthermore, the cell growth assay revealed that miR-26b inhibition promoted proliferation. Nevertheless, it had no effect on apoptosis. Taken together, these data indicate that miR-26b may be involved in adipogenesis and could be targeted for therapeutic intervention in obesity.     

Recovery of dicer-like 1-late flowering phenotype by miR172 expressed by the noncanonical DCL4-dependent biogenesis pathway

MicroRNAs (miRNAs) act as down-regulators of gene expression, and play a dominant role in eukaryote development. In Arabidopsis thaliana, DICER-LIKE 1 (DCL1) is the main processor in miRNA biogenesis, and dcl1 mutants show various developmental defects at the early stage of embryogenesis or at gamete formation. However, miRNAs responsible for the respective developmental stages of the dcl1 defects have not been identified. Here, we developed a DCL1-independent miRNA expression system using the unique DCL4-dependent miRNA, miR839. By replacing the mature sequence in the miR839 precursor sequence with that of miR172, one of the most widely conserved miRNAs in angiosperms, we succeeded in expressing miR172 from a chimeric miR839 precursor in dcl1-7 plants and observed the repression of miR172 target gene expression. In parallel, the DCL4-dependent miR172 expression rescued the late flowering phenotype of dcl1-7 by acceleration of flowering. We established the DCL1-independent miRNA expression system, and revealed that the reduction of miR172 expression is responsible for the dcl1-7 late flowering phenotype.     

MicroRNAs in Amoebozoa: Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs

The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.     

Identification of MicroRNAs in Helicoverpa armigera and Spodoptera litura Based on Deep Sequencing and Homology Analysis

The current identification of microRNAs (miRNAs) in insects is largely dependent on genome sequences. However, the lack of available genome sequences inhibits the identification of miRNAs in various insect species. In this study, we used a miRNA database of the silkworm Bombyx mori as a reference to identify miRNAs in Helicoverpa armigera and Spodoptera litura using deep sequencing and homology analysis. Because all three species belong to the Lepidoptera, the experiment produced reliable results. Our study identified 97 and 91 conserved miRNAs in H. armigera and S. litura, respectively. Using the genome of B. mori and BAC sequences of H. armigera as references, 1 novel miRNA and 8 novel miRNA candidates were identified in H. armigera, and 4 novel miRNA candidates were identified in S. litura. An evolutionary analysis revealed that most of the identified miRNAs were insect-specific, and more than 20 miRNAs were Lepidoptera-specific. The investigation of the expression patterns of miR-2a, miR-34, miR-2796-3p and miR-11 revealed their potential roles in insect development. miRNA target prediction revealed that conserved miRNA target sites exist in various genes in the 3 species. Conserved miRNA target sites for the Hsp90 gene among the 3 species were validated in the mammalian 293T cell line using a dual-luciferase reporter assay. Our study provides a new approach with which to identify miRNAs in insects lacking genome information and contributes to the functional analysis of insect miRNAs.     

An Intronic microRNA Links Rb/E2F and EGFR Signaling

The importance of microRNAs in the regulation of various aspects of biology and disease is well recognized. However, what remains largely unappreciated is that a significant number of miRNAs are embedded within and are often co-expressed with protein-coding host genes. Such a configuration raises the possibility of a functional interaction between a miRNA and the gene it resides in. This is exemplified by the Drosophila melanogaster dE2f1 gene that harbors two miRNAs, mir-11 and mir-998, within its last intron. miR-11 was demonstrated to limit the proapoptotic function of dE2F1 by repressing cell death genes that are directly regulated by dE2F1, however the biological role of miR-998 was unknown. Here we show that one of the functions of miR-998 is to suppress dE2F1-dependent cell death specifically in rbf mutants by elevating EGFR signaling. Mechanistically, miR-998 operates by repressing dCbl, a negative regulator of EGFR signaling. Significantly, dCbl is a critical target of miR-998 since dCbl phenocopies the effects of miR-998 on dE2f1-dependent apoptosis in rbf mutants. Importantly, this regulation is conserved, as the miR-998 seed family member miR-29 repressed c-Cbl, and enhanced MAPK activity and wound healing in mammalian cells. Therefore, the two intronic miRNAs embedded in the dE2f1 gene limit the apoptotic function of dE2f1, but operate in different contexts and act through distinct mechanisms. These results also illustrate that examining an intronic miRNA in the context of its host''s function can be valuable in elucidating the biological function of the miRNA, and provide new information about the regulation of the host gene itself.     

Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing

Salt is one of the main environmental factors limiting plant growth and a better understanding of mechanisms of salt stress would aid efforts to bolster plant salt tolerance. MicroRNAs are well known for their important regulatory roles in response to abiotic stress in plants. In this study, high-throughput sequencing was employed to identify miRNAs in Populus tomentosa plantlets treated or not with salt (200 mM for 10 h). We found 141 conserved miRNAs belonging to 31 families, 29 non-conserved but previously-known miRNAs belonging to 26 families, and 17 novel miRNAs. Under salt stress, 19 miRNAs belonging to seven conserved miRNA families were significantly downregulated, and two miRNAs belonging to two conserved miRNA families were upregulated. Of seven non-conserved miRNAs with significantly altered expression, five were downregulated and two were upregulated. Furthermore, eight miRNAs were validated by qRT-PCR and their dynamic differential expressions were analyzed. In addition, 269 target genes of identified miRNAs were predicted and categorized by function. These results provide new insights into salt-responsive miRNAs in Populus.