Seong Wook Yang

Rapid Detection of MicroRNA by a Silver Nanocluster DNA Probe

MicroRNAs (miRNAs) are regulatory small RNAs that have important roles in numerous developmental, metabolic, and disease processes of plants and animals. The individual levels of miRNAs can be useful biomarkers for cellular events or disease diagnosis. Thus, innovative new tools for rapid, specific, and sensitive detection of miRNAs are an important field of research. Using the fluorescence properties of DNA-nanosilver clusters (DNA/AgNC), we have designed a DNA/AgNC probe that can detect the presence of target miRNA. Here, we show that the red fluorescence of the DNA/AgNC probe is diminished upon the presence of target miRNA without pre- or postmodification, addition of extra enhancer molecules, and labeling. The DNA/AgNC probe emission was lowest when the complementary miRNA target was present and was significantly higher for four other control miRNA sequences. Also, when adding whole plant endogenous RNA to the DNA/AgNC probe, the emission was significantly higher for the mutant where miRNA was deficient. On the basis of these findings, we suggest that these DNA/AgNC probes could be developed into a new, simple, inexpensive, and instant technique for miRNAs detection.

Design aspects of bright red emissive silver nanoclusters/DNA probes for microRNA detection.

The influence of the nucleic acid secondary structure on the fast (1 h) formation of bright red emissive silver nanoclusters (AgNCs) in a DNA sequence (DNA-12nt-RED-160), designed for the detection of a microRNA sequence (RNA-miR160), was investigated. The findings show that especially the propensity for mismatch self-dimer formation of the DNA probes can be a good indicator for the creation and stabilization of red emissive AgNCs. Also, the role of the thermal stability of the secondary DNA structures (mismatch self-dimer and hairpin monomers) and the observed AgNC red emission intensity were investigated. These findings can form the basis for a rationale to design new red emissive AgNC-based probes. As an example, a bright red emissive AgNC-based DNA probe was designed for RNA-miR172 detection. The latter opens the possibility to create a variety of AgNC-based DNA probes for the specific detection of plant and animal miRNAs.

STA1, an Arabidopsis pre-mRNA processing factor 6 homolog, is a new player involved in miRNA biogenesis

MicroRNAs (miRNAs) are small regulatory RNAs that have important regulatory roles in numerous developmental and metabolic processes in most eukaryotes. In Arabidopsis, DICER-LIKE1 (DCL1), HYPONASTIC LEAVES 1, SERRATE, HUA ENHANCER1 and HASTY are involved in processing of primary miRNAs (pri-miRNAs) to yield precursor miRNAs (pre-miRNAs) and eventually miRNAs. In addition to these components, mRNA cap-binding proteins, CBP80/ABA HYPERSENSITIVE1 and CBP20, also participate in miRNA biogenesis. Here, we show that STABILIZED1 (STA1), an Arabidopsis pre-mRNA processing factor 6 homolog, is also involved in the biogenesis of miRNAs. Similar to other miRNA biogenesis-defective mutants, sta1-1 accumulated significantly lower levels of mature miRNAs and concurrently higher levels of pri-miRNAs than wild type. The dramatic reductions of mature miRNAs were associated with the accumulation of their target gene transcripts and developmental defects. Furthermore, sta1-1 impaired splicing of intron containing pri-miRNAs and decreased transcript levels of DCL1. These results suggest that STA1 is involved in miRNA biogenesis directly by functioning in pri-miRNA splicing and indirectly by modulating the DCL1 transcript level.

Arabidopsis DRB4 protein in antiviral defense against Turnip yellow mosaic virus infection

RNA silencing is an important antiviral mechanism in diverse eukaryotic organisms. In Arabidopsis DICER-LIKE 4 (DCL4) is the primary antiviral Dicer, required for the production of viral small RNAs from positive-strand RNA viruses. Here, we showed that DCL4 and its interacting partner dsRNA-binding protein 4 (DRB4) participate in the antiviral response to Turnip yellow mosaic virus (TYMV), and that both proteins are required for TYMV-derived small RNA production. In addition, our results indicate that DRB4 has a negative effect on viral coat protein accumulation. Upon infection DRB4 expression was induced and DRB4 protein was recruited from the nucleus to the cytoplasm, where replication and translation of viral RNA occur. DRB4 was associated with viral RNA in vivo and directly interacted in vitro with a TYMV RNA translational enhancer, raising the possibility that DRB4 might repress viral RNA translation. In plants the role of RNA silencing in viral RNA degradation is well established, but its potential function in the regulation of viral protein levels has not yet been explored. We observed that severe infection symptoms are not necessarily correlated with enhanced viral RNA levels, but might be caused by elevated accumulation of viral proteins. Our findings suggest that the control of viral protein as well as RNA levels might be important for mounting an efficient antiviral response.

Perturbation of NgTRF1 expression induces apoptosis-like cell death in tobacco BY-2 cells and implicates NgTRF1 in the control of telomere length and stability

Telomeres are specialized nucleoprotein complexes that are essential for preserving chromosome integrity in eukaryotic cells. Several potential telomere binding proteins have recently been identified in higher plants, but nothing is known about their in vivo functions. We previously identified NgTRF1 as a double-stranded telomeric repeat binding factor in tobacco (Nicotiana tabacum) and here show that the binding of NgTRF1 to telomeric repeats inhibits telomerase-mediated telomere extension. To determine whether NgTRF1 is involved in telomere length regulation, we established transgenic tobacco BY-2 cell lines that overexpress or suppress NgTRF1. Pulsed-field gel electrophoresis showed that 35S::NgTRF1 cells exhibited significantly shortened telomeres (45 to 10 kb), whereas 35S::antisense-NgTRF1 cells contained longer telomeres (80 to 25 kb) compared with wild-type and 35S::GUS control cells (65 to 15 kb), indicating that telomere length inversely correlates with the amount of functional NgTRF1 in BY-2 cells. 35S::NgTRF1 cells with shorter telomeres displayed a progressive reduction in cell viability and stopped dividing after 25 to 40 successive rounds of 12-d batch subculture, in sharp contrast with control cells, which have an unlimited capacity for division. Internucleosomal DNA fragmentation, mitochondrial release of cytochrome c, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling positive nuclei were detected in 35S::NgTRF1 cells during prolonged subculture, indicating that enhanced cell death was attributable to an apoptosis-like mechanism. 35S::antisense-NgTRF1 cells containing low levels of NgTRF1 also exhibited a progressive decrease in cell viability and apoptotic cell death, but less so than did 35S::NgTRF1 cells, suggesting that the level of NgTRF1 is critically associated with cell viability. Taken together, these data indicate that perturbation of NgTRF1 expression results in changes in telomere length and stability, which in turn causes apoptotic cell death in transgenic BY-2 cells. These results are discussed in light of the suggestion that NgTRF1 is involved in the mechanism by which telomere length and stability are maintained. We further suggest that the structural stability of telomeres, in addition to length maintenance, is essential for their function and for the immortality of BY-2 cells.

In-solution multiplex miRNA detection using DNA-templated silver nanocluster probes

MicroRNAs (miRNAs) are small regulatory RNAs (size ∼21nt to ∼25nt) that can be used as biomarkers of disease diagnosis, and efforts have been directed towards the invention of a rapid, simple and sequence-selective detection method for miRNAs. We recently developed a DNA/silver nanoclusters (AgNCs)-based turn-off fluorescence method in the presence of target miRNA. To further advance our method toward multiplex miRNA detection in solution, the design of various fluorescent DNA/AgNCs probes was essential. Therefore, tethering of DNA-12nt scaffolds with 9 different AgNCs emitters to target-sensing DNA sequences was investigated. Interestingly, for the creation of spectrally different DNA/AgNCs probes, not only were the emitters encapsulated in 9 different DNA-12nt scaffolds necessary but the tethered target-sensing DNA sequences are also crucial to tune the fluorescence across the visible to infra-red region. In this study, we obtained three spectrally distinctive emitters of each DNA/AgNCs probes such as green, red, and near-infrared (NIR) fluorescence. Using these DNA/AgNCs probes, we here show a proof of concept for a rapid, one-step, in-solution multiplex miRNA detection method.

COP1 E3 ligase protects HYL1 to retain microRNA biogenesis

Constitutive photomorphogenic 1 (COP1) is a RING-finger E3 ligase that plays a central role in photomorphogenesis by destabilizing many light-regulated transcription factors and photoreceptors. Here, we reveal a novel function for COP1 E3 ligase in controlling global miRNA biogenesis in Arabidopsis thaliana. In cop1 mutants, the level of miRNAs is dramatically reduced because of the diminution of HYPONASTIC LEAVES 1 (HYL1), an RNA-binding protein required for precise miRNA processing. HYL1 is destabilized by an unidentified protease, which we tentatively call protease X, that specifically cleaves the N-terminal region from HYL1, thus neutralizing its function. Our results further show that the cytoplasmic partitioning of COP1 under light is essential to protect HYL1 against protease X. Taken together, we suggest a novel regulatory network involving HYL1, protease X, COP1 and light signalling that is indispensable for miRNA biogenesis in Arabidopsis thaliana.

Multiplexed microRNA Detection Using Lanthanide-Labeled DNA Probes and Laser Ablation Inductively Coupled Plasma Mass Spectrometry

In the past decade, microRNAs (miRNAs) have drawn increasing attention due to their role in regulation of gene expression. Especially, their potential as biomarkers in disease diagnostics has motivated miRNA research, including the development of simple, accurate, and sensitive detection methods. The narrow size range of miRNAs (20-24 nucleotides) combined with the chemical properties of conventional reporter tags has hampered the development of multiplexed miRNA assays. In this study, we have used lanthanide-labeled DNA probes for the detection of miRNAs on membranes using laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS). Three miRNAs from Arabidopsis thaliana were analyzed simultaneously with high specificity, and the sensitivity of the method was comparable to radioactive detection (low femtomol range). The perspective of the developed method is highly multiplexed and quantitative miRNA analysis with high specificity and sensitivity.

Regulation of MIR165/166 by class II and class III homeodomain leucine zipper proteins establishes leaf polarity

Significance Leaves, being the prime photosynthetic organ of plants, are critical in many ways to our current biosphere. A defining characteristic, which also optimizes their function, is their flat shape that depends on the correct patterning of their upper and lower tissues during development. Here, we show that the correct patterning of upper and lower leaf tissues depends on two types of transcription factors (class II and class III homeodomain leucine zipper (HD-ZIPs) that act together to repress a set of miRNAs (MIR165/166), which in turn, represses the activity of these transcription factors (class III HD-ZIPs). This three-way interaction maintains the balance of tissue identities during growth, leading to the formation of a flat leaf. A defining feature of plant leaves is their flattened shape. This shape depends on an antagonism between the genes that specify adaxial (top) and abaxial (bottom) tissue identity; however, the molecular nature of this antagonism remains poorly understood. Class III homeodomain leucine zipper (HD-ZIP) transcription factors are key mediators in the regulation of adaxial–abaxial patterning. Their expression is restricted adaxially during early development by the abaxially expressed microRNA (MIR)165/166, yet the mechanism that restricts MIR165/166 expression to abaxial leaf tissues remains unknown. Here, we show that class III and class II HD-ZIP proteins act together to repress MIR165/166 via a conserved cis-element in their promoters. Organ morphology and tissue patterning in plants, therefore, depend on a bidirectional repressive circuit involving a set of miRNAs and its targets.

Locking-to-unlocking system is an efficient strategy to design DNA/silver nanoclusters (AgNCs) probe for human miRNAs

MicroRNAs (miRNAs), small non-coding RNA molecules, are important biomarkers for research and medical purposes. Here, we describe the development of a fast and simple method using highly fluorescent oligonucleotide-silver nanocluster probes (DNA/AgNCs) to efficiently detect specific miRNAs. Due to the great sequence diversity of miRNAs in humans and other organisms, a uniform strategy for miRNA detection is attractive. The concept presented is an oligonucleotide-based locking-to-unlocking system that can be endowed with miRNA complementarity while maintaining the same secondary structure. The locking-to-unlocking system is based on fold-back anchored DNA templates that consist of a cytosine-rich loop for AgNCs stabilization, an miRNA recognition site and an overlap region for hairpin stabilization. When an miRNA is recognized, fluorescence in the visible region is specifically extinguished in a concentration-dependent manner. Here, the exact composition of the fold-back anchor for the locking-to-unlocking system has been systematically optimized, balancing propensity for loop-structure formation, encapsulation of emissive AgNCs and target sensitivity. It is demonstrated that the applied strategy successfully can detect a number of cancer related miRNAs in RNA extracts from human cancer cell lines.