Dual Catalytic Hairpin Assembly-Based Automatic Molecule Machine for Amplified Detection of Auxin Response Factor-Targeted MicroRNA-160

MicroRNA160 plays a crucial role in plant development by negatively regulating the auxin response factors (ARFs). In this manuscript, we design an automatic molecule machine (AMM) based on the dual catalytic hairpin assembly (D-CHA) strategy for the signal amplification detection of miRNA160. The detection system contains four hairpin-shaped DNA probes (HP1, HP2, HP3, and HP4). For HP1, the loop is designed to be complementary to miRNA160. A fragment of DNA with the same sequences as miRNA160 is separated into two pieces that are connected at the 3′ end of HP2 and 5′ end of HP3, respectively. In the presence of the target, four HPs are successively dissolved by the first catalytic hairpin assembly (CHA1), forming a four-way DNA junction (F-DJ) that enables the rearrangement of separated DNA fragments at the end of HP2 and HP3 and serving as an integrated target analogue for initiating the second CHA reaction, generating an enhanced fluorescence signal. Assay experiments demonstrate that D-CHA has a better performance compared with traditional CHA, achieving the detection limit as low as 10 pM for miRNA160 as deduced from its corresponding DNA surrogates. Moreover, non-target miRNAs, as well as single-base mutation targets, can be detected. Overall, the D-CHA strategy provides a competitive method for plant miRNAs detection.

Plant Microrna Potential in Targeting COVID-19 Genome Offering Efficient Antiviral Mirna-based Therapies

In 2019, the seventh member of the Coronaviridae named severe acute respiratory coronavirus II (or COVID-19) emerged in Wuhan, China. It could rapidly turn into a global pandemic. Therefore, searching for a natural/ novel therapy is urgently essential to prevent disease distribution. Based on the principle of cross-kingdom interaction, plants are a candidate as a potential source of exogenous miRNAs that can efficiently affect the host cell gene expression by promote target mRNA degradation or repress protein translation, and, or virus RNA translation/replication. The possibility of taking up the diet-derived plant-originated/artificially miRNA(s) in addition to their biological role in the host provides a pivotal clue for the study of synthesized/artificially expressed active therapeutic plant-originated miRNA(s) to promote antiviral activity. In this study, plant miRNAs, which can potentially interact with the COVID-19 genome within the 3’-UTR region and prompt antiviral function, were searched using bioinformatics approaches. RNAHybrid, RNA22, and STarMir miRNA/target detection tools were served for the possible plant miRNA/target recognition on the 3’-UTR flanking region of the COVID-19 genome by different algorithms. The RNAHybrid algorithm resulted in 63 plant miRNAs having hybridization energy with less or equal to -25 kcal/mol. They interact with diverse classes of miRNA/target binding patterns. However, each RNA22 and STarMir tools identified eight probable miRNA/target interaction candidates, in which pvu-miR159a.2 and sbi-miR5387b detected by both the RNA22 and STarMir tools at the same position. For us, they are suitable plant-derived miRNA candidates, which have the great chance of targeting the COVID-19 genome in the 3’-UTR region in vitro to induce the virus degradation and translational repression and for antiviral miRNA-based therapies without any side effects in vivo.

PlantMirP2: An Accurate, Fast and Easy-To-Use Program for Plant Pre-miRNA and miRNA Prediction

MicroRNAs (miRNAs) are a kind of short non-coding ribonucleic acid molecules that can regulate gene expression. The computational identification of plant miRNAs is of great significance to understanding biological functions. In our previous studies, we have put firstly forward and further developed a set of knowledge-based energy features to construct two plant pre-miRNA prediction tools (plantMirP and riceMirP). However, these two tools cannot be used for miRNA prediction from NGS (Next-Generation Sequencing) data. In addition, for further improving the prediction performance and accessibility, plantMirP2 has been developed. Based on the latest dataset, plantMirP2 achieves a promising performance: 0.9968 (Area Under Curve, AUC), 0.9754 (accuracy), 0.9675 (sensitivity) and 0.9876 (specificity). Additionally, the comparisons with other plant pre-miRNA tools show that plantMirP2 performs better. Finally, the webserver and stand-alone version of plantMirP2 are available.

Diet-derived transmission of MicroRNAs from host plant into honey bee Midgut

Background MicroRNA (miRNA) is a class of small noncoding RNAs, which targets on thousands of mRNA and thus plays important roles in many biological processes. It has been reported that miRNA has cross-species regulation functions between parasitoid-host, or plant-animal, etc. For example, several plant miRNAs enter into the honey bees and regulate gene expression. However, whether cross-species regulation function of miRNAs is a universal mechanism remains a debate question. Results We have evaluated transmission of miRNAs from sunflower and sedr plants into the midgut of honey bee using RNA-Seq analyses complemented with confirmation by RT-qPCR. The results showed that at least 11 plant miRNAs were found in the midgut of honey bee feeding by sunflower and sedr pollen. Among which, nine miRNAs, including miR-30d, miR-143, miR-148a, miR-21, let-7 g, miR-26a, miR-126, miR-27a, and miR-203, were shared between the sunflower- and sedr-fed honey bees, suggesting they might have essential roles in plant-insect interactions. Moreover, existence of these co-shared miRNAs presents a strong evidence to support the successful transmission of miRNAs into the midgut of the insect. In total, 121 honeybee mRNAs were predicted to be the target of these 11 plant-derived miRNAs. Interestingly, a sedr-derived miRNA, miR-206, targets on 53 honeybee genes. Kyoto Encyclopedia of Genes and Genome (KEGG) analyses showed that these target genes are significantly involved in hippo signaling pathway-fly, Wnt signaling pathway, and N-Glycan biosynthesis. Conclusions In summary, these results provide evidence of cross-species regulation function of miRNA between honeybee and flowering host plants, extending our understanding of the molecular interactions between plants and animals.

A Timely Review of Cross-Kingdom Regulation of Plant-Derived MicroRNAs

MicroRNAs (miRNAs) belong to a class of non-coding RNAs that suppress gene expression by complementary oligonucleotide binding to the sites in target messenger RNAs. Numerous studies have demonstrated that miRNAs play crucial role in virtually all cellular processes of both plants and animals, such as cell growth, cell division, differentiation, proliferation and apoptosis. The study of rice MIR168a has demonstrated for the first time that exogenous plant MIR168a influences cholesterol transport in mice by inhibiting low-density lipoprotein receptor adapter protein 1 expression. Inspired by this finding, the cross-kingdom regulation of plant-derived miRNAs has drawn a lot of attention because of its capability to provide novel therapeutic agents in the treatment of miRNA deregulation-related diseases. Notably, unlike mRNA, some plant miRNAs are robust because of their 3′ end modification, high G, C content, and the protection by microvesicles, miRNAs protein cofactors or plant ingredients. The stability of these small molecules guarantees the reliability of plant miRNAs in clinical application. Although the function of endogenous miRNAs has been widely investigated, the cross-kingdom regulation of plant-derived miRNAs is still in its infancy. Herein, this review summarizes the current knowledge regarding the anti-virus, anti-tumor, anti-inflammatory, anti-apoptosis, immune modulation, and intestinal function regulation effects of plant-derived miRNAs in mammals. It is expected that exploring the versatile role of plant-derived miRNAs may lay the foundation for further study and application of these newly recognized, non-toxic, and inexpensive plant active ingredients.

Uptake of MicroRNAs from Exosome-Like Nanovesicles of Edible Plant Juice by Rat Enterocytes

MicroRNAs (miRNAs) are small RNAs present in extracellular vesicles (EVs) that, when transferred to a target cell, affect its biological functions. Plant miRNAs regulate the expression of certain mammalian genes. Here, we characterized EVs in fruit and vegetable juice, and their miRNA cargo, and investigated whether such miRNA-containing EVs could be taken up by mammalian enterocytes in vitro. Using filtration and ultra-centrifugation methods, EVs were purified from commercially available and manually squeezed plant juice. EV morphological features and subcellular localization were analyzed using the NanoSight tracking system and electron microscopy. Plant EV miRNA levels were evaluated using quantitative reverse transcription PCR. For the in vitro EV uptake experiments, rat intestinal epithelial cells (IEC6) were used. Plant EVs shared morphological features with mammalian EVs and contained miR156a-5p, miR166a-3p, and miR168a-5p. EVs were present in the cell sap-filled central vacuoles and were taken up by IEC6 cells. Edible plant cells produce EVs that contain various miRNAs and release them into the central vacuole. The exogenous plant EVs are taken up by mammalian enterocytes in vitro. These findings suggest the possibility that exogenous plant miRNAs carried by EVs can be absorbed via the gastrointestinal tract.

MicroRNA annotation in plants: current status and challenges

Last two decades, the studies on microRNAs (miRNAs) and the numbers of annotated miRNAs in plants and animals have surged. Herein, we reviewed the current progress and challenges of miRNA annotation in plants. Via the comparison of plant and animal miRNAs, we pinpointed out the difficulties on plant miRNA annotation and proposed potential solutions. In terms of recalling the history of methods and criteria in plant miRNA annotation, we detailed how the major progresses made and evolved. By collecting and categorizing bioinformatics tools for plant miRNA annotation, we surveyed their advantages and disadvantages, especially for ones with the principle of mimicking the miRNA biogenesis pathway by parsing deeply sequenced small RNA (sRNA) libraries. In addition, we summarized all available databases hosting plant miRNAs, and posted the potential optimization solutions such as how to increase the signal-to-noise ratio (SNR) in these databases. Finally, we discussed the challenges and perspectives of plant miRNA annotations, and indicated the possibilities offered by an all-in-one tool and platform according to the integration of artificial intelligence.