DNA self-assembly-boosted transcription amplification coupled with CRISPR/Cas13a system for plant microRNA analysis.

MicroRNAs (miRNAs) play important roles in the growth process of plants, and some food-originated plant miRNAs have potential impacts on human health, which makes the detection of plant miRNAs of great significance. However, plant miRNAs are naturally modified with 2'-O-methyl at the 3'-terminal, which is difficult to be directly quantified by enzyme-catalyzed terminal polymerization protocols. Herein, we have proposed a simple strategy by coupling DNA self-assembly-boosted transcription amplification with CRISPR/Cas13a platform (termed as Cas13a-SATA) for the specific and sensitive detection of plant miRNA. In the Cas13a-SATA, the plant miRNA will mediate DNA self-assembly on the surface of microbeads and then trigger efficient transcription amplification to yield numerous single-stranded RNA (ssRNA) molecules, which can effectively activate the Cas13a trans-cleavage activity to generate intense fluorescence signal in a plant miRNA dosage-responsive manner. Using the Cas13a-SATA, we have realized the sensitive detection of plant miR156a with the limit of detection (LOD) down to 3.8 fM. Furthermore, Cas13a-SATA has been successfully applied to the accurate quantification of miR156a in Arabidopsis and maize, demonstrating its feasibility in analyzing plant miRNAs in real biological samples.

Evidence of Cross-Kingdom Gene Regulation by Plant MicroRNAs and Possible Reasons for Inconsistencies.

The debate on whether cross-kingdom gene regulation by orally acquired plant miRNAs is possible has been ongoing for nearly 10 years without a conclusive answer. In this study, we categorized plant miRNAs into different groups, namely, extracellular vesicle (EV)-borne plant miRNAs, extracted plant miRNAs, herbal decoction-borne plant miRNAs, synthetic plant miRNA mimics, and plant tissue/juice-borne plant miRNAs. This categorization aimed to simplify the analysis and address the question more specifically. Our evidence suggests that EV-borne plant miRNAs, extracted plant miRNAs, herbal decoction-borne plant miRNAs, and synthetic plant miRNA mimics consistently facilitate cross-kingdom gene regulation. However, the results regarding the cross-kingdom gene regulation by plant tissue- and juice-borne plant miRNAs are inconclusive. This inconsistency may be due to variations in study methods, a low absorption rate of miRNAs and the selective absorption of plant miRNAs in the gastrointestinal tract. Overall, it is deduced that cross-kingdom gene regulation by orally acquired plant miRNAs can occur under certain circumstances, depending on factors such as the types of plant miRNAs, the delivery mechanism, and their concentrations in the plant.

Genome-wide analysis and identification of microRNAs in Medicago truncatula under aluminum stress.

Numerous studies have shown that plant microRNAs (miRNAs) play key roles in plant growth and development, as well as in response to biotic and abiotic stresses; however, the role of miRNA in legumes under aluminum (Al) stress have rarely been reported. Therefore, here, we aimed to investigate the role of miRNAs in and their mechanism of Al tolerance in legumes. To this end, we sequenced a 12-strand-specific library of Medicago truncatula under Al stress. A total of 195.80 M clean reads were obtained, and 876 miRNAs were identified, of which, 673 were known miRNAs and 203 were unknown. A total of 55 miRNAs and their corresponding 2,502 target genes were differentially expressed at various time points during Al stress. Further analysis revealed that mtr-miR156g-3p was the only miRNA that was significantly upregulated at all time points under Al stress and could directly regulate the expression of genes associated with root cell growth. Three miRNAs, novel_miR_135, novel_miR_182, and novel_miR_36, simultaneously regulated the expression of four Al-tolerant transcription factors, GRAS, MYB, WRKY, and bHLH, at an early stage of Al stress, indicating a response to Al stress. In addition, legume-specific miR2119 and miR5213 were involved in the tolerance mechanism to Al stress by regulating F-box proteins that have protective effects against stress. Our results contribute to an improved understanding of the role of miRNAs in Al stress in legumes and provide a basis for studying the molecular mechanisms of Al stress regulation.

Plant MicroRNA Identification and Annotation Using Deep Sequencing Data.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs, which regulate gene expression at the post-transcriptional level. A large number of studies have revealed that they play key roles in diverse life activities, such as growth and development. In the last decade, deep sequencing technology has generated substantial small RNA sequencing (sRNA-Seq) data. Meanwhile, numerous tools have been developed to identify miRNAs from these sRNA-Seq data, resulting in a surge of miRNA annotations. Among these tools, the series of miRDeep-P and miRDeep-P2 have been widely used in plant miRNA annotation. Here, we employed miRDeep-P2 to demonstrate the plant miRNA annotation processes step by step using the deep sequencing data.

A Small RNA-Seq Protocol with Less Bias and Improved Capture of 2'-O-Methyl RNAs.

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNAs such as plant microRNAs (miRNAs) carry a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This modification adds another level of difficulty as it inhibits 3' adapter ligation. We previously demonstrated that modified versions of the "TruSeq (TS)" protocol have less bias and an improved detection of 2'-OMe RNAs. Here we describe in detail protocol "TS5," which showed the best overall performance. We also provide guidelines for bioinformatics analysis of the sequencing data.

Novel approaches on identification of conserved miRNAs for broad-spectrum Potyvirus control measures.

Potyviridae comprises more than 200 ssRNA viruses, many of which have a broad host range and geographical distributions. Potyvirids (members of Potyviridae) infect several economically important plants such as saffron, cardamom, cucumber, pepper, potato, tomato, yam, etc. Cumulatively, potyvirids cause a substantial economic loss. The major bottleneck in developing an efficient antiviral strategy is that viruses quickly evade host immunity owing to their higher mutation and recombination rates. Due to this reason, the emergence of newer and improved broad-spectrum approaches to combat viral infections is essential. The use of microRNA's (miRNA) to circumvent viral infection against animal viruses has been successfully employed. Fewer studies reported the development of efficient miRNA-based antivirus resistant strategies against plant viruses and none focused on multiple virus resistance. We focused on potyviruses since studies are limited and identification of conserved miRNAs among various host plants would be an initiative to design broad-spectrum antivirus strategies. In this study, we predicted evolutionarily conserved miRNAs by BLAST searching of reported miRNAs from 15 plants against the GSS and EST sequences of banana. A total of nine miRNAs were predicted and screened in nine diverse potyvirids' hosts (Banana, Tomato, Green gram, Jasmine, Chilli, Coriander, Onion, Rose and Colocasia) belonging to eight different orders (Zingiberales, Solanales, Fabales, Lamiales, Apiales, Asperagales, Rosales and Alismatales). Results suggested that miR168 and miR162 are conserved among all the selected plants. This comprehensive study laid the foundations to design broad-spectrum antivirus resistance using miRNAs. To conclude miR168 and miR162 are conserved among many plants and play a crucial role in evading virus infection which could be used as a potential candidate for developing antiviral strategies against potyvirid infections.

Recent advances in the regulation of plant miRNA biogenesis.

MicroRNAs (miRNAs) are essential non-coding riboregulators of gene expression in plants and animals. In plants, miRNAs guide their effector protein named ARGONAUTE (AGO) to find target RNAs for gene silencing through target RNA cleavage or translational inhibition. miRNAs are derived from primary miRNA transcripts (pri-miRNAs), most of which are transcribed by the DNA-dependent RNA polymerase II. In plants, an RNase III enzyme DICER-LIKE1-containing complex processes pri-miRNAs in the nucleus into miRNAs. To ensure proper function of miRNAs, plants use multiple mechanisms to control miRNA accumulation. On one hand, pri-miRNA levels are controlled through transcription and stability. On the other hand, the activities of the DCL1 complex are regulated by many protein factors at transcriptional, post-transcriptional and post-translational levels. Notably, recent studies reveal that pri-miRNA structure/sequence features and modifications also play important roles in miRNA biogenesis. In this review, we summarize recent progresses on the mechanisms regulating miRNA biogenesis.

In Situ Detection of Mature miRNAs in Plants Using LNA-Modified DNA Probes.

MicroRNAs (miRNAs) play important roles in development in plants, and some miRNAs show developmentally regulated organ- and tissue-specific expression patterns. Therefore, in situ detection of mature miRNAs is important for understanding the functions for both miRNAs and their targets. The construction of promoter-reporter fusions and examination of their in planta expression has been widely used and the results obtained thus far are rather informative; however, in some cases, the length of promoter that contains entire regulatory elements is difficult to determine. In addition, traditional in situ hybridization with the antisense RNA fragment as the probe usually fails to detect miRNAs, because the mature miRNAs are too short (~21-nucleotides) to exhibit stable hybridization signals. In recent years, the Locked nucleic acid (LNA) modified DNA probe has been successfully used in animals and plants to detect small RNAs. Here, we describe a modified protocol using LNA-modified DNA probes to detect mature miRNAs in plant tissues, including the design of LNA probes and detailed steps for the in situ hybridization experiment, using Arabidopsis miR165 as an example.

Plant miRNAs Reduce Cancer Cell Proliferation by Targeting MALAT1 and NEAT1: A Beneficial Cross-Kingdom Interaction.

MicroRNAs (miRNAs) are ubiquitous regulators of gene expression, evolutionarily conserved in plants and mammals. In recent years, although a growing number of papers debate the role of plant miRNAs on human gene expression, the molecular mechanisms through which this effect is achieved are still not completely elucidated. Some evidence suggest that this interaction might be sequence specific, and in this work, we investigated this possibility by transcriptomic and bioinformatics approaches. Plant and human miRNA sequences from primary databases were collected and compared for their similarities (global or local alignments). Out of 2,588 human miRNAs, 1,606 showed a perfect match of their seed sequence with the 5' end of 3,172 plant miRNAs. Further selections were applied based on the role of the human target genes or of the miRNA in cell cycle regulation (as an oncogene, tumor suppressor, or a biomarker for prognosis, or diagnosis in cancer). Based on these criteria, 20 human miRNAs were selected as potential functional analogous of 7 plant miRNAs, which were in turn transfected in different cell lines to evaluate their effect on cell proliferation. A significant decrease was observed in colorectal carcinoma HCT116 cell line. RNA-Seq demonstrated that 446 genes were differentially expressed 72 h after transfection. Noteworthy, we demonstrated that the plant mtr-miR-5754 and gma-miR4995 directly target the tumor-associated long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and nuclear paraspeckle assembly transcript 1 (NEAT1) in a sequence-specific manner. In conclusion, according to other recent discoveries, our study strengthens and expands the hypothesis that plant miRNAs can have a regulatory effect in mammals by targeting both protein-coding and non-coding RNA, thus suggesting new biotechnological applications.

Small RNAs in eucaryotes: new clues for amplifying microRNA benefits.

miRNAs, the smallest nucleotide molecules able to regulate gene expression at post transcriptional level, are found in both animals and plants being involved in fundamental processes for growth and development of living organisms. The number of miRNAs has been hypothesized to increase when some organisms specialized the process of mastication and grinding of food. Further to the vertical transmission, miRNAs can undergo horizontal transmission among different species, in particular between plants and animals. In the last years, an increasing number of studies reported that miRNA passage occurs through feeding, and that in animals, plant miRNAs can survive the gastro intestinal digestion and transferred by blood into host cells, where they can exert their functions modulating gene expression. The present review reports studies on miRNAs during evolution, with particular focus on biogenesis and mechanisms regulating their stability in plants and animals. The different biogenesis and post biogenesis modifications allow to discriminate miRNAs of plant origin from those of animal origin, and make it possible to better clarify the controversial question on whether a possible cross-kingdom miRNA transfer through food does exist. The majority of human medicines and supplements derive from plants and a regular consumption of plant food is suggested for their beneficial effects in the prevention of metabolic diseases, cancers, and dietary related disorders. So far, these beneficial effects have been generally attributed to the content of secondary metabolites, whereas mechanisms regarding other components remain unclear. Therefore, in light of the above reported studies miRNAs could result another component for the medical properties of plants. miRNAs have been mainly studied in mammals characterizing their sequences and molecular targets as available in public databases. The herein presented studies provide evidences that miRNA situation is much more complex than the static situation reported in databases. Indeed, miRNAs may have redundant activities, variable sequences, different methods of biogenesis, and may be differently influenced by external and environmental factors. In-depth knowledge of mechanisms of synthesis, regulation and transfer of plant miRNAs to other species can open new frontiers in the therapy of many human diseases, including cancer.

Plant Small RNAs Responsive to Fungal Pathogen Infection.

Accumulating evidence indicates that small noncoding RNAs (sRNAs) can be transferred across species for interkingdom communication. In addition to the artificial transgene-derived small interfering RNAs (siRNAs), endogenous microRNAs (miRNAs) can also influence interacting organisms to execute a regulatory function. For instance, we have recently found that, in response to infection with Verticillium dahliae (V. dahliae), cotton plants increase accumulation of miR166 and miR159, which can be exported to the fungal hyphae for specific silencing of virulence genes. These findings suggest a great potential for applying interkingdom mobile miRNAs for crop protection against fungal pathogens. The methods described here provide an approach to identify plant miRNAs and their potential targets in invading fungal pathogens, which will help in revealing the underlying mechanisms of these crosstalk phenomena.

Small but powerful: function of microRNAs in plant development.

MicroRNAs (miRNAs) are a group of endogenous noncoding small RNAs frequently 21 nucleotides long. miRNAs act as negative regulators of their target genes through sequence-specific mRNA cleavage, translational repression, or chromatin modifications. Alterations of the expression of a miRNA or its targets often result in a variety of morphological and physiological abnormalities, suggesting the strong impact of miRNAs on plant development. Here, we review the recent advances on the functional studies of plant miRNAs. We will summarize the regulatory networks of miRNAs in a series of developmental processes, including meristem development, establishment of lateral organ polarity and boundaries, vegetative and reproductive organ growth, etc. We will also conclude the conserved and species-specific roles of plant miRNAs in evolution and discuss the strategies for further elucidating the functional mechanisms of miRNAs during plant development.

Plant and Animal microRNAs (miRNAs) and Their Potential for Inter-kingdom Communication.

microRNAs (miRNAs) comprise a class of ~18-25 nucleotide (nt) single-stranded non-coding RNAs (sncRNAs) that are the smallest known carriers of gene-encoded, post-transcriptional regulatory information in both plants and animals. There are many fundamental similarities between plant and animal miRNAs-the miRNAs of both kingdoms play essential roles in development, aging and disease, and the shaping of the transcriptome of many cell types. Both plant and animal miRNAs appear to predominantly exert their genetic and transcriptomic influences by regulating gene expression at the level of messenger RNA (mRNA) stability and/or translational inhibition. Certain miRNA species, such as miRNA-155, miRNA-168, and members of the miRNA-854 family may be expressed in both plants and animals, suggesting a common origin and functional selection of specific miRNAs over vast periods of evolution (for example, Arabidopsis thaliana-Homo sapiens divergence ~1.5 billion years). Although there is emerging evidence for cross-kingdom miRNA communication-that plant-enriched miRNAs may enter the diet and play physiological and/or pathophysiological roles in human health and disease-some research reports repudiate this possibility. This research paper highlights some recent, controversial, and remarkable findings in plant- and animal-based miRNA signaling research with emphasis on the intriguing possibility that dietary miRNAs and/or sncRNAs may have potential to contribute to both intra- and inter-kingdom signaling, and in doing so modulate molecular-genetic mechanisms associated with human health and disease.

Detection of Plant miRNAs Abundance in Human Breast Milk.

Breast milk is a natural food and important component of infant nutrition. Apart from the alimentary substances, breast milk contains many important bioactive compounds, including endogenous microRNA molecules (miRNAs). These regulatory molecules were identified in various mammalian biological fluids and were shown to be mostly packed in exosomes. Recently, it was revealed that plant food-derived miRNAs are stably present in human blood and regulate the expression of specific human genes. Since then, the scientific community has focused its efforts on contradicting or confirming this discovery. With the same intention, qRT-PCR experiments were performed to evaluate the presence of five plant food-derived miRNAs (miR166a, miR156a, miR157a, miR172a and miR168a) in breast milk (whole milk and exosomes) from healthy volunteers. In whole milk samples, all examined miRNAs were identified, while only two of these miRNAs were confirmed to be present in exosomes. The plant miRNA concentration in the samples ranged from 4 to 700 fM. Complementary bioinformatics analysis suggests that the evaluated plant miRNAs may potentially influence several crucial biological pathways in the infant organism.

miRTour: Plant miRNA and target prediction tool.

UNLABELLED: MicroRNAs (miRNAs) are important negative regulators of gene expression in plant and animals, which are endogenously produced from their own genes. Computational comparative approach based on evolutionary conservation of mature miRNAs has revealed a number of orthologs of known miRNAs in different plant species. The homology-based plant miRNA discovery, followed by target prediction, comprises several steps, which have been done so far manually. Here, we present the bioinformatics pipeline miRTour which automates all the steps of miRNA similarity search, miRNA precursor selection, target prediction and annotation, each of them performed with the same set of input sequences. AVAILABILITY: The database is available for free at http://bio2server.bioinfo.uni-plovdiv.bg/miRTour/