Yuanji Zhang

Lack of detectable oral bioavailability of plant microRNAs after feeding in mice

965 chow preparation where we observed the expected distribution and abundance of rice miRNAs. Oryza sativa (rice) osa-miR168a was among the most abundant miRNAs (Supplementary Table 1) in both ricecontaining chow and rice grain, consistent with previous reports9,12. Diet composition had no impact on food consumption (Supplementary Fig. 1). Following completion of the feeding regimen, small RNAs were sequenced from mouse liver and plasma samples using the Illumina (San Diego) HiSeq system. Details of the experimental protocols can be found in Supplementary Methods. We observed the expected endogenous miRNA profile and miRNA length distribution in mouse plasma and liver and rice samples, indicating consistent quality of the small RNA sequencing procedure (Supplementary Fig. 2a,b). Analysis of small RNAs from plasma and liver of mice fed on balanced rice chow and rice chow did not reveal measurable uptake of any rice grain miRNAs, including osa-miR168a. Of >10 million total sequence reads per library, fewer than ten reads identical to known rice miRNAs per library were noted in five out of eight samples from mice fed on rice-containing chow and four out of five samples from mice fed on synthetic chow (Table 1). Synthetic chow did not contain any grain or forage from plants (all plant-derived ingredients were highly purified isolates, for example, cornstarch and soybean oil); therefore, these low number of rice miRNAmappable reads could be explained by sequence errors or by cross-contamination. Mapping of mouse small RNA data to all annotated rice miRNAs in miRBase v19 identified a low number of mouse small RNA reads identical to several rice miRNA sequences (Table 1). Even so, most of the rice-like sequences were identical to the miR414 sequence (Supplementary Table 1), which is not detectable in rice grain12. In addition, these plant-like sequences were present in similar quantities in all mouse To the Editor: Human therapeutics based on nucleic acid targeting rely on sequence-specific interactions between effector and target molecules to achieve beneficial effects through specific modification to the expression of targeted genes. A variety of such compounds are being tested in laboratories and in clinical trials to treat a range of genetic and acquired diseases. Efficient, safe and cost-effective delivery of nucleic acid–based drug candidates is required to enable therapeutic levels of targeted gene regulation and overall success of this exciting new class of therapeutics. For several types of compounds in this class, effective drug delivery relies on injection of formulated nucleic acids at the site of action or into the bloodstream. Oral delivery would excel as a treatment strategy as it could offer convenient and patient-friendly features, however, progress in this approach has been hampered by substantial challenges associated with biological barriers that limit oral activity of nucleic acid therapeutics (e.g., stability within and uptake of nucleic acids from the mammalian gastrointestinal tract, nucleases and membrane barriers)1. Considerable effort has been applied to improve the stability and uptake efficiency of orally administered nucleic acids by introducing chemical modifications and formulating with excipients; however, limited success has been reported thus far2,3. The naturally occurring RNA interference (RNAi) response has been extensively reported after feeding double-stranded RNA (dsRNA) in some invertebrates, such as the model organism Caenorhabditis elegans4 and some agricultural pests5,6 (e.g., corn rootworm and cotton bollworm). Yet, despite responsiveness to ingested dsRNA, a recent survey revealed substantial variation in sensitivity to dsRNA in other Caenorhabditis nematodes7 and other invertebrate species8. In addition, despite major efforts in academic and pharmaceutical laboratories to activate the RNA silencing pathway in response to ingested RNA, the phenomenon had not been reported in mammals until a recent publication by Zhang et al.9 in Cell Research. This report described the uptake of plant-derived microRNAs (miRNA) into the serum, liver and a few other tissues in mice following consumption of rice, as well as apparent gene regulatory activity in the liver. The observation provided a potentially groundbreaking new possibility that RNAbased therapies could be delivered to mammals through oral administration and at the same time opened a discussion on the evolutionary impact of environmental dietary nucleic acid effects across broad phylogenies. A recently reported survey of a large number of animal small RNA datasets from public sources has not revealed evidence for any major plant-derived miRNA accumulation in animal samples10. Given the number of questions evoked by these analyses, the limited success with oral RNA delivery for pharmaceutical development, the history of safe consumption for dietary small RNAs11 and lack of evidence for uptake of plant-derived dietary small RNAs, we felt further evaluation of miRNA uptake and the potential for cross-kingdom gene regulation in animals was warranted to assess the prevalence, impact and robustness of the phenomenon. To address this question, we conducted a well-controlled mouse feeding study with rice-containing chow diets or with a purified synthetic chow devoid of plant grain or forage. After a two-week acclimation on synthetic chow (modified AIN93-G), animals were fasted for 12 h and then placed on synthetic chow; a nutritionally balanced, rice-containing chow (modified AIN93-G with 40.8% rice); or rice-based chow (75% rice), for 1, 3 and 7 days (Supplementary Methods). These groups are referred to herein as synthetic chow, balanced rice chow and rice chow, respectively. To confirm rice miRNA availability in feeding material, we first sequenced rice small RNAs from ricecontaining chow and rice grains used for Lack of detectable oral bioavailability of plant microRNAs after feeding in mice correspondence

Analysis of plant-derived miRNAs in animal small RNA datasets

BackgroundPlants contain significant quantities of small RNAs (sRNAs) derived from various sRNA biogenesis pathways. Many of these sRNAs play regulatory roles in plants. Previous analysis revealed that numerous sRNAs in corn, rice and soybean seeds have high sequence similarity to animal genes. However, exogenous RNA is considered to be unstable within the gastrointestinal tract of many animals, thus limiting potential for any adverse effects from consumption of dietary RNA. A recent paper reported that putative plant miRNAs were detected in animal plasma and serum, presumably acquired through ingestion, and may have a functional impact in the consuming organisms.ResultsTo address the question of how common this phenomenon could be, we searched for plant miRNAs sequences in public sRNA datasets from various tissues of mammals, chicken and insects. Our analyses revealed that plant miRNAs were present in the animal sRNA datasets, and significantly miR168 was extremely over-represented. Furthermore, all or nearly all (>96%) miR168 sequences were monocot derived for most datasets, including datasets for two insects reared on dicot plants in their respective experiments. To investigate if plant-derived miRNAs, including miR168, could accumulate and move systemically in insects, we conducted insect feeding studies for three insects including corn rootworm, which has been shown to be responsive to plant-produced long double-stranded RNAs.ConclusionsOur analyses suggest that the observed plant miRNAs in animal sRNA datasets can originate in the process of sequencing, and that accumulation of plant miRNAs via dietary exposure is not universal in animals.

Characterization of Unique Small RNA Populations from Rice Grain

Small RNAs (∼20 to 24 nucleotides) function as naturally occurring molecules critical in developmental pathways in plants and animals [1], [2]. Here we analyze small RNA populations from mature rice grain and seedlings by pyrosequencing. Using a clustering algorithm to locate regions producing small RNAs, we classified hotspots of small RNA generation within the genome. Hotspots here are defined as 1 kb regions within which small RNAs are significantly overproduced relative to the rest of the genome. Hotspots were identified to facilitate characterization of different categories of small RNA regulatory elements. Included in the hotspots, we found known members of 23 miRNA families representing 92 genes, one trans acting siRNA (ta-siRNA) gene, novel siRNA-generating coding genes and phased siRNA generating genes. Interestingly, over 20% of the small RNA population in grain came from a single foldback structure, which generated eight phased 21-nt siRNAs. This is reminiscent of a newly arising miRNA derived from duplication of progenitor genes [3], [4]. Our results provide data identifying distinct populations of small RNAs, including phased small RNAs, in mature grain to facilitate characterization of small regulatory RNA expression in monocot species.

Regulation of Gene Expression in Plants through miRNA Inactivation

Eukaryotic organisms possess a complex RNA-directed gene expression regulatory network allowing the production of unique gene expression patterns. A recent addition to the repertoire of RNA-based gene regulation is miRNA target decoys, endogenous RNA that can negatively regulate miRNA activity. miRNA decoys have been shown to be a valuable tool for understanding the function of several miRNA families in plants and invertebrates. Engineering and precise manipulation of an endogenous RNA regulatory network through modification of miRNA activity also affords a significant opportunity to achieve a desired outcome of enhanced plant development or response to environmental stresses. Here we report that expression of miRNA decoys as single or heteromeric non-cleavable microRNA (miRNA) sites embedded in either non-protein-coding or within the 3′ untranslated region of protein-coding transcripts can regulate the expression of one or more miRNA targets. By altering the sequence of the miRNA decoy sites, we were able to attenuate miRNA inactivation, which allowed for fine regulation of native miRNA targets and the production of a desirable range of plant phenotypes. Thus, our results demonstrate miRNA decoys are a flexible and robust tool, not only for studying miRNA function, but also for targeted engineering of gene expression in plants. Computational analysis of the Arabidopsis transcriptome revealed a number of potential miRNA decoys, suggesting that endogenous decoys may have an important role in natural modulation of expression in plants.

Environmental RNAi in herbivorous insects

Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a receptive organism by exogenous double-stranded RNA (dsRNA). Although demonstrated under artificial dietary conditions and via transgenic plant presentations in several herbivorous insects, the magnitude and consequence of exogenous dsRNA uptake and the role of eRNAi remains unknown under natural insect living conditions. Our analysis of coleopteran insects sensitive to eRNAi fed on wild-type plants revealed uptake of plant endogenous long dsRNAs, but not small RNAs. Subsequently, the dsRNAs were processed into 21 nt siRNAs by insects and accumulated in high quantities in insect cells. No accumulation of host plant-derived siRNAs was observed in lepidopteran larvae that are recalcitrant to eRNAi. Stability of ingested dsRNA in coleopteran larval gut followed by uptake and transport from the gut to distal tissues appeared to be enabling factors for eRNAi. Although a relatively large number of distinct coleopteran insect-processed plant-derived siRNAs had sequence complementarity to insect transcripts, the vast majority of the siRNAs were present in relatively low abundance, and RNA-seq analysis did not detect a significant effect of plant-derived siRNAs on insect transcriptome. In summary, we observed a broad genome-wide uptake of plant endogenous dsRNA and subsequent processing of ingested dsRNA into 21 nt siRNAs in eRNAi-sensitive insects under natural feeding conditions. In addition to dsRNA stability in gut lumen and uptake, dosage of siRNAs targeting a given insect transcript is likely an important factor in order to achieve measurable eRNAi-based regulation in eRNAi-competent insects that lack an apparent silencing amplification mechanism.

Endogenous small RNAs in grain: Semi-quantification and sequence homology to human and animal genes

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are effector molecules of RNA interference (RNAi), a highly conserved RNA-based gene suppression mechanism in plants, mammals and other eukaryotes. Endogenous RNAi-based gene suppression has been harnessed naturally and through conventional breeding to achieve desired plant phenotypes. The present study demonstrates that endogenous small RNAs, such as siRNAs and miRNAs, are abundant in soybean seeds, corn kernels, and rice grain, plant tissues that are traditionally used for food and feed. Numerous endogenous plant small RNAs were found to have perfect complementarity to human genes as well as those of other mammals. The abundance of endogenous small RNA molecules in grain from safely consumed food and feed crops such as soybean, corn, and rice and the homology of a number of these dietary small RNAs to human and animal genomes and transcriptomes establishes a history of safe consumption for dietary small RNAs.

Establishing an In Vivo Assay System to Identify Components Involved in Environmental RNA Interference in the Western Corn Rootworm

The discovery of environmental RNA interference (RNAi), in which gene expression is suppressed via feeding with double-stranded RNA (dsRNA) molecules, opened the door to the practical application of RNAi-based techniques in crop pest management. The western corn rootworm (WCR, Diabrotica virgifera virgifera) is one of the most devastating corn pests in North America. Interestingly, WCR displays a robust environmental RNAi response, raising the possibility of applying an RNAi-based pest management strategy to this pest. Understanding the molecular mechanisms involved in the WCR environmental RNAi process will allow for determining the rate limiting steps involved with dsRNA toxicity and potential dsRNA resistance mechanisms in WCR. In this study, we have established a two-step in vivo assay system, which allows us to evaluate the involvement of genes in environmental RNAi in WCR. We show that laccase 2 and ebony, critical cuticle pigmentation/tanning genes, can be used as marker genes in our assay system, with ebony being a more stable marker to monitor RNAi activity. In addition, we optimized the dsRNA dose and length for the assay, and confirmed that this assay system is sensitive to detect well-known RNAi components such as Dicer-2 and Argonaute-2. We also evaluated two WCR sid1- like (sil) genes with this assay system. This system will be useful to quickly survey candidate systemic RNAi genes in WCR, and also will be adaptable for a genome-wide RNAi screening to give us an unbiased view of the environmental/systemic RNAi pathway in WCR.

RNA decoys: an emerging component of plant regulatory networks?

The role of non-coding RNAs (ncRNAs), both short and long ncRNAs, in the regulation of gene expression has become evident in recent years. Non-coding RNA-based regulation is achieved through a variety of mechanisms; some are relatively well-characterized, while others are much less understood. MicroRNAs (miRNAs), a class of endogenous small RNAs, function as master regulators of gene expression in eukaryotic organisms. A notable, recently discovered role for long ncRNAs is that of miRNA decoys, also referred to as target mimics or sponges, in which long ncRNAs carry a short stretch of sequence sharing homology to miRNA-binding sites in endogenous targets. As a consequence, miRNA decoys are able to sequester and inactivate miRNA function. Engineered miRNA decoys are also efficacious and useful tools for studying gene function. We recently demonstrated that the potential of miRNA decoys to inactivate miRNAs in the model plants Arabidopsis thaliana and Nicotiana benthamiana is dependent on the level of sequence complementarity to miRNAs of interest. The flexibility of the miRNA decoy approach in sequence-dependent miRNA inactivation, backbone choice, ability to simultaneously inactivate multiple miRNAs, and more importantly, to achieve a desirable level of miRNA inactivation, makes it a potentially useful tool for crop improvement. This research addendum reports the functional extension of miRNA decoys from model plants to crops. Furthermore, endogenous miRNA decoys, first described in plants, have been proposed to play a significant role in regulating the transcriptome in eukaryotes. Using computational analysis, we have identified numerous endogenous sequences with potential miRNA decoy activity for conserved miRNAs in several plant species. Our data suggest that endogenous miRNA decoys can be widespread in plants and may be a component of the global gene expression regulatory network in plants.

Computational sequence analysis of predicted long dsRNA transcriptomes of major crops reveals sequence complementarity with human genes

Long double-stranded RNAs (long dsRNAs) are precursors for the effector molecules of sequence-specific RNA-based gene silencing in eukaryotes. Plant cells can contain numerous endogenous long dsRNAs. This study demonstrates that such endogenous long dsRNAs in plants have sequence complementarity to human genes. Many of these complementary long dsRNAs have perfect sequence complementarity of at least 21 nucleotides to human genes; enough complementarity to potentially trigger gene silencing in targeted human cells if delivered in functional form. However, the number and diversity of long dsRNA molecules in plant tissue from crops such as lettuce, tomato, corn, soy and rice with complementarity to human genes that have a long history of safe consumption supports a conclusion that long dsRNAs do not present a significant dietary risk.

Small RNA profiles from virus-infected fresh market vegetables.

Functional small RNAs, such as short interfering RNAs (siRNAs) and microRNAs (miRNAs), exist in freshly consumed fruits and vegetables. These siRNAs can be derived either from endogenous sequences or from viruses that infect them. Symptomatic tomatoes, watermelons, zucchini, and onions were purchased from grocery stores and investigated by small RNA sequencing. By aligning the obtained small RNA sequences to sequences of known viruses, four different viruses were identified as infecting these fruits and vegetables. Many of these virally derived small RNAs along with endogenous small RNAs were found to be highly complementary to human genes. However, the established history of safe consumption of these vegetables suggests that this sequence homology has little biological relevance. By extension, these results provide evidence for the safe use by humans and animals of genetically engineered crops using RNA-based suppression technologies, especially vegetable crops with virus resistance conferred by expression of siRNAs or miRNAs derived from viral sequences.