Suppression of microRNA168 enhances salt tolerance in rice (Oryza sativa L.).

BACKGROUND: Rice is a salt-sensitive crop. Complex gene regulatory cascades are likely involved in salinity stress in rice roots. microRNA168 (miR168) is a conserved miRNA among different plant species. It in-directly regulates the expression of all miRNAs by targeting gene ARGONAUTE1(AGO1). Short Tandem Target Mimic (STTM) technology is an ideal approach to study miRNA functions by in-activating mature miRNA in plants. RESULTS: In this study, rice miR168 was inactivated by STTM. The T3 generation seedlings of STTM168 exhibited significantly enhanced salt resistance. Direct target genes of rice miR168 were obtained by in silico prediction and further confirmed by degradome-sequencing. PINHEAD (OsAGO1), which was previously suggested to be a plant abiotic stress response regulator. RNA-Seq was performed in root samples of 150mM salt-treated STTM168 and control seedlings. Among these screened 481 differentially expressed genes within STTM168 and the control, 44 abiotic stress response related genes showed significant difference, including four known salt-responsive genes. CONCLUSION: Based on sequencing and qRT-PCR, a "miR168-AGO1-downstream" gene regulation model was proposed to be responsible for rice salt stress response. The present study proved miR168-AGO1 cascade to play important role in rice salinity stress responding, as well as to be applied in agronomic improvement in further.

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.

miR168 targets Argonaute1A mediated miRNAs regulation pathways in response to potassium deficiency stress in tomato.

BACKGROUND: Potassium (K+) is an essential ion for most plants, as it is involved in the regulation of growth and development. K+ homeostasis in plant cells has evolved to facilitate plant adaptation to K+-deficiency stress. Argonaute1 (AGO1) is regulated by miR168 to modulate the small RNA regulatory pathway by RNA silencing complex (RISC) in tomatoes. However, the role of miR168-mediated regulation of AGO1 in the context of K+ deficiency stress in tomatoes has not been elucidated yet. RESULTS: SlmiR168 and its target gene SlAGO1A were differentially expressed among low-K+-tolerant JZ34 and low-K+-sensitive JZ18 tomato plants. Transgenic tomato plants constitutively expressing pri-SlmiR168a showed stronger root system growth, better leaves development, and higher K+ contents in roots under K+-deficiency stress than those of the transgenic tomato lines expressing rSlAGO1A (SlmiR168-resistant) and the wild type (WT). Deep sequencing analysis showed that 62 known microRNAs (miRNAs) were up-regulated in 35S:rSlAGO1 compared with WT tomatoes. The same miRNAs were down-regulated in 35S:SlmiR168a compared with WT plants. The integrated analysis found 12 miRNA/mRNA pairs from the 62 miRNAs, including the root growth and cytokinin (CTK)/abscisic acid (ABA) pathways. CONCLUSIONS: The regulation mediated by SlmiR168 of SlAGO1A contributes to the plant development under low-K+ stress. Moreover, this regulation mechanism may influence downstream miRNA pathways in response to low-K+ stress through the CTK/ABA and root growth modulation pathways.

Malus hupehensis miR168 Targets to ARGONAUTE1 and Contributes to the Resistance against Botryosphaeria dothidea Infection by Altering Defense Responses.

MicroRNA (miRNA)-mediated post-transcriptional regulation plays a fundamental role in various plant physiological processes, including responses to pathogens. MicroRNA168 has been implicated as an essential factor of miRNA pathways by targeting ARGONAUTE1 (AGO1), the core component of the RNA-induced silencing complex (RISC). A fluctuation in AGO1 expression influences various plant-pathogen interactions, and the homeostasis of AGO1 and miR168 accumulation is maintained by a complicated feedback regulatory loop. In this study, the connection between miR168 and the resistance of Malus hupehensis to Botryosphaeria dothidea is revealed. The induction of both the mature miR168 and its precursor in plants subjected to B. dothidea infection indicate the transcriptional activation of MIR168a. MIR168a promoter analysis demonstrates that the promoter can be activated by B. dothidea and salicylic acid (SA). However, the direct target of miR168, M. hupehensis ARGONAUTE1 (MhAGO1), is shown to be induced under the infection. Expression and transcription activity analysis demonstrate the transcriptional activation and the post-transcriptional suppression of MhAGO1 in response to B. dothidea infection. By inhibiting reactive oxygen species (ROS) production and enhancing SA-mediated defense responses, miR168a delays the symptom development of leaves inoculated with B. dothidea and impedes the pathogen growth, while MhAGO1 is found to have the opposite effects. Collectively, these findings suggest that the expression of miR168 and MhAGO1 in M. hupehensis in response to B. dothidea infection is regulated by a complicated mechanism. Targeting to MhAGO1, a negative regulator, miR168 plays a positive role in the resistance by alterations in diverse defense responses.

HOS1 regulates Argonaute1 by promoting transcription of the microRNA gene MIR168b in Arabidopsis.

Proper accumulation and function of miRNAs is essential for plant growth and development. While core components of the miRNA biogenesis pathway and miRNA-induced silencing complex have been well characterized, cellular regulators of miRNAs remain to be fully explored. Here we report that High Expression Of Osmotically Responsive Genes1 (HOS1) is a regulator of an important miRNA, mi168a/b, that targets the Argonaute1 (AGO1) gene in Arabidopsis. HOS1 functions as an ubiquitin E3 ligase to regulate plant cold-stress responses, associates with the nuclear pores to regulate mRNA export, and regulates the circadian clock and flowering time by binding to chromatin of the flowering regulator gene Flowering Locus C (FLC). In a genetic screen for enhancers of sic-1, we isolated a loss-of-function Arabidopsis mutant of HOS1 that is defective in miRNA biogenesis. Like other hos1 mutant alleles, the hos1-7 mutant flowered early and was smaller in stature than the wild-type. Dysfunction in HOS1 reduced the abundance of miR168a/b but not of other miRNAs. In hos1 mutants, pri-MIR168b and pre-MIR168b levels were decreased, and RNA polymerase II occupancy was reduced at the promoter of MIR168b but not that of MIR168a. Chromatin immunoprecipitation assays revealed that HOS1 protein is enriched at the chromatin of the MIR168b promoter. The reduced miR168a/b level in hos1 mutants results in an increase in the mRNA and protein levels of its target gene, AGO1. Our results reveal that HOS1 regulates miR168a/b and AGO1 levels in Arabidopsis by maintaining proper transcription of MIR168b.

Mining of public sequencing databases supports a non-dietary origin for putative foreign miRNAs: underestimated effects of contamination in NGS.

The report that exogenous plant miRNAs are able to cross the mammalian gastrointestinal tract and exert gene-regulation mechanism in mammalian tissues has yielded a lot of controversy, both in the public press and the scientific literature. Despite the initial enthusiasm, reproducibility of these results was recently questioned by several authors. To analyze the causes of this unease, we searched for diet-derived miRNAs in deep-sequencing libraries performed by ourselves and others. We found variable amounts of plant miRNAs in publicly available small RNA-seq data sets of human tissues. In human spermatozoa, exogenous RNAs reached extreme, biologically meaningless levels. On the contrary, plant miRNAs were not detected in our sequencing of human sperm cells, which was performed in the absence of any known sources of plant contamination. We designed an experiment to show that cross-contamination during library preparation is a source of exogenous RNAs. These contamination-derived exogenous sequences even resisted oxidation with sodium periodate. To test the assumption that diet-derived miRNAs were actually contamination-derived, we sought in the literature for previous sequencing reports performed by the same group which reported the initial finding. We analyzed the spectra of plant miRNAs in a small RNA sequencing study performed in amphioxus by this group in 2009 and we found a very strong correlation with the plant miRNAs which they later reported in human sera. Even though contamination with exogenous sequences may be easy to detect, cross-contamination between samples from the same organism can go completely unnoticed, possibly affecting conclusions derived from NGS transcriptomics.

Identification and characterization of microRNAs in oilseed rape (Brassica napus) responsive to infection with the pathogenic fungus Verticillium longisporum using Brassica AA (Brassica rapa) and CC (Brassica oleracea) as reference genomes.

Verticillium longisporum, a soil-borne pathogenic fungus, causes vascular disease in oilseed rape (Brassica napus). We proposed that plant microRNAs (miRNAs) are involved in the plant-V. longisporum interaction. To identify oilseed rape miRNAs, we deep-sequenced two small RNA libraries made from V. longisporum infected/noninfected roots and employed Brassica rapa and Brassica oleracea genomes as references for miRNA prediction and characterization. We identified 893 B. napus miRNAs representing 360 conserved and 533 novel miRNAs, and mapped 429 and 464 miRNAs to the AA and CC genomes, respectively. Microsynteny analysis with the conserved miRNAs and their flanking protein coding sequences revealed 137 AA-CC genome syntenic miRNA pairs and 61 AA and 42 CC genome-unique miRNAs. Sixty-two miRNAs were responsive to the V. longisporum infection. We present data for specific interactions and simultaneously reciprocal changes in the expression levels of the miRNAs and their targets in the infected roots. We demonstrate that miRNAs are involved in the plant-fungus interaction and that miRNA168-Argonaute 1 (AGO1) expression modulation might act as a key regulatory module in a compatible plant-V. longisporum interaction. Our results suggest that V. longisporum may have evolved a virulence mechanism by interference with plant miRNAs to reprogram plant gene expression and achieve infection.

miR168 influences phase transition, leaf epinasty, and fruit development via SlAGO1s in tomato.

In Arabidopsis thaliana, Argonaute1 (AGO1) interacts with miR168 to modulate the small RNA regulatory pathway. However, the underlying mechanism of regulation and relationship between AGO1 and miR168 is poorly understood in the cash crop Solanum lycopersicum (tomato). We previously found that SlAGO1A and SlAGO1B were cleaved by miR168 in tomato. In this study, we show that SlAGO1A and SlAGO1B accumulate in miR168-sponge transgenic plants, and that expression of miR168-resistant SlAGO1A (4m-SlAGO1A) and SlAGO1B (4m-SlAGO1B) in tomato results in a series of defects affecting growth rate, floral timing, leaves, and fruit. Accumulation of miR156 was found when 4m-SlAGO1A was at an early developmental stage compared to the wild type and original SlAGO1A transgenic plants, and miR172 was highly expressed in adult 4m-SlAGO1A compared to the controls. In addition, the expression of multiple small RNAs was altered in 4m-SlAGO1A. Taken together, our data provide novel insights into the interaction between SlAGO1s and miR168 in determining growth rate, phase change, leaf epinasty, fruit initiation and expansion, and other developmental processes in tomato.

Mulberry-derived miR168a downregulates BmMthl1 to promote physical development and fecundity in silkworms.

Plant-derived miRNAs and their interactions with host organisms are considered important factors in regulating host physiological processes. In this study, we investigated the interaction between the silkworm, an oligophagous insect, and its primary food source, mulberry, to determine whether mulberry-derived miRNAs can penetrate silkworm cells and regulate their functions. Our results demonstrated that miR168a from mulberry leaves enters the silkworm hemolymph and binds to the silkworm Argonaute1 BmAGO1, which is transported via vesicles secreted by silkworm cells to exert its regulatory functions. In vivo and in vitro functional studies revealed that miR168a targets the mRNA of silkworm G protein-coupled receptor, BmMthl1, thereby inhibiting its expression and activating the JNK-FoxO pathway. This activation reduces oxidative stress responses, prolongs the lifespan of silkworms, and improves their reproductive capacity. These findings highlight the challenges of replacing mulberry leaves with alternative protein sources and provide a foundation for developing silkworm germplasms suitable for factory rearing.

New roles for microRNAs in cross-species communication.

Communication between cells ensures coordinated behavior. In prokaryotes, this signaling is typically referred to as quorum sensing, whereas in eukaryotic cells, communication occurs through hormones. In recent years, reports have shown that small noncoding RNAs, called microRNAs (miRNAs), can be transmitted from one species to another, inducing signal interference in distant species, even in a cross-kingdom manner. This new mode of cross-species communication might mediate symbiotic and pathogenic relationships between various organisms (e.g., microorganisms and their hosts). Here, we discuss several recent studies concerning miRNA-mediated cross-species gene regulation.

The Identification and Comparative Analysis of Non-Coding RNAs in Spores and Mycelia of Penicillium expansum.

Penicillium expansum is the most popular post-harvest pathogen and causes blue mold disease in pome fruit and leads to significant economic losses worldwide every year. However, the fundamental regulation mechanisms of growth in P. expansum are unclear. Recently, non-coding RNAs (ncRNAs) have attracted more attention due to critical roles in normalizing gene expression and maintaining cellular genotypes in organisms. However, the research related to ncRNAs in P. expansum have not been reported. Therefore, to provide an overview of ncRNAs on composition, distribution, expression changes, and potential targets in the growth process, a comparative transcriptomic analysis was performed on spores and mycelia of P. expansum in the present study. A total of 2595 novel mRNAs, 3362 long non-coding RNAs (lncRNAs), 10 novel microRNAs (miRNAs), 86 novel small interfering RNAs (siRNAs), and 11,238 circular RNAs (circRNAs) were predicted and quantified. Of these, 1482 novel mRNAs, 5987 known mRNAs, 2047 lncRNAs, 40 miRNAs, 38 novel siRNAs, and 9235 circRNAs were differentially expressed (DE) in response to the different development stages. Afterward, the involved functions and pathways of DE RNAs were revealed via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) database enrichment analysis. The interaction networks between mRNAs, lncRNAs, and miRNAs were also predicted based on their correlation coefficient of expression profiles. Among them, it was found that miR168 family members may play important roles in fungal growth due to their central location in the network. These findings will contribute to a better understanding on regulation machinery at the RNA level on fungal growth and provide a theoretical basis to develop novel control strategies against P. expansum.

The Translational Impact of Plant-Derived Xeno-miRNA miR-168 in Gastrointestinal Cancers and Preneoplastic Conditions.

INTRODUCTION: Diet is one of the most important factors contributing to the multistep process of carcinogenesis. The clinical relevance of exogenous food-derived xeno-microRNAs (miRNAs) in human diseases is poorly understood. In this study, we aimed to evaluate the potential clinical relevance of the xeno-miRNA miR-168 in the gastric mucosa along the preneoplastic conditions and gastric carcinogenesis. METHODS: For a systematic analysis, we included stomach tissues from patients with different pathologies, including normal mucosa (N), chronic non-atrophic (CNAG) and atrophic gastritis (CAG) and intestinal metaplasia (IM) (n = 72), matched non-tumorous (NT) and tumorous (T) gastric cancer (GC) tissues (n = 81), matched colorectal cancer (CRC) tissues (n = 40), and colon mucosa and faeces from controls and IBD patients. RESULTS: miR-168 was reproducibly detectable in all samples studied, with the highest levels in the proximal upper GI and in non-tumorous compared to tumorous tissues in both GC and CRC. There was no difference related to H. pylori positivity or inflammation grade, while higher miR-168 levels were observed in patients with moderate or severe AG/IM or OLGIM3/4. Survival analysis showed only a small, non-significant trend towards worse overall survival for patients with the highest to lowest miR-168 levels, while no differences were related to Lauren's classification. CONCLUSIONS: Food-derived xeno miRNAs are reproducibly detectable in the gastric and colonic mucosa. Although the clinically relevant function remains to be elucidated, higher levels of miR-168 in patients with moderate and severe IM merit further investigation.

Alfalfa Xeno-miR168b Target CPT1A to Regulate Milk Fat Synthesis in Bovine Mammary Epithelial Cells.

It was shown that microRNAs (miRNAs) play an important role in the synthesis of milk fat; thus, this manuscript evaluated whether exogenous miRNA (xeno-miRNAs) from alfalfa could influence the milk fat content in dairy cows. At first, mtr-miR168b was screened from dairy cow milk and blood. Then, EdU staining, flow cytometry, Oil Red O staining, qRT-PCR, and WB were applied to explore the effect of xeno-miR168b on the proliferation, apoptosis, and lipid metabolism of bovine mammary epithelial cells (BMECs). Finally, in order to clarify the pathway that regulated the lipid metabolism of BMECs using xeno-miR168b, a double-luciferase reporter assay was used to verify the target gene related to milk fat. These results showed that overexpression of xeno-miR168b inhibited cell proliferation but promoted apoptosis, which also decreased the expression of several lipid metabolism genes, including PPARγ, SCD1, C/EBPß, and SREBP1, significantly inhibited lipid droplet formation, and reduced triglyceride content in BMECs. Furthermore, the targeting relationship between CPT1A and xeno-miR168b was determined and it was confirmed that CPT1A silencing reduced the expression of lipid metabolism genes and inhibited fat accumulation in BMECs. These findings identified xeno-miR168b from alfalfa as a cross-kingdom regulatory element that could influence milk fat content in dairy cows by modulating CPT1A expression.

Rice miR168a-5p regulates seed length, nitrogen allocation and salt tolerance by targeting OsOFP3, OsNPF2.4 and OsAGO1a, respectively.

Rice microRNA168a (osa-miR168a) plays important roles in mediating flowering time, grain yield and vigor, seeding growth, and immunity by targeting the RNA-induced silencing complex component Argonaute 1 (AGO1). However, the functions of miR168a exerted by targeting other genes require further clarification before it could be used in rice molecular breeding. In this study, we identified a new target gene of osa-miR168a-5p (miR168a-5p) in rice called OsOFP3 (ovate family protein 3) and investigated the roles of miR168a-5p in response to brassinosteroids (BRs), salt stress, and nitrogen allocation. Up- and downregulated miR168a-5p expression respectively decreased and increased the expression of the BR-negative regulator OsOPF3. The results of RNA ligase-mediated rapid amplification of cDNA ends (5'RLM-RACE) revealed cleavage sites in OsOPF3 and OsNPF2.4 mRNAs. The phenotype of miR168a-5p transgenic rice was BR-associated and included the lamina bending response to BR, short seeds, and low 1000-grain weight. MicroRNA 168a-5p also regulated the expression of the nitrate transporter, OsNPF2.4, which affected nitrogen allocation, and regulated OsAGO1a expression in response to salt stress. Taken together, rice miR168a-5p regulates BR-associated pathways, nitrogen transport, and stress by targeting OsOFP3, OsNPF2.4, and OsAGO1a, respectively, resulting in a series of important agronomic traits for rice breeding.

Plant hvu-MIR168-3p enhances expression of glucose transporter 1 (SLC2A1) in human cells by silencing genes related to mitochondrial electron transport chain complex I.

Diet is a crucial factor for preventing most diseases. Edible plant extracts are known to contain exosome-like nanoparticles, in which food-derived plant microRNAs are included and may serve as a novel functional component in human health. Here, we demonstrated that hvu-MIR168-3p included in the nanoparticles of rice aleurone cells down-regulated the expression of the genes related to mitochondrial electron transport chain complex I in human cells. Subsequently, hvu-MIR168-3p enhanced protein and RNA expression levels of glucose transporter I and caused a decrease in the blood glucose level, which findings were obtained by in vitro and in vivo experiments, respectively. These findings suggest that a cross-kingdom relationship between plants and humans with respect to hvu-MIR168-3p exists and may contribute to preventive medicine for GLUT1-related dysfunctions including glucose metabolism, aging, and tumor immunology.

Common Variants of the Plant microRNA-168a Exhibit Differing Silencing Efficacy for Human Low-Density Lipoprotein Receptor Adaptor Protein 1 (LDLRAP1).

BACKGROUND: The discovery that a plant microRNA (miRNAs) from rice (Oryza sativa miR168a) can modify post-transcriptional expression of the mammalian. Low-Density Lipoprotein Receptor Adaptor Protein 1 (LDLRAP1) gene highlights the potential for cross-kingdom miRNAmRNA interactions. OBJECTIVE: To investigate whether common variants of the conserved miR168a family have the capability for similar cross-kingdom regulatory functions, we selected sequences from three dietary plant sources: rice (Oryza sativa), tomato (Solanum lycopersicum), apple (Malus domestica) and compared their ability to regulate human LDLRAP1 expression. METHODS: Target prediction software intaRNA and RNAhybrid were used to analyze and calculate the energy and alignment score between the miR168a variants and human LDLRAP1 mRNA. An in vitro cell-based Dual-Luciferase® Reporter Assay (pmirGLO, Promega), was then used to validate the miRNA-mRNA interaction experimentally. RESULTS: Computational analyses revealed that a single nucleotide difference at position 14 (from the 5' end of the miRNA) creates a G:U wobble in the miRNA-mRNA duplex formed by tomato and apple miR168a variants. This G:U wobble had only a small effect on the free energy score (-33.8-34.7 kcal/mol). However, despite reasonable hybridization energy scores (<-20 kcal/mol) for all miR168a variants, only the rice miR168a variant lacking a G:U wobble significantly reduced LDLRAP1 transcript expression by 25.8 + 7.3% (p<0.05), as measured by relative luciferase activity. CONCLUSION: In summary, single nucleotide differences at key positions can have a marked influence on regulatory function despite similar predicted energy scores and miRNA-mRNA duplex structures.

Structural Flexibility Enables Alternative Maturation, ARGONAUTE Sorting and Activities of miR168, a Global Gene Silencing Regulator in Plants.

In eukaryotes, the RNase-III Dicer often produces length/sequence microRNA (miRNA) variants, called "isomiRs", owing to intrinsic structural/sequence determinants of the miRNA precursors (pre-miRNAs). In this study, we combined biophysics, genetics and biochemistry approaches to study Arabidopsis miR168, the key feedback regulator of central plant silencing effector protein ARGONAUTE1 (AGO1). We identified a motif conserved among plant pre-miR168 orthologs, which enables flexible internal base-pairing underlying at least three metastable structural configurations. These configurations promote alternative, accurate Dicer cleavage events generating length and structural isomiR168 variants with distinctive AGO sorting properties and modes of action. Among these isomiR168s, a duplex with a 22-nt guide strand exhibits strikingly preferential affinity for AGO10, the closest AGO1 paralog. The 22-nt miR168-AGO10 complex antagonizes AGO1 accumulation in part via "transitive RNAi", a silencing-amplification process, to maintain appropriate AGO1 cellular homeostasis. Furthermore, we found that the tombusviral P19 silencing-suppressor protein displays markedly weaker affinity for the 22-nt form among its isomiR168 cargoes, thereby promoting AGO10-directed suppression of AGO1-mediated antiviral silencing. Taken together, these findings indicate that structural flexibility, a previously overlooked property of pre-miRNAs, considerably increases the versatility and regulatory potential of individual MIRNA genes, and that some pathogens might have evolved the capacity or mechanisms to usurp this property.

Position Based Nucleotide Analysis of miR168 Family in Higher Plants and its Targets in Mammalian Transcripts.

BACKGROUND: miRNA are the post transcriptional regulator of the genes. The conserved miR168 family is evaluated for position based nucleotide preference in higher plants. Low density lipoprotein receptor adaptor protein 1 (LDLRAP1) target validated for miR168a obtained from rice origin is reported. METHODS: The mature miRNA sequences include miR168-5p and miR168-3p, were obtained from miRBase (v21, June 2014) for 15 families (28 plants). The preferred position based nucleotide sequences were obtained using Data Analysis in Molecular Biology and Evolution software. The miR168-5p was subjected to cross kingdom analysis using psRNATarget. Target expression and functional annotation was analyzed by using Human Protein Atlas database WEB-based Gene SeTAnaLysis Toolkit. RESULTS: miR168-5p shows same nucleotides at positions 1-6, 8-9, 11-12, 15-17 and 19. Also, miR168-3p is present in 3 families (10 plants) shows the same nucleotide at position 1-11, 13-15 and 17-21. The 123 targets in human transcriptome were identified showing 58% cleavage and 41% translation repression. Low density lipoprotein receptor adaptor protein 1 (LDLRAP1) target validated for miR168a obtained from rice origin, could also be targeted from miR168 from any other plant sources. The randomly selected 10 targets include some important genes likeRPL34, ATXN1, AKAPI3 and ALS2 and is involved in transcription, cell trafficking, cell metabolism and neurodegenerative disorder. CONCLUSION: Our work suggests that miR168 family has conserved sequence in higher plants. The seed region position 2-8 shows 70-95% pairing with human targets. Cleavage site at position 10-14 and these were analysed for the base preference with the targets showed 80-96% Watson Crick pairing.

Design, Construction, and Validation of Artificial MicroRNA Vectors Using Agrobacterium-Mediated Transient Expression System.

Artificial microRNA (amiRNA) technology utilizes microRNA (miRNA) biogenesis pathway to produce artificially selected small RNAs using miRNA gene backbone. It provides a feasible strategy for inducing loss of gene function, and has been applied in functional genomics study, improvement of crop quality and plant virus disease resistance. A big challenge in amiRNA applications is the unpredictability of silencing efficacy of the designed amiRNAs and not all constructed amiRNA candidates would be expressed effectively in plant cells. We and others found that high efficiency and specificity in RNA silencing can be achieved by designing amiRNAs with perfect or almost perfect sequence complementarity to their targets. In addition, we recently demonstrated that Agrobacterium-mediated transient expression system can be used to validate amiRNA constructs, which provides a simple, rapid and effective method to select highly expressible amiRNA candidates for stable genetic transformation. Here, we describe the methods for design of amiRNA candidates with perfect or almost perfect base-pairing to the target gene or gene groups, incorporation of amiRNA candidates in miR168a gene backbone by one step inverse PCR amplification, construction of plant amiRNA expression vectors, and assay of transient expression of amiRNAs in Nicotiana benthamiana through agro-infiltration, small RNA extraction, and amiRNA Northern blot.