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Elevated CO2 along with drought is a serious global threat to crop productivity. Therefore, understanding the molecular mechanisms plants use to protect these stresses is the key for plant growth and development. In this study, we mimicked natural stress conditions under a controlled Soil-Plant-Atmosphere-Research (SPAR) system and provided the evidence for how miRNAs regulate target genes under elevated CO2 and drought conditions. Significant physiological and biomass data supported the effective utilization of source-sink (leaf to root) under elevated CO2. Additionally, elevated CO2 partially rescued the effect of drought on total biomass. We identified both known and novel miRNAs differentially expressed during drought, CO2, and combined stress, along with putative targets. A total of 32 conserved miRNAs belonged to 23 miRNA families, and 25 novel miRNAs were identified by deep sequencing. Using the existing sweet potato genome database and stringent analyses, a total of 42 and 22 potential target genes were predicted for the conserved and novel miRNAs, respectively. These target genes are involved in drought response, hormone signaling, photosynthesis, carbon fixation, sucrose and starch metabolism, etc. Gene ontology and KEGG ontology functional enrichment revealed that these miRNAs might target transcription factors (MYB, TCP, NAC), hormone signaling regulators (ARF, AP2/ERF), cold and drought factors (corA), carbon metabolism (ATP synthase, fructose-1,6-bisphosphate), and photosynthesis (photosystem I and II complex units). Our study is the first report identifying targets of miRNAs under elevated CO2 levels and could support the molecular mechanisms under elevated CO2 in sweet potato and other crops in the future.

The tobacco hornworm Manduca sexta has served as a model for insect biochemical and physiological research for decades. However, knowledge of the posttranscriptional regulation of gene expression by microRNAs is still rudimentary in this species. Our previous study (Zhang et al., 2012) identified 163 conserved and 13 novel microRNAs in M. sexta, most of which were present at low levels in pupae. To identify additional M. sexta microRNAs and more importantly to examine their possible roles in the expression regulation of immunity-related genes, we constructed four small RNA libraries using fat body and hemocytes from naïve or bacteria-injected larvae and obtained 32.9 million reads of 18-31 nucleotides by Illumina sequencing. Mse-miR-929 and mse-miR-1b (antisense microRNA of mse-miR-1) were predicted in the previous study and now found to be conserved microRNAs in the tissue samples. We also found four novel microRNAs, two of which result from a gene cluster. Mse-miR-281-star, mse-miR-965-star, mse-miR-31-star, and mse-miR-9b-star were present at higher levels than their respective mature strands. Abundance changes of microRNAs were observed after the immune challenge. Based on the quantitative data of mRNA levels in control and induced fat body and hemocytes as well as the results of microRNA target site prediction, we suggest that certain microRNAs and microRNA*s regulate gene expression for pattern recognition, prophenoloxidase activation, cellular responses, antimicrobial peptide synthesis, and conserved intracellular signal transduction (Toll, IMD, JAK-STAT, MAPK-JNK-p38, and small interfering RNA pathways). In summary, this study has enriched our knowledge on M. sexta microRNAs and how some of them may participate in the expression regulation of immunity-related genes.

MicroRNA395 (miR395) is a conserved miRNA that targets a low-affinity sulfate transporter (AST68) and three ATP sulfurylases (APS1, APS3 and APS4) in higher plants. In this study, At2g28780 was confirmed as another target of miR395 in Arabidopsis. Interestingly, several dicots contained genes homologous to At2g28780 and a cognate miR395 complementary site but possess a gradient of mismatches at the target site. It is well established that miR395 is induced during S deprivation in Arabidopsis; however, the signaling pathways that mediate this regulation are unknown. Several findings in the present study demonstrate that redox signaling plays an important role in induction of miR395 during S deprivation. These include the following results: (i) glutathione (GSH) supplementation suppressed miR395 induction in S-deprived plants (ii) miR395 is induced in Arabidopsis seedlings exposed to Arsenate or Cu(2+) , which induces oxidative stress (iii), S deprivation-induced oxidative stress, and (iv) compromised induction of miR395 during S deprivation in cad2 mutant (deficient in GSH biosynthesis) that is defective in glutaredoxin-dependent redox signaling and ntra/ntrb (defective in thioredoxin reductases a and b) double mutants that are defective in thioredoxin-dependent redox signaling. Collectively, these findings strongly support the involvement of redox signaling in inducing the expression of miR395 during S deprivation in Arabidopsis.

Wheat is the most highly cultivated plant species for its grain production throughout the world. Because small RNA-dependent gene regulation is critical for successful completion of plant life cycle including its productivity, identification of not only miRNAs but also confirming their targets in wheat is important. To identify small RNAs including novel miRNAs as well as miRNA targets in wheat, we constructed small RNA and degradome libraries from wheat seedlings. Small RNA analysis resulted in identification of most conserved miRNAs including novel miRNAs that can be grouped into 32 miRNA families. The sequence analysis also led to the characterization of two abundantly expressed rRNA-derived small RNAs. To identify miRNA targets, degradome library was sequenced and the bioinformatic analysis confirmed 53 genes as targets for miRNAs and Tas3-siRNAs. Degradome analysis also confirmed a conserved fine-tuning mechanism of Tas3-siRNA abundance by siRNA-mediated silencing of TAS3 transcripts in diverse plant species. These findings added additional information to the small RNA knowledge-base in wheat.

Sweet Sorghum is largely grown for grain production but also recently emerged as one of the model feedstock plants for biofuel production. In plants, microRNA (miRNA)-guided gene regulation plays a key role in diverse biological processes, thus, their identification in different plant species is essential to understand post-transcriptional gene regulation. To identify miRNAs in Sorghum, we sequenced a small RNA library. Sequence analysis revealed the identity of 29 conserved miRNA families. Importantly, 13 novel miRNAs are identified, seven of which are conserved in closely related monocots. Temporal expression analysis of conserved and novel miRNAs indicated differential expression of several miRNAs. Approximately 125 genes that play diverse roles have been predicted as targets and a few targets were experimentally validated. These results provided insights into miRNA-controlled processes in Sorghum and also laid the foundation for manipulating miRNAs or their targets for improving biomass production and stress tolerance in Sorghum.

In recent years, several plant species such as switchgrass, Miscanthus and Brachypodium have been recognized as potential model plant species for cellulosic bioenergy production. Of these, switchgrass has attracted much attention in the United States and worldwide because it can grow well on marginal lands and tolerate frequent drought spells. However, little is known about the basic biology of the traits that control these important characteristics in switchgrass. Genome-encoded approximately 21-24nt microRNAs (miRNAs) have emerged as critical regulators of gene expression important for normal growth and development and adaptation to abiotic stress, including nutrient-deprived conditions. To understand miRNA-guided post-transcriptional gene regulatory networks in this plant species, we sought to identify miRNAs in switchgrass. Using computational and experimental approaches, we identified approximately 20 conserved miRNA families. Temporal expression analysis indicated that some miRNAs have distinct tissue-specific expression, although most are ubiquitously expressed. Unlike in Arabidopsis and other plants, miR395 and miR399 were detected in plants grown on optimal levels of sulfate or phosphate in switchgrass, and were only slightly altered when exposed to sulfate or phosphate deficit conditions. Thirty-seven genes were predicted as targets for miRNAs, and 4 target mRNAs (Squamosa promoter binding-like factor, apetala 2-like, NAC domain containing transcription factor and HD-Zip homologs) were validated by 5'-RACE assays. These findings provide a snapshot of the miRNA component and possible targets in switchgrass.

Complex epithelial and stromal cell interactions are required during the development and progression of prostate cancer. Regulatory small non-coding microRNAs (miRNAs) participate in the spatiotemporal regulation of messenger RNA (mRNA) and regulation of translation affecting a large number of genes involved in prostate carcinogenesis. In this study, through deep-sequencing of size fractionated small RNA libraries we profiled the miRNAs of prostate epithelial (PrEC) and stromal (PrSC) cells. Over 50 million reads were obtained for PrEC in which 860,468 were unique sequences. Similarly, nearly 76 million reads for PrSC were obtained in which over 1 million were unique reads. Expression of many miRNAs of broadly conserved and poorly conserved miRNA families were identified. Sixteen highly expressed miRNAs with significant change in expression in PrSC than PrEC were further analyzed in silico. ConsensusPathDB showed the target genes of these miRNAs were significantly involved in adherence junction, cell adhesion, EGRF, TGF-β and androgen signaling. Let-7 family of tumor-suppressor miRNAs expression was highly pervasive in both, PrEC and PrSC cells. In addition, we have also identified several miRNAs that are unique to PrEC or PrSC cells and their predicted putative targets are a group of transcription factors. This study provides perspective on the miRNA expression in PrEC and PrSC, and reveals a global trend in miRNA interactome. We conclude that the most abundant miRNAs are potential regulators of development and differentiation of the prostate gland by targeting a set of growth factors. Additionally, high level expression of the most members of let-7 family miRNAs suggests their role in the fine tuning of the growth and proliferation of prostate epithelial and stromal cells.

Background Regulatory function of small non-coding RNAs (sRNA) in response to environmental and developmental cues has been established. Additionally, sRNA, also plays an important role in maintaining the heterochromatin and centromere structures of the chromosome. Papaya, a trioecious species with recently evolved sex chromosomes, has emerged as an excellent model system to study sex determination and sex chromosome evolution in plants. However, role of small RNA in papaya sex determination is yet to be explored. Results We analyzed the high throughput sRNAs reads in the Illumina libraries prepared from male, female, and hermaphrodite flowers of papaya. Using the sRNA reads, we identified 29 miRNAs that were not previously reported from papaya. Including this and two previous studies, a total of 90 miRNAs has been identified in papaya. We analyzed the expression of these miRNAs in each sex types. A total of 65 miRNAs, including 31 conserved and 34 novel mirNA, were detected in at least one library. Fourteen of the 65 miRNAs were differentially expressed among different sex types. Most of the miRNA expressed higher in male flowers were related to the auxin signaling pathways, whereas the miRNAs expressed higher in female flowers were the potential regulators of the apical meristem identity genes. Aligning the sRNA reads identified the sRNA hotspots adjacent to the gaps of the X and Y chromosomes. The X and Y chromosomes sRNA hotspots has a 7.8 and 4.4 folds higher expression of sRNA, respectively, relative to the chromosome wide average. Approximately 75% of the reads aligned to the X chromosome hotspot was identical to that of the Y chromosome hotspot. Conclusion By analyzing the large-scale sRNA sequences from three sex types, we identified the sRNA hotspots flanking the gaps of papaya X, Y, and Yh chromosome. The sRNAs expression patterns in these regions were reminiscent of the pericentromeric region indicating that the only remaining gap in each of these chromosomes is likely the centromere. We also identified 14 differentially expressed miRNAs in male, female and hermaphrodite flowers of papaya. Our results provide valuable information toward understanding the papaya sex determination. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-20) contains supplementary material, which is available to authorized users.

Endogenous small RNAs can be grouped into several distinct classes of 21-nt-long microRNAs (miRNAs), short interfering RNAs (siRNAs), trans-acting siRNAs (tasiRNAs), and 24-nt long heterochromatic siRNAs. miRNAs are increasingly being recognized as significant effectors of gene regulation in a wide range of organisms. These molecules are typically ∼21-nt long and are amenable for cloning by streamlined protocols. Here we detail the methodology for cloning small RNAs in rice to identify novel miRNAs and other important small RNAs. Briefly, small RNA molecules are size fractionated, attached to adaptors, and subsequently converted into cDNA and PCR amplified. Current high-throughput sequencing technologies allow sequencing of the PCR products directly.

Background MicroRNAs (miRNAs) are a class of non-coding small RNAs involved in post-transcriptional regulation of gene expression critical for plant growth and development, stress responses and other diverse biological processes in plants. The Cucurbitaceae or cucurbit family represents some of economically important species, particularly those with edible and medicinal fruits. Genomic tools for the molecular analysis of members of this family are just emerging. Partial draft genome sequence became available recently for cucumber and watermelon facilitating investigation of the small RNA component of the transcriptomes in cucurbits. Results We generated four small RNA libraries from bottle gourd (Lagenaria siceraria), Cucurbita moschata, Cucurbita pepo, and, watermelon (Citrullus lanatus var. lanatus) in order to identify conserved and novel lineage specific miRNAs in these cucurbits. Deep sequencing of small RNA libraries from these species resulted in 1,597,263, 532,948, 601,388, and 493,384 unique sRNA reads from bottle gourd, moschata, pepo and watermelon, respectively. Sequence analysis of these four libraries resulted in identification of 21 miRNA families that are highly conserved and 8 miRNA families that are moderately conserved in diverse dicots. We also identified 4 putative novel miRNAs in these plant species. Furthermore, the tasiRNAs were identified and their biogenesis was determined in these cucurbits. Small RNA blot analysis or q-PCR analyses of leaf and fruit tissues of these cucurbits showed differential expression of several conserved miRNAs. Interestingly, the abundance of several miRNAs in leaves and fruits of closely related C. moschata and C. pepo was also distinctly different. Target genes for the most conserved miRNAs are also predicted. Conclusion High-throughput sequencing of small RNA libraries from four cucurbit species has provided a glimpse of small RNA component in their transcriptomes. The analysis also showed considerable variation within four cucurbit species with regards to expression of individual miRNAs.

MicroRNAs (miRNAs) are a group of small RNAs involved in translation inhibition or mRNA degradation. Due to its large size, Manduca sexta has long been used as a model to study insect physiology and biochemistry. While transcriptome studies have greatly enriched our knowledge on M. sexta structural genes, little is known about posttranscriptional regulation by miRNAs in this lepidopteran species. We constructed four small RNA libraries from embryos, 4th instar feeding larvae, pupae, and adults, obtained 21 million reads of 18-31 nucleotides by Illumina sequencing, and found 163 conserved and 13 novel miRNAs. By searching the M. sexta genome assembly, we identified precursors of 82 conserved miRNAs, 76 of which had mapped reads in one or more of these libraries. After normalization, we compared numbers of miRNA and miRNA-star reads in these libraries and observed abundance changes during development. Interestingly, mse-miR-281-star, mse-miR-31-star, mse-miR-965-star, mse-miR-9a-star, mse-miR-9b-star, mse-miR-2a-star, mse-miR-92b-star and mse-miR-279c-star are either more abundant or maintained at similar levels compared to respective mature miRNA strand. Expression profiling of the first set of miRNAs provided insights to their possible involvement in developmental regulation. This study will aid in the annotation of miRNA genes in the genome.

The discovery of microRNAs (miRNAs) as gene regulators has led to a paradigm shift in the understanding of post-transcriptional gene regulation in plants and animals. miRNAs have emerged as master regulators of plant growth and development. Evidence suggesting that miRNAs play a role in plant stress responses arises from the discovery that miR398 targets genes with known roles in stress tolerance. In addition, the expression profiles of most miRNAs that are implicated in plant growth and development are significantly altered during stress. These later findings imply that attenuated plant growth and development under stress may be under the control of stress-responsive miRNAs. Here we review recent progress in the understanding of miRNA-mediated plant stress tolerance.

MicroRNA (miRNA)-guided target RNA expression is vital for a wide variety of biological processes in eukaryotes. Currently, miRBase (version 13) lists 142 and 353 miRNAs from Arabidopsis and rice (Oryza sativa), respectively. The integration of miRNAs in diverse biological networks relies upon the confirmation of their RNA targets. In contrast with the well-characterized miRNA targets that are cleaved in Arabidopsis, only a few such targets have been confirmed in rice. To identify small RNA targets in rice, we applied the 'degradome sequencing' approach, which globally identifies the remnants of small RNA-directed target cleavage by sequencing the 5' ends of uncapped RNAs. One hundred and sixty targets of 53 miRNA families (24 conserved and 29 rice-specific) and five targets of TAS3-small interfering RNAs (siRNAs) were identified. Surprisingly, an additional conserved target for miR398, which has not been reported so far, has been validated. Besides conserved homologous transcripts, 23 non-conserved genes for nine conserved miRNAs and 56 genes for 29 rice-specific miRNAs were also identified as targets. Besides miRNA targets, the rice degradome contained fragments derived from MIRNA precursors. A closer inspection of these fragments revealed a unique pattern distinct from siRNA-producing loci. This attribute can serve as one of the ancillary criteria for separating miRNAs from siRNAs in plants.

Background In eukaryotes, microRNAs (miRNAs) have emerged as critical regulators of gene expression. The Silkworm (Bombyx mori L.) is one of the most suitable lepidopteran insects for studying the molecular aspects of metamorphosis because of its large size, availability of mutants and genome sequence. Besides, this insect also has been amply studied from a physiological and biochemical perspective. Deep sequencing of small RNAs isolated from different stages of silkworm is a powerful tool not only for measuring the changes in miRNA profile but also for discovering novel miRNAs. Results We generated small RNA libraries from feeding larvae, spinning larvae, pupae and adults of B. mori and obtained ~2.5 million reads of 18-30 nt. Sequence analysis identified 14 novel and 101 conserved miRNAs. Most novel miRNAs are preferentially expressed in pupae, whereas more than 95% of the conserved miRNAs are dynamically regulated during different developmental stages. Remarkably, the miRNA-star (miR*) of four miRNAs are expressed at much higher levels than their corresponding miRNAs, and their expression profiles are distinct from their corresponding miRNA profiles during different developmental stages. Additionally, we detected two antisense miRNA loci (miR-263-S and miR-263-AS; miR-306-S and miR-306-AS) that are expressed in sense and antisense directions. Interestingly, miR-263 and miR-306 are preferentially and abundantly expressed in pupae and adults, respectively. Conclusions We identified 101 homologs of conserved miRNAs, 14 species-specific and two antisense miRNAs in the silkworm. Our results provided deeper insights into changes in conserved and novel miRNA and miRNA* accumulation during development.

MicroRNAs (miRNAs) and small-interfering RNAs (siRNAs) have emerged as important regulators of gene expression in higher eukaryotes. Recent studies indicate that genomes in higher plants encode lineage-specific and species-specific miRNAs in addition to the well-conserved miRNAs. Leguminous plants are grown throughout the world for food and forage production. To date the lack of genomic sequence data has prevented systematic examination of small RNAs in leguminous plants. Medicago truncatula, a diploid plant with a near-completely sequenced genome has recently emerged as an important model legume. We sequenced a small RNA library generated from M. truncatula to identify not only conserved miRNAs but also novel small RNAs, if any. Eight novel small RNAs were identified, of which four (miR1507, miR2118, miR2119 and miR2199) are annotated as legume-specific miRNAs because these are conserved in related legumes. Three novel transcripts encoding TIR-NBS-LRR proteins are validated as targets for one of the novel miRNA, miR2118. Small RNA sequence analysis coupled with the small RNA blot analysis, confirmed the expression of around 20 conserved miRNA families in M. truncatula. Fifteen transcripts have been validated as targets for conserved miRNAs. We also characterized Tas3-siRNA biogenesis in M. truncatula and validated three auxin response factor (ARF) transcripts that are targeted by tasiRNAs. These findings indicate that M. truncatula and possibly other related legumes have complex mechanisms of gene regulation involving specific and common small RNAs operating post-transcriptionally.

MicroRNA398 targets two Cu/Zn superoxide dismutases (CSD1 and CSD2) in higher plants. Previous investigations revealed both decreased miR398 expression during high Cu(2+) or paraquat stress and increased expression under low Cu(2+) or high sucrose in the growth medium. Here, we show that additional abiotic stresses such as ozone and salinity also affect miR398 levels. Ozone fumigation decreased miR398 levels that were gradually restored to normal levels after relieved from the stress. Furthermore, miR398 levels decreased in Arabidopsis leaves infiltrated with avirulent strains of Pseudomonas syringae pv. tomato, Pst DC3000 (avrRpm1 or avrRpt2) but not the virulent strain Pst DC3000. To our knowledge, miR398 is the first miRNA shown to be down-regulated in response to biotic stress (P. syringae). CSD1, but not CSD2, mRNA levels were negatively correlated with miR398 levels during ozone, salinity and biotic stress, suggesting that CSD2 regulation is not strictly under miR398 control during diverse stresses. Overall, this study further establishes a link between oxidative stress and miR398 in Arabidopsis.

Background MicroRNAs (miRNAs) are small ~22-nt regulatory RNAs that can silence target genes, by blocking their protein production or degrading the mRNAs. Pig is an important animal in the agriculture industry because of its utility in the meat production. Besides, pig has tremendous biomedical importance as a model organism because of its closer proximity to humans than the mouse model. Several hundreds of miRNAs have been identified from mammals, humans, mice and rats, but little is known about the miRNA component in the pig genome. Here, we adopted an experimental approach to identify conserved and unique miRNAs and characterize their expression patterns in diverse tissues of pig. Results By sequencing a small RNA library generated using pooled RNA from the pig heart, liver and thymus; we identified a total of 120 conserved miRNA homologs in pig. Expression analysis of conserved miRNAs in 14 different tissue types revealed heart-specific expression of miR-499 and miR-208 and liver-specific expression of miR-122. Additionally, miR-1 and miR-133 in the heart, miR-181a and miR-142-3p in the thymus, miR-194 in the liver, and miR-143 in the stomach showed the highest levels of expression. miR-22, miR-26b, miR-29c and miR-30c showed ubiquitous expression in diverse tissues. The expression patterns of pig-specific miRNAs also varied among the tissues examined. Conclusion Identification of 120 miRNAs and determination of the spatial expression patterns of a sub-set of these in the pig is a valuable resource for molecular biologists, breeders, and biomedical investigators interested in post-transcriptional gene regulation in pig and in related mammals, including humans.

Background MicroRNAs (miRNAs) are recently discovered small non-coding RNAs that play pivotal roles in gene expression, specifically at the post-transcriptional level in plants and animals. Identification of miRNAs in large number of diverse plant species is important to understand the evolution of miRNAs and miRNA-targeted gene regulations. Now-a-days, publicly available databases play a central role in the in-silico biology. Because, at least ~21 miRNA families are conserved in higher plants, a homology based search using these databases can help identify orthologs or paralogs in plants. Results We searched all publicly available nucleotide databases of genome survey sequences (GSS), high-throughput genomics sequences (HTGS), expressed sequenced tags (ESTs) and nonredundant (NR) nucleotides and identified 682 miRNAs in 155 diverse plant species. We found more than 15 conserved miRNA families in 11 plant species, 10 to14 families in 10 plant species and 5 to 9 families in 29 plant species. Nineteen conserved miRNA families were identified in important model legumes such as Medicago, Lotus and soybean. Five miRNA families – miR319, miR156/157, miR169, miR165/166 and miR394 – were found in 51, 45, 41, 40 and 40 diverse plant species, respectively. miR403 homologs were found in 16 dicots, whereas miR437 and miR444 homologs, as well as the miR396d/e variant of the miR396 family, were found only in monocots, thus providing large-scale authenticity for the dicot- and monocot-specific miRNAs. Furthermore, we provide computational and/or experimental evidence for the conservation of 6 newly found Arabidopsis miRNA homologs (miR158, miR391, miR824, miR825, miR827 and miR840) and 2 small RNAs (small-85 and small-87) in Brassica spp. Conclusion Using all publicly available nucleotide databases, 682 miRNAs were identified in 155 diverse plant species. By combining the expression analysis with the computational approach, we found that 6 miRNAs and 2 small RNAs that have been identified only in Arabidopsis thus far, are also conserved in Brassica spp. These findings will be useful for tracing the evolution of small RNAs by examining their expression in common ancestors of the Arabidopsis-Brassica lineage.

Among legumes, chickpea (Cicer arietinum L.) is the second most important crop after soybean. MicroRNAs (miRNAs) play important roles by regulating target gene expression important for plant development and tolerance to stress conditions. Additionally, recently discovered phased siRNAs (phasiRNAs), a new class of small RNAs, are abundantly produced in legumes. Nevertheless, little is known about these regulatory molecules in chickpea. The small RNA population was sequenced from leaves and flowers of chickpea to identify conserved and novel miRNAs as well as phasiRNAs/phasiRNA loci. Bioinformatics analysis revealed 157 miRNA loci for the 96 highly conserved and known miRNA homologs belonging to 38 miRNA families in chickpea. Furthermore, 20 novel miRNAs belonging to 17 miRNA families were identified. Sequence analysis revealed approximately 60 phasiRNA loci. Potential target genes likely to be regulated by these miRNAs were predicted and some were confirmed by modified 5′ RACE assay. Predicted targets are mostly transcription factors that might be important for developmental processes, and others include superoxide dismutases, plantacyanin, laccases and F-box proteins that could participate in stress responses and protein degradation. Overall, this study provides an inventory of miRNA–target gene interactions for chickpea, useful for the comparative analysis of small RNAs among legumes.