miRNA-mediated Cleavage Research Articles

Identification of microRNAs involved in ectomycorrhizal formation in Populus tomentosa.

The majority of woody plants are able to form ectomycorrhizal (ECM) symbioses with fungi. During symbiotic development, plants undergo a complex re-programming process involving a series of physiological and morphological changes. MicroRNAs (miRNAs) are important components of the regulatory network underlying symbiotic development. To elucidate the mechanisms of miRNAs and miRNA-mediated mRNA cleavage during symbiotic development, we conducted high-throughput sequencing of small RNAs and degradome tags from roots of Populus tomentosa inoculated with Cenococcum geophilum. This process led to the annotation of 51 differentially expressed miRNAs between non-mycorrhizal and mycorrhizal roots of P. tomentosa, including 13 novel miRNAs. Increased or decreased accumulation of several novel and conserved miRNAs in ECM roots, including miR162, miR164, miR319, miR396, miR397, miR398, novel-miR44 and novel-miR47, suggests essential roles for these miRNAs in ECM formation. The degradome analysis identified root transcripts as miRNA-mediated mRNA cleavage targets, which was confirmed using real-time quantitative PCR. Several of the identified miRNAs and corresponding targets are involved in arbuscular mycorrhizal symbioses. In summary, increased or decreased accumulation of specific miRNAs and miRNA-mediated cleavage of symbiosis-related genes indicate that miRNAs play important roles in the regulatory network underlying symbiotic development.

Multi-omics of Circular RNAs and Their Responses to Hormones in Moso Bamboo (Phyllostachys edulis)

Circular RNAs (circRNAs) are endogenous non-coding RNAs with covalently closed structures, which have important functions in plants. However, their biogenesis, degradation, and function upon treatment with gibberellins (GAs) and auxins (1-naphthaleneacetic acid, NAA) remain unknown. Here, we systematically identified and characterized the expression patterns, evolutionary conservation, genomic features, and internal structures of circRNAs using RNase R-treated libraries from moso bamboo (Phyllostachys edulis) seedlings. Moreover, we investigated the biogenesis of circRNAs dependent on both cis- and trans-regulation. We explored the function of circRNAs, including their roles in regulating microRNA (miRNA)-related genes and modulating the alternative splicing of their linear counterparts. Importantly, we developed a customized degradome sequencing approach to detect miRNA-mediated cleavage of circRNAs. Finally, we presented a comprehensive view of the participation of circRNAs in the regulation of hormone metabolism upon treatment of bamboo seedlings with GA and NAA. Collectively, our study provides insights into the biogenesis, function, and miRNA-mediated degradation of circRNAs in moso bamboo.

Genetically Encoded Light-Up RNA Amplifier Dissecting MicroRNA Activity in Live Cells.

Live cell dissection of microRNA activities is crucial for basic and translational medicine, but current hybridization-based strategies may fail to dissect surrounding-dependent activities. Here, we develop a genetically encoded miRNA-induced light-up RNA amplifier (iLAMP) that enables fast-activated, signal-amplified, fluorogenic imaging of miRNA activities in live cells. iLAMP responds to miRNA targets in the mode of "activation upon cleavage", in which the light-up RNA aptamer restores its fluorescence rapidly upon cleavage by the RNA-induced silencing complex. We demonstrate that iLAMP affords substantial signal amplification of ∼100-fold and high specificity in single nucleotide discrimination because of the miRNA-mediated cyclic cleavage. Combined with a Mango RNA aptamer reference module and a pseudoknot terminal stabilizer, iLAMP is shown for quantitative ratiometric imaging and dynamic monitoring of miRNA activities under exogenous stimulations. iLAMP is featured by a modular "plug and play" design and can be readily adapted to the detection of other miRNAs, highlighting its potential in tracking cell differentiation and screening miRNA therapeutics.

Degradome comparison between wild and cultivated rice identifies differential targeting by miRNAs

BackgroundSmall non-coding (s)RNAs are involved in the negative regulation of gene expression, playing critical roles in genome integrity, development and metabolic pathways. Targeting of RNAs by ribonucleoprotein complexes of sRNAs bound to Argonaute (AGO) proteins results in cleaved RNAs having precise and predictable 5` ends. While tools to study sliced bits of RNAs to confirm the efficiency of sRNA-mediated regulation are available, they are sub-optimal. In this study, we provide an improvised version of a tool with better efficiency to accurately validate sRNA targets.ResultsHere, we improvised the CleaveLand tool to identify additional micro (mi)RNA targets that belong to the same family and also other targets within a specified free energy cut-off. These additional targets were otherwise excluded during the default run. We employed these tools to understand the sRNA targeting efficiency in wild and cultivated rice, sequenced degradome from two rice lines, O. nivara and O. sativa indica Pusa Basmati-1 and analyzed variations in sRNA targeting. Our results indicate the existence of multiple miRNA-mediated targeting differences between domesticated and wild species. For example, Os5NG4 was targeted only in wild rice that might be responsible for the poor secondary wall formation when compared to cultivated rice. We also identified differential mRNA targets of secondary sRNAs that were generated after miRNA-mediated cleavage of primary targets.ConclusionsWe identified many differentially targeted mRNAs between wild and domesticated rice lines. In addition to providing a step-wise guide to generate and analyze degradome datasets, we showed how domestication altered sRNA-mediated cascade silencing during the evolution of indica rice.

Widespread occurrence of microRNA-mediated target cleavage on membrane-bound polysomes

BackgroundSmall RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) serve as core players in gene silencing at transcriptional and post-transcriptional levels in plants, but their subcellular localization has not yet been well studied, thus limiting our mechanistic understanding of sRNA action.ResultsWe investigate the cytoplasmic partitioning of sRNAs and their targets globally in maize (Zea mays, inbred line “B73”) and rice (Oryza sativa, cv. “Nipponbare”) by high-throughput sequencing of polysome-associated sRNAs and 3′ cleavage fragments, and find that both miRNAs and a subset of 21-nucleotide (nt)/22-nt siRNAs are enriched on membrane-bound polysomes (MBPs) relative to total polysomes (TPs) across different tissues. Most of the siRNAs are generated from transposable elements (TEs), and retrotransposons positively contributed to MBP overaccumulation of 22-nt TE-derived siRNAs (TE-siRNAs) as opposed to DNA transposons. Widespread occurrence of miRNA-mediated target cleavage is observed on MBPs, and a large proportion of these cleavage events are MBP-unique. Reproductive 21PHAS (21-nt phasiRNA-generating) and 24PHAS (24-nt phasiRNA-generating) precursors, which were commonly considered as noncoding RNAs, are bound by polysomes, and high-frequency cleavage of 21PHAS precursors by miR2118 and 24PHAS precursors by miR2275 is further detected on MBPs. Reproductive 21-nt phasiRNAs are enriched on MBPs as opposed to TPs, whereas 24-nt phasiRNAs are nearly completely devoid of polysome occupancy.ConclusionsMBP overaccumulation is a conserved pattern for cytoplasmic partitioning of sRNAs, and endoplasmic reticulum (ER)-bound ribosomes function as an independent regulatory layer for miRNA-induced gene silencing and reproductive phasiRNA biosynthesis in maize and rice.

Integration of small RNA, degradome and proteome sequencing in Oryza sativa reveals a delayed senescence network in tetraploid rice seed.

Seed of rice is an important strategic resource for ensuring the security of China's staple food. Seed deterioration as a result of senescence is a major problem during seed storage, which can cause major economic losses. Screening among accessions in rice germplasm resources for traits such as slow senescence and increased seed longevity during storage is, therefore, of great significance. However, studies on delayed senescence in rice have been based mostly on diploid rice seed to date. Despite better tolerance have been verified by the artificial aging treatment for polyploid rice seed, the delayed senescence properties and delayed senescence related regulatory mechanisms of polyploid rice seed are rarely reported, due to the lack of polyploid rice materials with high seed set. High-throughput sequencing was applied to systematically investigate variations in small RNAs, the degradome, and the proteome between tetraploid and diploid rice seeds. Degradome sequencing analysis of microRNAs showed that expression of miR-164d, which regulates genes encoding antioxidant enzymes, was changed significantly, resulting in decreased miRNA-mediated cleavage of target genes in tetraploid rice. Comparisons of the expression levels of small RNAs (sRNAs) in the tetraploid and diploid libraries revealed that 12 sRNAs changed significantly, consistent with the findings from degradome sequencing. Furthermore, proteomics also showed that antioxidant enzymes were up-regulated in tetraploid rice seeds, relative to diploids.

A dynamic degradome landscape on miRNAs and their predicted targets in sugarcane caused by Sporisorium scitamineum stress

BackgroundSugarcane smut is a fungal disease caused by Sporisorium scitamineum. Cultivation of smut-resistant sugarcane varieties is the most effective way to control this disease. The interaction between sugarcane and S. scitamineum is a complex network system. However, to date, there is no report on the identification of microRNA (miRNA) target genes of sugarcane in response to smut pathogen infection by degradome technology.ResultsTaqMan qRT-PCR detection and enzyme activity determination showed that S. scitamineum rapidly proliferated and incurred significant enzyme activity changes in the reactive oxygen species metabolic pathway and phenylpropanoid metabolic pathway at 2 d and 5 d after inoculation, which was the best time points to study target gene degradation during sugarcane and S. scitamineum interaction. A total of 122.33 Mb of raw data was obtained from degradome sequencing analysis of YC05–179 (smut-resistant) and ROC22 (smut-susceptible) after inoculation. The Q30 of each sample was > 93%, and the sequence used for degradation site analysis exactly matched the sugarcane reference sequence. A total of 309 target genes were predicted in sugarcane, corresponding to 97 known miRNAs and 112 novel miRNAs, and 337 degradation sites, suggesting that miRNAs can efficiently direct cleavage at multiple sites in the predicted target mRNAs. Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the predicted target genes were involved in various regulatory processes, such as signal transduction mechanisms, inorganic ion transport and metabolism, defense mechanisms, translation, posttranslational modifications, energy production and conversion, and glycerolipid metabolism. qRT-PCR analysis of the expression level of 13 predicted target genes and their corresponding miRNAs revealed that there was no obvious negative regulatory relationship between miRNAs and their target genes. In addition, a number of putative resistance-related target genes regulated by miRNA-mediated cleavage were accumulated in sugarcane during S. scitamineum infection, suggesting that feedback regulation of miRNAs may be involved in the response of sugarcane to S. scitamineum infection.ConclusionsThis study elucidates the underlying response of sugarcane to S. scitamineum infection, and also provides a resource for miRNAs and their predicted target genes for smut resistance improvement in sugarcane.

Degradome Sequencing in Plants.

Degradome sequencing provides large amounts of data regarding RNA degradation. The degradome library construction described here is modified from the 5'-rapid amplification of cDNA ends (5'-RACE), and each degradome cDNA is sequenced by next-generation sequencing (NGS). Degradome profiles provide information confirming miRNA-mediated cleavage of target genes and allow the identification of novel targets. Furthermore, degradome sequencing provides additional information for the study of RNA processing, such as information regarding RNA-binding proteins. In this chapter, we describe a detailed optimized protocol for constructing a degradome library with high yield and quality, along with NGS and data mining procedures. We hope that the degradome approach will be applied to further studies of non-model organisms.

Mimicry Technology: A Versatile Tool for Small RNA Suppression.

A decade ago the discovery of the target mimicry regulatory process on the activity of a mature microRNA (miRNA) enabled for the first time the customized attenuation of miRNA activity in plants. That powerful technology was named MIMIC and was based on engineering the IPS1 long noncoding transcript to become complementary to the miRNA under study. In order to avoid IPS1 degradation, the predicted miRNA-mediated cleavage site was interrupted by three additional nucleotides giving rise to the so-called MIMIC decoy. Since then, MIMIC technology has been used in several plant species and in basic and translational research. We here provide a detailed guide to produce custom-designed MIMIC decoys to facilitate the study of sRNA functions in plants.

A Short Indel-Lacking-Resistance Gene Triggers Silencing of the Photosynthetic Machinery Components Through TYLCSV-Associated Endogenous siRNAs in Tomato.

Plant viruses modify gene expression in infected tissues by altering the micro (mi)RNA-mediated regulation of genes. Among conserved miRNA targets there are transcripts coding for transcription factors, RNA silencing core, and disease-resistance proteins. Paralogs in these gene families are widely present in plant genomes and are known to respond differently to miRNA-mediated regulation during plant virus infections. Using genome-wide approaches applied to Solanum lycopersicum infected by a nuclear-replicating virus, we highlighted miRNA-mediated cleavage events that could not be revealed in virus-free systems. Among them we confirmed miR6024 targeting and cleavage of RX-coiled-coil (RX-CC), nucleotide binding site (NBS), leucine-rich (LRR) mRNA. Cleavage of paralogs was associated with short indels close to the target sites, indicating a general functional significance of indels in fine-tuning gene expression in plant–virus interaction. miR6024-mediated cleavage, uniquely in virus-infected tissues, triggers the production of several 21–22 nt secondary siRNAs. These secondary siRNAs, rather than being involved in the cascade regulation of other NBS–LRR paralogs, explained cleavages of several mRNAs annotated as defence-related proteins and components of the photosynthetic machinery. Outputs of these data explain part of the phenotype plasticity in plants, including the appearance of yellowing symptoms in the viral pathosystem.

Biogenesis and regulatory hierarchy of phased small interfering RNAs in plants.

SummarySeveral varieties of small RNAs including microRNAs (miRNAs) and small interfering RNAs (siRNAs) are generated in plants to regulate development, genome stability and response to adverse environments. Phased siRNA (phasiRNA) is a type of secondary siRNA that is processed from a miRNA‐mediated cleavage of RNA transcripts, increasing silencing efficiency or simultaneously suppressing multiple target genes. Trans‐acting siRNAs (ta‐siRNAs) are a particular class of phasiRNA produced from noncoding transcripts that silence targets in trans. It was originally thought that ‘one‐hit’ and ‘two‐hit’ models were essential for processing distinct TAS precursors; however, a single hit event was recently shown to be sufficient at triggering all types of ta‐siRNAs. This review discusses the findings about biogenesis, targeting modes and regulatory networks of plant ta‐siRNAs. We also summarize recent advances in the generation of other phasiRNAs and their possible biological benefits to plants.

Activity and roles of Arabidopsis thaliana XRN family exoribonucleases in noncoding RNA pathways.

RNA metabolism is mediated by several sophisticated exo- or endo- ribonucleases. XRN family proteins are the conserved 5'-3' exoribonucleases in eukaryotes. A. thaliana genome encodes three XRN homologs (AtXRN2, AtXRN3 and AtXRN4) and their independent or redundant roles, which are possibly plant-specific in some cases, have been reported. AtXRN2 acts in maturation of ribosomal RNAs partially with AtXRN3. AtXRN3 is also involved in elimination of 3' remnants of microRNA precursors and in termination of mRNA transcription events. AtXRN4 degrades not only a small fraction of mRNAs in stress response but also 3' cleavage products of miRNA-mediated cleavage of target mRNAs. Moreover, all AtXRNs are important factors to suppress unexpected RNA silencing occurrence. Thus, this review summarizes and discusses multiple roles of AtXRN exoribonucleases and their relationship with noncoding RNA pathways including RNA silencing pathways.

Unravelling the complexity of microRNA-mediated gene regulation in black pepper (Piper nigrum L.) using high-throughput small RNA profiling

Analysis of high-throughput small RNA deep sequencing data, in combination with black pepper transcriptome sequences revealed microRNA-mediated gene regulation in black pepper ( Piper nigrum L.). Black pepper is an important spice crop and its berries are used worldwide as a natural food additive that contributes unique flavour to foods. In the present study to characterize microRNAs from black pepper, we generated a small RNA library from black pepper leaf and sequenced it by Illumina high-throughput sequencing technology. MicroRNAs belonging to a total of 303 conserved miRNA families were identified from the sRNAome data. Subsequent analysis from recently sequenced black pepper transcriptome confirmed precursor sequences of 50 conserved miRNAs and four potential novel miRNA candidates. Stem-loop qRT-PCR experiments demonstrated differential expression of eight conserved miRNAs in black pepper. Computational analysis of targets of the miRNAs showed 223 potential black pepper unigene targets that encode diverse transcription factors and enzymes involved in plant development, disease resistance, metabolic and signalling pathways. RLM-RACE experiments further mapped miRNA-mediated cleavage at five of the mRNA targets. In addition, miRNA isoforms corresponding to 18 miRNA families were also identified from black pepper. This study presents the first large-scale identification of microRNAs from black pepper and provides the foundation for the future studies of miRNA-mediated gene regulation of stress responses and diverse metabolic processes in black pepper.

Identification, evolution, and expression partitioning of miRNAs in allopolyploid Brassica napus.

The recently published genome of Brassica napus offers for the first time the opportunity to gain insights into the genomic organization and the evolution of miRNAs in oilseed rape. In this study, 12 small RNA libraries from two B. napus cultivars (Tapidor and Ningyou7) and their four double-haploid lines were sequenced, employing the newly sequenced B. napus genome, together with genomes of its progenitors Brassica rapa and Brassica oleracea. A total of 645 miRNAs including 280 conserved and 365 novel miRNAs were identified. Comparative analysis revealed a high level of genomic conservation of MIRNAs (75.9%) between the subgenomes of B. napus and its two progenitors’ genomes, and MIRNA lost/gain events (133) occurred in B. napus after its speciation. Furthermore, significant partitioning of miRNA expressions between the two subgenomes in B. napus was detected. The data of degradome sequencing, miRNA-mediated cleavage, and expression analyses support specific interactions between miRNAs and their targets in the modulation of diverse physiological processes in roots and leaves, as well as in biosynthesis of, for example, glucosinolates and lipids in oilseed rape. These data provide a first genome-wide view on the origin, evolution, and genomic organization of B. napus MIRNAs.

Abstract 196: A SNP in the 3′-untranslated region of FZD4 linked to lung cancer survival modulates a miRNA-mediated FZD4 transcript binding, cleavage, expression, and Wnt-signaling in NSCLC cells

Abstract Single nucleotide polymorphisms (SNPs) in miRNA genes and miRNA-associated pathways (miR-SNPs) are predicted to have significant effects on gene expression and cellular processes by disrupting miRNA biogenesis and modulating miRNA-mRNA interactions. We identified several germline SNPs at potential miRNA-binding sites in the 3′ untranslated regions (3′UTRs) of genes in the wingless (Wnt) signaling pathway to be significantly associated with overall survival in NSCLC patients. SNP (rs713065 with a C allele) in the 3′UTR of the FZD4 gene in the Wnt canonical pathway displayed a significant association with decreased risk of death in early NSCLC patients. We aimed to determine the biological function and clinical relevance of this novel epidemiological FZD4-miR-SNP (rs713065) in NSCLC by identifying specific miRNAs that could directly interact with the FZD4-miR-SNP locus and assessing the effect of such a SNP and associated miRNAs on target gene regulation and NSCLC prognosis. We used a novel stem-loop array-reverse transcription-PCR (SLA-RT-PCR) assay to assess miRNA:target mRNA interaction at the specific SNP locus and detected miRNA-mediated FZD4 mRNA cleavage and 3′-uridylation activities in FZD4-SNP (C, rs713065) allele-bearing NSCLC cells H1299 and H322, but not in FZD4-WT (T) allele-containing A549 and normal human bronchial epithelial cells. A significant down regulation of GFP and Luciferase (Luc) gene and protein expression in the presence of FZD4-SNP was detected in NSCLC cell lines using both GFP-SNP and Dual-Luc-SNP reporter systems. The presence of the FZD4-SNP in the 3′UTR also down-regulated the ectopic expression of the host FZD4 gene and protein and inhibited tumor colony formation and tumor cell mobility in NSCLC cells. We identified miR-204 as a potential miRNA candidate that differentially interacts with and targets allelic variants at FZD4-miR-SNP (rs713065) loci. Target mRNA cleavage was detected at the SNP C allele site but not at the WT-allele T site in H1299 cells after co-transfection with a GFP-FZD4-SNP reporter and miR-204 expression constructs by SLA-RT-PCR. A significant correlation of miR-204 with FZD4 gene expression was also detected in NSCLC cell lines and primary tumor specimens in various miRNA expression profiling databases. Our findings suggest that the miR-204 binding site at the SNP (rs713065) loci in the 3′UTR of FZD4 may influence survival in NSCLC by modulating FZD4 expression and the Wnt/FZD4-signaling driven tumor cell proliferation and progression. (This study is partially supported by NIH/NCI grants Lung SPORE 5P50CA070907 and R01CA176568, a CPRIT Grant and a MDACC Moonshot Program Grant) Citation Format: Jing Lin, Roza Zandi, Jian Gu, Yuanqin Ye, Alexander Pertsemlidis, Xifeng Wu, Jack A. Roth, Lin Ji. A SNP in the 3′-untranslated region of FZD4 linked to lung cancer survival modulates a miRNA-mediated FZD4 transcript binding, cleavage, expression, and Wnt-signaling in NSCLC cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 196. doi:10.1158/1538-7445.AM2015-196

High-throughput sequencing-based genome-wide identification of microRNAs expressed in developing cotton seeds.

MicroRNAs (miRNAs) have been shown to play critical regulatory roles in gene expression in cotton. Although a large number of miRNAs have been identified in cotton fibers, the functions of miRNAs in seed development remain unexplored. In this study, a small RNA library was constructed from cotton seeds sampled at 15 days post-anthesis (DPA) and was subjected to high-throughput sequencing. A total of 95 known miRNAs were detected to be expressed in cotton seeds. The expression pattern of these identified miRNAs was profiled and 48 known miRNAs were differentially expressed between cotton seeds and fibers at 15 DPA. In addition, 23 novel miRNA candidates were identified in 15-DPA seeds. Putative targets for 21 novel and 87 known miRNAs were successfully predicted and 900 expressed sequence tag (EST) sequences were proposed to be candidate target genes, which are involved in various metabolic and biological processes, suggesting a complex regulatory network in developing cotton seeds. Furthermore, miRNA-mediated cleavage of three important transcripts in vivo was validated by RLM-5' RACE. This study is the first to show the regulatory network of miRNAs that are involved in developing cotton seeds and provides a foundation for future studies on the specific functions of these miRNAs in seed development.

MicroRNA396-Targeted SHORT VEGETATIVE PHASE Is Required to Repress Flowering and Is Related to the Development of Abnormal Flower Symptoms by the Phyllody Symptoms1 Effector.

Leafy flowers are the major symptoms of peanut witches' broom (PnWB) phytoplasma infection in Catharanthus roseus. The orthologs of the phyllody symptoms1 (PHYL1) effector of PnWB from other species of phytoplasma can trigger the proteasomal degradation of several MADS box transcription factors, resulting in leafy flower formation. In contrast, the flowering negative regulator gene SHORT VEGETATIVE PHASE (SVP) was up-regulated in PnWB-infected C. roseus plants, but most microRNA (miRNA) genes had repressed expression. Coincidentally, transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the PHYL1 gene of PnWB (PHYL1 plants), which show leafy flower phenotypes, up-regulate SVP of Arabidopsis (AtSVP) but repress a putative regulatory miRNA of AtSVP, miR396. However, the mechanism by which PHYL1 regulates AtSVP and miR396 is unknown, and the evidence of miR396-mediated AtSVP degradation is lacking. Here, we show that miR396 triggers AtSVP messenger RNA (mRNA) decay using genetic approaches, a reporter assay, and high-throughput degradome profiles. Genetic evidence indicates that PHYL1 plants and atmir396a-1 mutants have higher AtSVP accumulation, whereas the transgenic plants overexpressing MIR396 display lower AtSVP expression. The reporter assay indicated that target-site mutation results in decreasing the miR396-mediated repression efficiency. Moreover, degradome profiles revealed that miR396 triggers AtSVP mRNA decay rather than miRNA-mediated cleavage, implying that AtSVP caused miR396-mediated translation inhibition. We hypothesize that PHYL1 directly or indirectly interferes with miR396-mediated AtSVP mRNA decay and synergizes with other effects (e.g. MADS box transcription factor degradation), resulting in abnormal flower formation. We anticipate our findings to be a starting point for studying the posttranscriptional regulation of PHYL1 effectors in symptom development.

Transcriptome-Wide Identification of miRNA Targets under Nitrogen Deficiency in Populus tomentosa Using Degradome Sequencing.

miRNAs are endogenous non-coding small RNAs with important regulatory roles in stress responses. Nitrogen (N) is an indispensable macronutrient required for plant growth and development. Previous studies have identified a variety of known and novel miRNAs responsive to low N stress in plants, including Populus. However, miRNAs involved in the cleavage of target genes and the corresponding regulatory networks in response to N stress in Populus remain largely unknown. Consequently, degradome sequencing was employed for global detection and validation of N-responsive miRNAs and their targets. A total of 60 unique miRNAs (39 conserved, 13 non-conserved, and eight novel) were experimentally identified to target 64 mRNA transcripts and 21 precursors. Among them, we further verified the cleavage of 11 N-responsive miRNAs identified previously and provided empirical evidence for the cleavage mode of these miRNAs on their target mRNAs. Furthermore, five miRNA stars (miRNA*s) were shown to have cleavage function. The specificity and diversity of cleavage sites on the targets and miRNA precursors in P. tomentosa were further detected. Identification and annotation of miRNA-mediated cleavage of target genes in Populus can increase our understanding of miRNA-mediated molecular mechanisms of woody plants adapted to low N environments.

Transcriptionally and post-transcriptionally regulated microRNAs in heat stress response in barley.

Heat stress is one of the major abiotic factors that can induce severe plant damage, leading to a decrease in crop plant productivity. Despite barley being a cereal of great economic importance, few data are available concerning its thermotolerance mechanisms. In this work microRNAs (miRNAs) involved in heat stress response in barley were investigated. The level of selected barley mature miRNAs was examined by hybridization. Quantitative real-time PCR (RT-qPCR) was used to monitor the changes in the expression profiles of primary miRNA (pri-miRNA) precursors, as well as novel and conserved target genes during heat stress. The miRNA-mediated cleavage sites in the target transcripts were confirmed by degradome analysis and the 5' RACE (rapid amplification of cDNA ends) approach. Four barley miRNAs (miR160a, 166a, 167h, and 5175a) were found which are heat stress up-regulated at the level of both mature miRNAs and precursor pri-miRNAs. Moreover, the splicing of introns hosting miR160a and miR5175a is also heat induced. The results demonstrate transcriptional and post-transcriptional regulation of heat-responsive miRNAs in barley. The observed induction of miRNA expression is correlated with the down-regulation of the expression level of their experimentally identified new and conservative target genes.

MicroRNAs in a multicellular green alga Volvox carteri

microRNAs (miRNAs) have emerged as key components in the eukaryotic gene regulatory network. We and others have previously identified many miRNAs in a unicellular green alga, Chlamydomonas reinhardtii. To investigate whether miRNA-mediated gene regulation is a general mechanism in green algae and how miRNAs have been evolved in the green algal lineage, we examined small RNAs in Volvox carteri, a multicellular species in the same family with Chlamydomonas reinhardtii. We identified 174 miRNAs in Volvox, with many of them being highly enriched in gonidia or somatic cells. The targets of the miRNAs were predicted and many of them were subjected to miRNA-mediated cleavage in vivo, suggesting that miRNAs play regulatory roles in the biology of green algae. Our catalog of miRNAs and their targets provides a resource for further studies on the evolution, biological functions, and genomic properties of miRNAs in green algae.