A global profiling of uncapped mRNAs under cold stress reveals specific decay patterns and endonucleolytic cleavages in Brachypodium
ABSTRACT: PARE (parallel analysis of RNA ends) was performed to study the change of uncapped mRNAs before and after cold treatment in Brachypodium. Different change patterns were identified. We have provided a complete view of uncapped transcriptome under cold stress condition, which will deepen our understanding of gene expression regulation in cold stress response as well as cold stress response mechanism for monocot plants. The uncapped mRNA profiles of 12-day-old Brachypodium seedlings with and without cold treatment (4 °C for 24 h) were generated by deep sequencing using Illumina GAIIx
Project description:RNA-Seq was performed to study the change of gene expression before and after cold treatment in Brachypodium. Different change patterns were identified. We have provided a complete view of transcriptome under cold stress condition, which will deepen our understanding of gene expression regulation in cold stress response as well as cold stress response mechanism for monocot plants. The mRNA profiles of 12-day-old Brachypodium seedlings with and without cold treatment (4 °C for 24 h) were generated by deep sequencing using Illumina HiSeq™ 2000.
Project description:ABSTRACT: Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional 70 and post-transcriptional levels. Small RNA (smRNA) pathways and splicing both function as crucial mechanisms regulating stress-responsive gene expression. However, examples of smRNAs regulating gene expression remain largely limited to effects on mRNA stability, translation, and epigenetic regulation. Also, our understanding of the networks controlling plant gene expression in response to environmental changes, and examples of these regulatory pathways intersecting, remains limited. Here, to investigate the role of smRNAs in stress responses we examined smRNA transcriptomes of Brachypodium distachyon plants subjected to various abiotic stresses. We found that exposure to different abiotic stresses specifically induced a group 75 of novel, endogenous small interfering RNAs (stress-induced, UTR-derived siRNAs, or sutr-siRNAs) that originate from the 3′ UTRs of a subset of coding genes. Our bioinformatics analyses predicted that sutr-siRNAs have potential regulatory functions and that over 90% of sutr-siRNAs target intronic regions of many mRNAs in trans. Importantly, a subgroup of these sutr- siRNAs target the important intron regulatory regions, such as branch point sequences, that could affect splicing. Our study indicates that in Brachypodium, sutr-siRNAs may affect splicing by masking or changing accessibility of specific cis-elements 80 through base-pairing interactions to mediate gene expression in response to stresses. We hypothesize that this mode of regulation of gene expression may also serve as a general mechanism for regulation of gene expression in plants and potentially in other eukaryotes. Analysis of smRNA populations in Brachypodium plants challenged by abiotic stresses: To profile the populations of smRNAs in the model monocot Brachypodium distachyon and examine their regulation in response to abiotic stresses, we conducted high-throughput sequencing of small RNAs from plants exposed to four different abiotic stress conditions, cold, heat (air), heat (water immersion), and salt, in the wild type Brachypodium cultivar Bd21. For our experiments we used information from the literature to select two time-points for stress durations, short and long, which differed for each stress: cold (6 and 24 hours), heat-air (1 and 3 hours), heat-water (1 and 3 hours), and salt (48 hours). We generated small RNA libraries for Illumina sequencing (GAII) from the leaves of Brachypodium plants subjected to stresses and selected smRNAs between 15 and 40 nt in length, which we mapped to the Brachypodium genome.
Project description:The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using Parallel Analysis of RNA Ends (PARE) data is lacking in B. distachyon. B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset to analyze small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner. B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants. Examination of various tissues and stresses in Brachypodium by high throughput sequencing for small RNA profiling and PARE (Parallel Analysis of RNA Ends)
Project description:Purpose: The goal of this study is to compare the transcriptomes expressed during submergence stress of two Brachypodium distachyon ecotypes with contrasting survival under this stress. Bd21 is a submergence sensitive ecotype with EC50 of 2.5 days and Bd2-3 is a tolerant ecotype with EC50 of 4 days. Methods (Stress): Brachypodium Bd21 and Bd2-3 plants (14-day-old, 6 leaves stage) were submerged in a water column of 30 cm inside opaque-wall plastic tanks. Light still reached the plants at 40 uE m-2 s-1. Ecotypes were submerged side-by-side in a randomized manner; only plants submerged in the same tank were compared. Controls were grown in plastic tanks without a water column. Submergence stress started at ZT14 (2h before night, long-day regime 16h light, 8h dark). Above ground tissue was collected after 48 h submergence stress in liquid nitrogen and stored at -80C in an ultra freezer until further processing. Tissue was ground to powder with mortar, pestle and liquid nitrogen avoiding thawing. Control and submerged total RNA was extracted with TRIzol reagent (Invitrogen, 15596018), purified with Direct-zol RNA mini prep columns (Zymo Research, R2050) and digested in-column with DNAse I (ThermoScientific, EN0521). RNA integrity and concentration was verified in denaturing 1.0% agarose gels, Nanodrop 2000 (ThermoScientific) and in a Bioanalyzer 2100 (Agilent) with the integrated software 2100 Expert, samples had a RNA Integrity Number (RIN) between 6.4-7.2 characteristic of aerial plant tissue (Babu and Gassman, 2011). Total RNA extracted from control and submerged tissue from three independent experiments consisting each of four individuals were used to construct cDNA indexed libraries and sequenced in a HiSeq2500 (Illumina) at 1x50 format, making a total of 12 sequenced libraries (tolerant and intolerant ecotype, control and submerged, all experimental triplicates) in a 2-lane format. RNA integrity, library construction and sequencing was performed as a service at the Unidad Universitaria de Secuenciacion Masiva, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico (IBT-UNAM). Differential Gene Expression (DGE) analysis was performed with edgeR using a generalized linear model and false discovery rate <0.05 (FDR). To group differentially expressed transcripts a logFC value of 1.5 (up-regulated) or -1.5 (down-regulated) and a FDR <0.05x10-5 were selected. GO analysis of differential transcripts was performed at phytozome.org Results: We identified commonly up-regulated genes (317) and exclusively up-regulated in Bd2-3 (466) or Bd21 (706). Regarding down-regulation, 330 transcripts were common, an exclusively 851 and 1026 for Bd2-3 and Bd21, respectively. GO analysis indicated that oxidative stress, pathogen responses and nitric oxide homeostasis were the most differential characteristics of tolerant ecotype Bd2-3. Conclusions: The use of triplicate RNAseq data of transcriptomes expressed in ecotypes with contrasting tolerance to submergence under long-day light regime, allowed us to identify common responsive routes such as SUSY, glycolysis, anaerobic routes (alanine, ethanol, lactate, GABA) and glyoxylate cycle. It also enabled us to discover integrated oxidative stress and NO homeostasis pathways that are differentially expressed in the tolerant ecotype. We expect that this information can be translated to agricultural relevant plants to increase our knowledge and biotechnological possibilities on plant submergence stress. Sequenced libraries (triplicates, HiSeq2500 Illumina, 1x50 format) of aerial tissue (control and 48h submergence stress) of Brachypodium distachyon Bd21 (sensitive) and Bd2-3 (tolerant).
Project description:The small RNA transcriptomes of bread wheat (Triticum aestivum L.) and its emerging model (Brachypodium distachyon (L.) Beauv) were obtained by using deep sequencing technology. Small RNA compositions were analyzed in these two species. In addition to 70 conserved microRNAs (miRNA) from 25 families, 23 novel wheat miRNAs were identified. For Brachypodium, 12 putative miRNAs were predicted from a limited number of ESTs, of which one was a potential novel miRNA. Also, 94 conserved miRNAs from 28 families were identified in this species. Expression validation was performed for several novel wheat miRNAs. RNA ligase-mediated 5' RACE experiments demonstrated their capability to cleave predicted target genes including three disease resistant gene analogs. Differential expression of miRNAs was observed between Brachypodium vegetative and reproductive tissues, suggesting their different roles at the two growth stages. Our work significantly increases the novel miRNA numbers in wheat and provides the first set of small RNAs in Brachypodium distachyon. Keywords: Small RNA One wheat small RNA library (Tae) and two Brachypodium small RNA libraries (BdR and BdV) were sequenced.
Project description:We present “centered sites,” a class of microRNA target sites that lacks both perfect seed pairing and 3'-compensatory pairing and instead has 11–12 contiguous Watson–Crick pairs to the center of the microRNA. In elevated Mg2+, centered sites impart mRNA cleavage, but in cells, centered sites repress protein output without consequential Agronaute-catalyzed cleavage. Our study also identified novel extensively paired sites that are cleavage substrates in cultured cells and human brain. This expanded repertoire of cleavage targets and the identification of the centered site type help explain why central regions of many microRNAs are evolutionarily conserved. To study centered sites and identify miRNA cleavage targets, mRNA degradomes were sequenced from human brain and HeLa cells, and smallRNAs were sequenced from human brain and zebrafish embryo at 24 hours post fertilization (hpf). Replicates were combined before the analysis. Fastq files are not available for GSM548638 and GSM548639.
Project description:Deep sequencing of the 5' ends of uncapped, polyA-enriched mRNA from two biological replicate samples from Arabidopsis thaliana inflorescences, as well as two biological replicates of Arabidopsis lyrata inflorescences. These data were used to experimentally identify sliced microRNA targets from the two species. Two biological replicate samples of the 5' ends of uncapped, polyA+ RNAs from both A. thaliana and A. lyrata
Project description:Stress acclimation is an effective mechanism that plants acquired for adaption to dynamic environmental conditions. After undergoing cold acclimation, plants become more tolerant to cold stress. In order to understand the mechanism of cold acclimation, we performed a systematic, comprehensive study of cold response and acclimation in Cassava (Manihot esculenta), a staple crop and major food source in the tropical regions of the world. We profiled mRNA genes and small-RNA species, using next generation sequencing, and performed an integrative analysis of the transcriptome and microRNAome of Cassava across the normal condition, a moderate cold stress at 14°C, a harsh stress at 4°C after cold acclimation at 14°C, and a cold shock from 24°C to 4°C. Two results from the analysis were striking. First, the moderate stress and cold shock, despite a difference of 10°C between the two, triggered comparable degrees of perturbation to the transcriptome; in contrary, further harsh stress after cold acclimation resulted in a much smaller degree of transcriptome variation. Second and more importantly, about two thirds of the up- or down-regulated genes after moderate stress reversed their expression to down- or up-regulation, respectively, under harsh stress after cold acclimation, resulting in a genome-wide rewiring of regulatory networks. MicroRNAs, which are key post-transcriptional gene regulators, were major players in this massive rewiring of genetic circuitry. Further, a function enrichment analysis of the perturbed genes revealed that cold acclimation helped the plant to develop immunity to further harsh stress by exclusively inducing genes with functions of nutrient reservoir; in contrast, many genes with functions of viral reproduction were induced by cold shock. Our study revealed, for the first time, the molecular basis of stress acclimation in plants, and shed lights on the role of microRNA gene regulation in cold response and acclimation in Euphorbia. Three organs/tissues (folded leaf, fully expanded leaf and roots) of Cassava cultivar SC124 harvested at 6h, 24h and 5d for three cold treatments of CA, CCA and CS, for gene expression profiling at the stages of initial response, secondary response, and functional adaption to cold stresses. Total RNA of each sample was isolated individually, and then pooled with an equal amount from each sample into one for profiling. As a result, four mRNA libraries and four small-RNA libraries, corresponding to the conditions of CA, CCA, CS and NC, were constructed.
Project description:miRNA levels depend on both biogenesis and turnover. The methyltransferase HEN1 stabilizes plant miRNAs, animal piRNAs, and siRNAs in both kingdoms via 3' terminal methylation. Loss of HEN1 in plants results in non-templated oligo-uridylation and accelerated degradation of miRNAs. In hen1 mutants from Arabidopsis and rice, we found that the patterns of miRNA truncation and uridylation differ substantially among miRNA families, but such patterns for the same miRNA are conserved between species. miR166 and miR163 are truncated predominantly to ~17 and ~16 nt, and subsequently recover via uridylation to approximately their original sizes, 21 and 24 nt, suggesting that in these cases miRNA truncation triggers uridylation. miR171 is untruncated but uridylated to 22 nt in hen1 mutants, gaining the ability to trigger production of phased, secondary siRNAs. Truncated and tailed variants were bound by ARGONAUTE1 (AGO1) in hen1, implying that these events occur while miRNAs are still bound by AGO1. Unexpectedly, a portion of miR158 in wildtype remains unmethylated and thus subject to uridylation and destabilization, suggesting that plants naturally utilize miRNA methylation to modulate miRNA accumulation. Our results suggest that the AGO1-containing RISC complex may undergo programming to reflect each bound miRNA, determining a defined, distinct decay destiny. In this analysis, we sequenced sRNAs from two hen1 mutant alleles in Arabidopsis and three hen1 alleles in rice. In Arabidopsis, the strong hen1-1 allele in the Landsberg erecta (Ler) ecotype is the first hen1 mutant and emerged from an enhancer screen in the hua1-1/hua2-1 background, and hen1-8 in the Columbia (Col) background is a weak allele. In rice, WAVY LEAF1 (WAF1) is the ortholog of Arabidopsis HEN1, and two mutant alleles waf1-1 and waf1-2 each bear a single-base substitution leading to a premature stop codon in the second exon and a non-functional splicing site of the fourth intron, respectively. We identified a third mutant allele of the rice HEN1 gene (Oshen1-3 from the Korean (POSTEC) rice T-DNA mutant population).
Project description:Two small RNA libraries were generated from micropropagated ‘Muscat Hamburg’ (Vitis vinifera) plantlets under normal and low temperatures (4 °C). A total of 163 known miRNAs and 299 putative novel miRNAs were detected from two small RNA libraries by Solexa sequencing. Forty-four cold-inducible miRNAs were identified through differentially expressed miRNAs (DEMs) analysis; among which, 13 belonged to upregulated DEMs while 31 belonged downregulated DEMs. This study indicated that a diverse set of miRNAs in V. vinifera are cold-inducible and may play an important role in cold stress response. Examination of small RNA populations in grapevine under cold treatment and none cold treatment.