Stress-induced endogenous siRNAs targeting regulatory intron sequences in Brachypodium
ABSTRACT: 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:Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional 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 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 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.
Project description:BACKGROUND: Small non-coding RNAs (smRNAs) are known to have major roles in gene regulation in eukaryotes. In plants, knowledge of the biogenesis and mechanisms of action of smRNA classes including microRNAs (miRNAs), short interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs) has been gained mostly through studies with Arabidopsis. In recent years, high throughput sequencing of smRNA populations has enabled extension of knowledge from model systems to plants with larger, more complex genomes. Soybean (Glycine max) now has many genomics resources available including a complete genome sequence and predicted gene models. Relatively little is known, however, about the full complement of its endogenous smRNAs populations and the silenced genes. RESULTS: Using Illumina sequencing and computational analysis, we characterized eight smRNA populations from multiple tissues and organs of soybean including developing seed and vegetative tissues. A total of 41 million raw sequence reads collapsed into 135,055 unique reads were mapped to the soybean genome and its predicted cDNA gene models. Bioinformatic analyses were used to distinguish miRNAs and siRNAs and to determine their genomic origins and potential target genes. In addition, we identified two soybean TAS3 gene homologs, the miRNAs that putatively guide cleavage of their transcripts, and the derived tasiRNAs that could target soybean genes annotated as auxin response factors. Tissue-differential expression based on the flux of normalized miRNA and siRNA abundances in the eight smRNA libraries was evident, some of which was confirmed by smRNA blotting. Our global view of these smRNA populations also revealed that the size classes of smRNAs varied amongst different tissues, with the developing seed and seed coat having greater numbers of unique smRNAs of the 24-nt class compared to the vegetative tissues of germinating seedlings. The 24-nt class is known to be derived from repetitive elements including transposons. Detailed analysis of the size classes associated with ribosomal RNAs and transposable element families showed greater diversity of smRNAs in the 22- and 24-nt size classes. CONCLUSIONS: The flux of endogenous smRNAs within multiple stages and tissues of seed development was contrasted with vegetative tissues of soybean, one of the dominant sources of protein and oil in world markets. The smRNAs varied in size class, complexity of origins, and possible targets. Sequencing revealed tissue-preferential expression for certain smRNAs and expression differences among closely related miRNA family members.
Project description:As important components of small RNA (smRNA) pathways, Argonaute (AGO) proteins mediate the interaction of incorporated smRNAs with their targets. Arabidopsis contains 10 AGO proteins with specialized or redundant functions. Among them, AGO1 mainly acts in microRNA (miRNA) and small-interfering RNA (siRNA) pathways for post-transcriptional gene silencing (PTGS), whereas AGO4 regulates transcriptional gene silencing (TGS) via endogenous 24-nucleotide (nt) smRNAs. To fully characterize smRNAs associated with AGO1 and AGO4, we developed a two-step protocol to purify AGO/smRNA complexes from flowers, leaves, roots and seedlings with enhanced purity, and sequenced the smRNAs by Illumina technology. Besides recovering most previously annotated smRNAs, we also identified some additional miRNAs, phased smRNA clusters and small-interfering RNAs derived from the overlapping region of natural antisense transcript pairs (NAT) (nat-siRNAs). We also identified a smRNA distribution feature on miRNA precursors which may help to identify authentic miRNAs. Organ-specific sequencing provided digital expression profiles of all obtained smRNAs, especially miRNAs. The presence and conservation of collateral miRNAs on known miRNA precursors were also investigated. Intriguingly, about 30% of AGO1-associated smRNAs were 24-nt long and unrelated to the 21-nt species. Further analysis showed that DNA-dependent RNA polymerase IV (Pol IV)-dependent smRNAs were mainly 24?nt and associated with AGO4, whereas the majority of the potential Pol V-dependent ones were 21-nt smRNAs and bound to AGO1, suggesting the potential involvement of AGO1 in Pol V-related pathways.
Project description:RNA silencing is an evolutionarily conserved process in eukaryotes that represses gene expression by using 21- to 24-nt guide RNAs to mediate mRNA cleavage or translational inhibition. Plants have two distinct groups of silencing-associated small RNAs (smRNAs): the micro RNAs (miRNAs) and the small interfering RNAs (siRNAs). A recent report by Yu et al. [Yu, B., Yang, Z., Li, J., Minakhina, S., Yang, M., Padgett, R. W., Steward, R. & Chen, X. (2005) Science 307, 932-935] has shown that plant miRNAs are modified at their 3' termini with a methyl group. Here, we show that a large fraction of all silencing-associated smRNAs in tobacco are modified; this modification occurs on the 2' hydroxyl of the terminal ribose and significantly reduces the cloning efficiency of these modified smRNAs. Expression of the strong silencing suppressor P1/helper-component proteinase results in a marked decrease in the 3'-terminal modification of viral siRNAs but does not significantly affect the modification of endogenous miRNAs and 24-nt siRNAs. The differential modification mediated by helper-component proteinase expression implies that exogenous and endogenous smRNAs are processed through independent pathways that are isolated by subcellular compartmentalization and/or the association with distinct Dicer complexes. The degree of terminal modification may play an important role in regulating the extent to which primary smRNA signals can be amplified by RNA-dependent RNA polymerases.
Project description:Small RNAs (smRNAs) in plants, mainly microRNAs and small interfering RNAs, play important roles in both transcriptional and post-transcriptional gene regulation. The broad application of high-throughput sequencing technology has made routinely generation of bulk smRNA sequences in laboratories possible, thus has significantly increased the need for batch analysis tools. PsRobot is a web-based easy-to-use tool dedicated to the identification of smRNAs with stem-loop shaped precursors (such as microRNAs and short hairpin RNAs) and their target genes/transcripts. It performs fast analysis to identify smRNAs with stem-loop shaped precursors among batch input data and predicts their targets using a modified Smith-Waterman algorithm. PsRobot integrates the expression data of smRNAs in major plant smRNA biogenesis gene mutants and smRNA-associated protein complexes to give clues to the smRNA generation and functional processes. Besides improved specificity, the reliability of smRNA target prediction results can also be evaluated by mRNA cleavage (degradome) data. The cross species conservation statuses and the multiplicity of smRNA target sites are also provided. PsRobot is freely accessible at http://omicslab.genetics.ac.cn/psRobot/.
Project description:The exosome functions throughout eukaryotic RNA metabolism and has a prominent role in gene silencing in yeast. In Arabidopsis, exosome regulates expression of a "hidden" transcriptome layer from centromeric, pericentromeric, and other heterochromatic loci that are also controlled by small (sm)RNA-based de novo DNA methylation (RdDM). However, the relationship between exosome and smRNAs in gene silencing in Arabidopsis remains unexplored. To investigate whether exosome interacts with RdDM, we profiled Arabidopsis smRNAs by deep sequencing in exosome and RdDM mutants and also analyzed RdDM-controlled loci. We found that exosome loss had a very minor effect on global smRNA populations, suggesting that, in contrast to fission yeast, in Arabidopsis the exosome does not control the spurious entry of RNAs into smRNA pathways. Exosome defects resulted in decreased histone H3K9 dimethylation at RdDM-controlled loci, without affecting smRNAs or DNA methylation. Exosome also exhibits a strong genetic interaction with RNA Pol V, but not Pol IV, and physically associates with transcripts produced from the scaffold RNAs generating region. We also show that two Arabidopsis rrp6 homologues act in gene silencing. Our data suggest that Arabidopsis exosome may act in parallel with RdDM in gene silencing, by epigenetic effects on chromatin structure, not through siRNAs or DNA methylation.
Project description:BACKGROUND: Endogenous small (sm) RNAs (primarily si- and miRNAs) are important trans/cis-acting regulators involved in diverse cellular functions. In plants, the RNA-dependent RNA polymerases (RDRs) are essential for smRNA biogenesis. It has been established that RDR2 is involved in the 24 nt siRNA-dependent RNA-directed DNA methylation (RdDM) pathway. Recent studies have suggested that RDR1 is involved in a second RdDM pathway that relies mostly on 21 nt smRNAs and functions to silence a subset of genomic loci that are usually refractory to the normal RdDM pathway in Arabidopsis. Whether and to what extent the homologs of RDR1 may have similar functions in other plants remained unknown. RESULTS: We characterized a loss-of-function mutant (Osrdr1) of the OsRDR1 gene in rice (Oryza sativa L.) derived from a retrotransposon Tos17 insertion. Microarray analysis identified 1,175 differentially expressed genes (5.2% of all expressed genes in the shoot-tip tissue of rice) between Osrdr1 and WT, of which 896 and 279 genes were up- and down-regulated, respectively, in Osrdr1. smRNA sequencing revealed regional alterations in smRNA clusters across the rice genome. Some of the regions with altered smRNA clusters were associated with changes in DNA methylation. In addition, altered expression of several miRNAs was detected in Osrdr1, and at least some of which were associated with altered expression of predicted miRNA target genes. Despite these changes, no phenotypic difference was identified in Osrdr1 relative to WT under normal condition; however, ephemeral phenotypic fluctuations occurred under some abiotic stress conditions. CONCLUSIONS: Our results showed that OsRDR1 plays a role in regulating a substantial number of endogenous genes with diverse functions in rice through smRNA-mediated pathways involving DNA methylation, and which participates in abiotic stress response.
Project description:To identifiy osmotic stress responsive smRNAs, we used a deep-sequencing technique to profile small RNA populations in leaf and root tissues of plants under high osmotic stress and control conditions. We treated 30day-old plants with high osmotic stress and sampled leaves and roots from the same plant with 3 biologial replicates. Then, 3 replicates were pooled and total RNA was extracted and prepared for smRNA deep sequencing. After normalization and annotation, we selected potential osmotic stress responsive smRNAs.
Project description:To fully characterize smRNAs associated with AGO1 and AGO4, we developed a two-step protocol to purify AGO/smRNA complexes from flowers, leaves, roots and seedlings with enhanced purity, and sequenced the smRNAs by Illumina’s technology. We identified some additional miRNAs, collateral miRNAs encoded in known miRNA precursors, phased smRNA clusters and nat-siRNAs. Organ specific sequencing provided digital expression profiles of all obtained smRNAs, especially miRNAs. We used extracts from Arabidopsis flowers, leaves and roots as well as ten-day old seedlings to purify smRNAs associated with AGO1 and AGO4 protein complexes using a two-step immunoprecipitation method.
Project description:BACKGROUND:Animal and plant genomes produce numerous small RNAs (smRNAs) that regulate gene expression post-transcriptionally affecting metabolism, development, and epigenetic inheritance. In order to characterize the repertoire of endogenous smRNAs and potential gene targets in dinoflagellates, we conducted smRNA and mRNA expression profiling over 9 experimental treatments of cultures from Symbiodinium microadriaticum, a photosynthetic symbiont of scleractinian corals. RESULTS:We identified a set of 21 novel smRNAs that share stringent key features with functional microRNAs from other model organisms. smRNAs were predicted independently over all 9 treatments and their putative gene targets were identified. We found 1,720 animal-like target sites in the 3'UTRs of 12,858 mRNAs and 19 plant-like target sites in 51,917 genes. We assembled a transcriptome of 58,649 genes and determined differentially expressed genes (DEGs) between treatments. Heat stress was found to produce a much larger number of DEGs than other treatments that yielded only few DEGs. Analysis of DEGs also revealed that minicircle-encoded photosynthesis proteins seem to be common targets of transcriptional regulation. Furthermore, we identified the core RNAi protein machinery in Symbiodinium. CONCLUSIONS:Integration of smRNA and mRNA expression profiling identified a variety of processes that could be under microRNA control, e.g. protein modification, signaling, gene expression, and response to DNA damage. Given that Symbiodinium seems to have a paucity of transcription factors and differentially expressed genes, identification and characterization of its smRNA repertoire establishes the possibility of a range of gene regulatory mechanisms in dinoflagellates acting post-transcriptionally.