Bioinformatics analysis of small RNAs in pima (Gossypium barbadense L.).
ABSTRACT: Small RNAs (sRNAs) are ~20 to 24 nucleotide single-stranded RNAs that play crucial roles in regulation of gene expression. In plants, sRNAs are classified into microRNAs (miRNAs), repeat-associated siRNAs (ra-siRNAs), phased siRNAs (pha-siRNAs), cis and trans natural antisense transcript siRNAs (cis- and trans-nat siRNAs). Pima (Gossypium barbadense L.) is one of the most economically important fiber crops, producing the best and longest spinnable fiber. Although some miRNAs are profiled in Pima, little is known about siRNAs, the largest subclass of plant sRNAs. In order to profile these gene regulators in Pima, a comprehensive analysis of sRNAs was conducted by mining publicly available sRNA data, leading to identification of 678 miRNAs, 3,559,126 ra-siRNAs, 627 pha-siRNAs, 136,600 cis-nat siRNAs and 79,994 trans-nat siRNAs. The 678 miRNAs, belonging to 98 conserved and 402 lineage-specific families, were produced from 2,138 precursors, of which 297 arose from introns, exons, or intron/UTR-exon junctions of protein-coding genes. Ra-siRNAs were produced from various repeat loci, while most (97%) were yielded from retrotransposons, especially LTRs (long terminal repeats). The genes encoding auxin-signaling-related proteins, NBS-LRRs and transcription factors were major sources of pha-siRNAs, while two conserved TAS3 homologs were found as well. Most cis-NATs in Pima overlapped in enclosed and convergent orientations, while a few hybridized in divergent and coincided orientations. Most cis- and trans-nat siRNAs were produced from overlapping regions. Additionally, characteristics of length and the 5'-first nucleotide of each sRNA class were analyzed as well. Results in this study created a valuable molecular resource that would facilitate studies on mechanism of controlling gene expression.
Project description:MicroRNAs (miRNAs) and secondary small interfering RNAs (principally phased siRNAs or trans-acting siRNAs) are two distinct subfamilies of small RNAs (sRNAs) that are emerging as key regulators of posttranscriptional gene expression in plants. Both miRNAs and secondary-siRNAs (sec-siRNAs) are processed from longer RNA precursors by DICER-LIKE proteins (DCLs). Gossypium arboreum L., also known as tree cotton or Asian cotton, is a diploid, possibly ancestral relative of tetraploid Gossypium hirsutum L., the predominant type of commercially grown cotton worldwide known as upland cotton. To understand the biological significance of these gene regulators in G. arboreum, a bioinformatics analysis was performed on G. arboreum small RNAs produced from G. arboreum leaf, flower, and boll tissues. Consequently, 263 miRNAs derived from 353 precursors, including 155 conserved miRNAs (cs-miRNAs) and 108 novel lineage-specific miRNAs (ls-miRNAs). Along with miRNAs, 2,033 miRNA variants (isomiRNAs) were identified as well. Those isomiRNAs with variation at the 3'-miRNA end were expressed at the highest levels, compared to other types of variants. In addition, 755 pha-siRNAs derived 319 pha-siRNA gene transcripts (PGTs) were identified, and the potential pha-siRNA initiators were predicted. Also, 2,251 non-phased siRNAs were found as well, of which 1,088 appeared to be produced by so-called cis- or trans-cleavage of the PGTs observed at positions differing from pha-siRNAs. Of those sRNAs, 148 miRNAs/isomiRNAs and 274 phased/non-phased siRNAs were differentially expressed in one or more pairs of tissues examined. Target analysis revealed that target genes for both miRNAs and pha-siRNAs are involved a broad range of metabolic and enzymatic activities. We demonstrate that secondary siRNA production could result from initial cleavage of precursors by both miRNAs or isomiRNAs, and that subsequently produced phased and unphased siRNAs could result that also serve as triggers of a second round of both cis- and trans-cleavage of additional siRNAs, leading to the formation of complex sRNA regulatory networks mediating posttranscriptional gene silencing. Results from this study extended our knowledge on G. arboreum sRNAs and their biological importance, which would facilitate future studies on regulatory mechanism of tissue development in cotton and other plant species.
Project description:Trans-acting small interfering RNAs (ta-siRNAs) and natural cis-antisense siRNAs (cis-nat-siRNAs) are recently discovered small RNAs (sRNAs) involved in post-transcriptional gene silencing. ta-siRNAs are transcribed from genomic loci and require processing by microRNAs (miRNAs). cis-nat-siRNAs are derived from antisense RNAs produced by the simultaneous transcription of overlapping antisense genes. Their roles in many plant processes, including pathogen response, are mostly unknown. In this work, we employed a bioinformatic approach to identify ta-siRNAs and cis-nat-siRNAs in cassava from two sRNA libraries, one constructed from healthy cassava plants and one from plants inoculated with the bacterium Xanthomonas axonopodis pv. manihotis (Xam). A total of 54 possible ta-siRNA loci were identified in cassava, including a homolog of TAS3, the best studied plant ta-siRNA. Fifteen of these loci were induced, while 39 were repressed in response to Xam infection. In addition, 15 possible cis-natural antisense transcript (cis-NAT) loci producing siRNAs were identified from overlapping antisense regions in the genome, and were found to be differentially expressed upon Xam infection. Roles of sRNAs were predicted by sequence complementarity and our results showed that many sRNAs identified in this work might be directed against various transcription factors. This work represents a significant step toward understanding the roles of sRNAs in the immune response of cassava.
Project description:In plants, small RNAs (sRNAs) usually refer to non-coding RNAs (ncRNAs) with lengths of 20-24 nucleotides. sRNAs are involved in the regulation of many essential processes related to plant development and environmental responses. sRNAs in plants are mainly grouped into microRNAs (miRNAs) and small interfering RNAs (siRNAs), and the latter can be further classified into trans-acting siRNAs (ta-siRNAs), repeat-associated siRNAs (ra-siRNAs), natural anti-sense siRNAs (nat-siRNAs), etc. Many sRNAs exhibit a clustered distribution pattern in the genome. Here, we summarize the features and functions of cluster-distributed sRNAs, aimed to not only provide a thorough picture of sRNA clusters (SRCs) in plants, but also shed light on the identification of new classes of functional sRNAs.
Project description:BACKGROUND:Cold stress causes dynamic changes in gene expression that are partially caused by small non-coding RNAs since they regulate protein coding transcripts and act in epigenetic gene silencing pathways. Thus, a detailed analysis of transcriptional changes of small RNAs (sRNAs) belonging to all known sRNA classes such as microRNAs (miRNA) and small interfering RNA (siRNAs) in response to cold contributes to an understanding of cold-related transcriptome changes. RESULT:We subjected A. thaliana plants to cold acclimation conditions (4?°C) and analyzed the sRNA transcriptomes after 3?h, 6?h and 2 d. We found 93 cold responsive differentially expressed miRNAs and only 14 of these were previously shown to be cold responsive. We performed miRNA target prediction for all differentially expressed miRNAs and a GO analysis revealed the overrepresentation of miRNA-targeted transcripts that code for proteins acting in transcriptional regulation. We also identified a large number of differentially expressed cis- and trans-nat-siRNAs, as well as sRNAs that are derived from long non-coding RNAs. By combining the results of sRNA and mRNA profiling with miRNA target predictions and publicly available information on transcription factors, we reconstructed a cold-specific, miRNA and transcription factor dependent gene regulatory network. We verified the validity of links in the network by testing its ability to predict target gene expression under cold acclimation. CONCLUSION:In A. thaliana, miRNAs and sRNAs derived from cis- and trans-NAT gene pairs and sRNAs derived from lncRNAs play an important role in regulating gene expression in cold acclimation conditions. This study provides a fundamental database to deepen our knowledge and understanding of regulatory networks in cold acclimation.
Project description:BACKGROUND: Natural antisense transcripts (NATs) are a class of RNAs that contain a sequence complementary to other transcripts. NATs occur widely in eukaryotes and play critical roles in post-transcriptional regulation. Soybean NAT sequences are predicted in the PlantNATsDB, but detailed analyses of these NATs remain to be performed. RESULTS: A total of 26,216 NATs, including 994 cis-NATs and 25,222 trans-NATs, were predicted in soybean. Each sense transcript had 1-177 antisense transcripts. We identified 21 trans-NATs using RT-PCR amplification. Additionally, we identified 179 cis-NATs and 6,629 trans-NATs that gave rise to small RNAs; these were enriched in the NAT overlapping region. The most abundant small RNAs were 21, 22, and 24 nt in length. The generation of small RNAs was biased to one stand of the NATs, and the degradation of NATs was biased. High-throughput sequencing of the degradome allowed for the global identification of NAT small interfering RNAs (nat-siRNAs) targets. 446 target genes for 165 of these nat-siRNAs were identified. The nat-siRNA target could be one transcript of a given NAT, or from other gene transcripts. We identified five NAT transcripts containing a hairpin structure that is characteristic of pre-miRNA. We identified a total of 86 microRNA (miRNA) targets that had antisense transcripts in soybean. CONCLUSIONS: We globally identified nat-siRNAs, and the targets of nat-siRNAs in soybean. It is likely that the cis-NATs, trans-NATs, nat-siRNAs, miRNAs, and miRNA targets form complex regulatory networks.
Project description:BACKGROUND: Brassica rapa includes several important leaf vegetable crops whose production is often damaged by high temperature. Cis-natural antisense transcripts (cis-NATs) and cis-NATs-derived small interfering RNAs (nat-siRNAs) play important roles in plant development and stress responses. However, genome-wide cis-NATs in B. rapa are not known. The NATs and nat-siRNAs that respond to heat stress have never been well studied in B. rapa. Here, we took advantage of RNA-seq and small RNA (sRNA) deep sequencing technology to identify cis-NATs and heat responsive nat-siRNAs in B. rapa. RESULTS: Analyses of four RNA sequencing datasets revealed 1031 cis-NATs B. rapa ssp. chinensis cv Wut and B. rapa ssp. pekinensis cv. Bre. Based on sequence homology between Arabidopsis thaliana and B. rapa, 303 conserved cis-NATs in B. rapa were found to correspond to 280 cis-NATs in Arabidopsis; the remaining 728 novel cis-NATs were identified as Brassica-specific ones. Using six sRNA libraries, 4846 nat-siRNAs derived from 150 cis-NATs were detected. Differential expression analysis revealed that nat-siRNAs derived from 12 cis-NATs were responsive to heat stress, and most of them showed strand bias. Real-time PCR indicated that most of the transcripts generating heat-responsive nat-siRNAs were upregulated under heat stress, while the transcripts from the opposite strands of the same loci were downregulated. CONCLUSIONS: Our results provide the first subsets of genome-wide cis-NATs and heat-responsive nat-siRNAs in B. rapa; these sRNAs are potentially useful for the genetic improvement of heat tolerance in B. rapa and other crops.
Project description:Natural antisense transcripts (NATs) are endogenous transcripts that can form double-stranded RNA structures. Many protein-coding genes (PCs) and non-protein-coding genes (NPCs) tend to form cis-NATs and trans-NATs, respectively. In this work, we identified 4,080 cis-NATs and 2,491 trans-NATs genome-widely in Arabidopsis. Of these, 5,385 NAT-siRNAs were detected from the small RNA sequencing data. NAT-siRNAs are typically 21nt, and are processed by Dicer-like 1 (DCL1)/DCL2 and RDR6 and function in epigenetically activated situations, or 24nt, suggesting these are processed by DCL3 and RDR2 and function in environment stress. NAT-siRNAs are significantly derived from PC/PC pairs of trans-NATs and NPC/NPC pairs of cis-NATs. Furthermore, NAT pair genes typically have similar pattern of epigenetic status. Cis-NATs tend to be marked by euchromatic modifications, whereas trans-NATs tend to be marked by heterochromatic modifications.
Project description:Small noncoding RNA (sncRNA), including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs) are key gene regulators in eukaryotes, playing critical roles in plant development and stress tolerance. Trans-acting siRNAs (ta-siRNAs), which are secondary siRNAs triggered by miRNAs, and siRNAs from natural antisense transcripts (nat-siRNAs) are two well-studied classes of endo-siRNAs.In order to understand sncRNAs' roles in plant chilling response and stress acclimation, we performed a comprehensive study of miRNAs and endo-siRNAs in Cassava (Manihot esculenta), a major source of food for the world populations in tropical regions. Combining Next-Generation sequencing and computational and experimental analyses, we profiled and characterized sncRNA species and mRNA genes from the plants that experienced severe and moderate chilling stresses, that underwent further severe chilling stress after chilling acclimation at moderate stress, and that grew under the normal condition. We also included castor bean (Ricinus communis) in our study to understand conservation of sncRNAs. In addition to known miRNAs, we identified 32 (22 and 10) novel miRNAs as well as 47 (26 and 21) putative secondary siRNA-yielding and 8 (7 and 1) nat-siRNA-yielding candidate loci in Cassava and castor bean, respectively. Among the expressed sncRNAs, 114 miRNAs, 12 ta-siRNAs and 2 nat-siRNAs showed significant expression changes under chilling stresses.Systematic and computational analysis of microRNAome and experimental validation collectively showed that miRNAs, ta-siRNAs, and possibly nat-siRNAs play important roles in chilling response and chilling acclimation in Cassava by regulating stress-related pathways, e.g. Auxin signal transduction. The conservation of these sncRNA might shed lights on the role of sncRNA-mediated pathways affected by chilling stress and stress acclimation in Euphorbiaceous plants.
Project description:Many eukaryotic genomes encode cis-natural antisense transcripts (cis-NATs). Sense and antisense transcripts may form double-stranded RNAs that are processed by the RNA interference machinery into small interfering RNAs (siRNAs). A few so-called nat-siRNAs have been reported in plants, mammals, Drosophila, and yeasts. However, many questions remain regarding the features and biogenesis of nat-siRNAs.Through deep sequencing, we identified more than 17,000 unique siRNAs corresponding to cis-NATs from biotic and abiotic stress-challenged Arabidopsis thaliana and 56,000 from abiotic stress-treated rice. These siRNAs were enriched in the overlapping regions of NATs and exhibited either site-specific or distributed patterns, often with strand bias. Out of 1,439 and 767 cis-NAT pairs identified in Arabidopsis and rice, respectively, 84 and 119 could generate at least 10 siRNAs per million reads from the overlapping regions. Among them, 16 cis-NAT pairs from Arabidopsis and 34 from rice gave rise to nat-siRNAs exclusively in the overlap regions. Genetic analysis showed that the overlapping double-stranded RNAs could be processed by Dicer-like 1 (DCL1) and/or DCL3. The DCL3-dependent nat-siRNAs were also dependent on RNA-dependent RNA polymerase 2 (RDR2) and plant-specific RNA polymerase IV (PolIV), whereas only a fraction of DCL1-dependent nat-siRNAs was RDR- and PolIV-dependent. Furthermore, the levels of some nat-siRNAs were regulated by specific biotic or abiotic stress conditions in Arabidopsis and rice.Our results suggest that nat-siRNAs display distinct distribution patterns and are generated by DCL1 and/or DCL3. Our analysis further supported the existence of nat-siRNAs in plants and advanced our understanding of their characteristics.
Project description:BACKGROUND: Small non-coding RNAs (sRNAs) play key roles in plant development, growth and responses to biotic and abiotic stresses. At least four classes of sRNAs have been well characterized in plants, including repeat-associated siRNAs (rasiRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs) and natural antisense transcript-derived siRNAs. Chinese fir (Cunninghamia lanceolata) is one of the most important coniferous evergreen tree species in China. No sRNA from Chinese fir has been described to date. RESULTS: To obtain sRNAs in Chinese fir, we sequenced a sRNA library generated from seeds, seedlings, leaves, stems and calli, using Illumina high throughput sequencing technology. A comprehensive set of sRNAs were acquired, including conserved and novel miRNAs, rasiRNAs and tasiRNAs. With BLASTN and MIREAP we identified a total of 115 conserved miRNAs comprising 40 miRNA families and one novel miRNA with precursor sequence. The expressions of 16 conserved and one novel miRNAs and one tasiRNA were detected by RT-PCR. Utilizing real time RT-PCR, we revealed that four conserved and one novel miRNAs displayed developmental stage-specific expression patterns in Chinese fir. In addition, 209 unigenes were predicted to be targets of 30 Chinese fir miRNA families, of which five target genes were experimentally verified by 5' RACE, including a squamosa promoter-binding protein gene, a pentatricopeptide (PPR) repeat-containing protein gene, a BolA-like family protein gene, AGO1 and a gene of unknown function. We also demonstrated that the DCL3-dependent rasiRNA biogenesis pathway, which had been considered absent in conifers, existed in Chinese fir. Furthermore, the miR390-TAS3-ARF regulatory pathway was elucidated. CONCLUSIONS: We unveiled a complex population of sRNAs in Chinese fir through high throughput sequencing. This provides an insight into the composition and function of sRNAs in Chinese fir and sheds new light on land plant sRNA evolution.