High-throughput pyrosequencing of endogenous small RNAs in O. Europaea
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ABSTRACT: Small RNAs of 20 to 25 nucleotides in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the small RNA component of more than 40 plant species. Here, use deep sequencing and molecular methods to report the first inventory of small RNAs in olive (Olea europaea). Small RNAs of 24 nts dominate the small RNA transcriptome and atypically accumulate to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. By contrast, small RNAs of 20 to 22 nts were poorly represented in the population at levels lower than those found in most plant species tested. A total of 14 known miRNA families were identified in two libraries prepared from growing and dormant lateral buds. We found that some known miRNAs showed tissue- and/or developmental-specific expression. Also, seven novel, olive-specific miRNA candidates were found in our sequenced set of which 1 were supported by their star strands. Potential precursors for these miRNA candidates with intramolecular folding capacities were found in the olive EST database. Target mRNAs of conserved miRNAs and new olive-specific miRNA were computationally predicted among the olive EST collection and experimentally validated through endonucleolytic cleavage assays. Two samples analyzed: growing (juvenile) and dormant (adult) lateral buds from the progeny of a genetic cross between the Picual and Arbequina olive varieties
Project description:Melon (Cucumis melo L.) is a commercially important fruit crop that is cultivated worldwide. The melon research community has recently benefited from the determination of a complete draft genome sequence and the development of associated genomic tools, which have allowed us to focus on small RNAs (sRNAs). These are short, non-coding RNAs 21â24 nucleotides in length with diverse physiological roles. In plants, they regulate gene expression and heterochromatin assembly, and control protection against virus infection. Much remains to be learned about the role of sRNAs in melon. We constructed 10 sRNA libraries from two stages of developing ovaries, fruits and photosynthetic cotyledons infected with viruses, and carried out high-throughput pyrosequencing. We catalogued and analyzed the melon sRNAs, resulting in the identification of 26 known miRNA families (many conserved with other species), the prediction of 84 melon-specific miRNA candidates, the identification of trans-acting siRNAs, and the identification of chloroplast, mitochondrion and transposon-derived sRNAs. In silico analysis revealed more than 400 potential targets for the conserved and novel miRNAs. This analysis provides insight into the composition and function of the melon small RNAome, and paves the way towards an understanding of sRNA-mediated processes that regulate melon fruit development and melonâvirus interactions. 11 small RNA libraries from several tissues of melon are included en the raw data. 2 samples from ovary, 2 samples from fruit, 1 sample from healthy cotyledons (Cultivar Tendral), 1 samples from healthy cotyledons (genotype TGR-1551), 1 sample from cotyledons (cultivar Tendral) infected with Watermelon mosaic virus (WMV), 1 sample from cotyledons (cultivar TGR-1551) infected with WMV, 1 sample from cotyledons (cultivar Tendral) infected with Melon necrotic spot virus (MNSV, Malfa5 isolate), 1 sample from cotyledons (cultivar Tendral) infected with MNSV (chimeric virus with Malfa5-264 isolates), 1 library from synthetic RNA oligos. Raw reads were obtained from two independent 454 runs, ~22,000 reads each one, to a total of 447,180 reads
Project description:Plant virus infection involves the production of viral small RNAs (vsRNAs) with the potential to associate with distinct Argonaute (AGO)-containing silencing complexes and mediate diverse silencing effects on RNA and chromatin. We used multiplexed, high-throughput pyrosequencing to profile populations of vsRNAs from plants infected with viruses from different genera. Sense and antisense vsRNAs of 20 to 24 nucleotides (nts) spread throughout the entire viral genomes in an overlapping configuration; virtually all genomic nucleotide positions were represented in the dataset. We present evidence to suggest that every genomic position could be a putative cleavage site for vsRNA formation, although viral genomes contain specific regions that serve as preferential sources of vsRNA production. Hotspots for vsRNAs of 21-, 22-, and 24-nt usually coincide in the same genomic regions, indicating similar target affinities among Dicer-like (DCL) enzymes. In the light of our results, the overall contribution of perfectly base paired double-stranded RNA and imperfectly base paired structures within single-stranded RNA to vsRNA formation is discussed. Our census of vsRNAs extends the current view of the distribution and composition of vsRNAs in virus-infected plants, and contributes to define a more comprehensive scenario of vsRNA biogenesis and their regulatory functions in plants Raw data files are available on our FTP site: ftp://ftp.ncbi.nlm.nih.gov/pub/geosup/Series/GSE16996 10 samples examined: Arabidopsis plants infected with TRV, TuMV or CMV; Nicothiana benthamiana plants infected with CymRSV, PVX or PMMoV; Cucumis melo plants infected with MNSV, quimeric MNSV or WMV and Solanum lycopersicum plants infected with TYLCV.
Project description: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. 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. 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. The aim of this study is to investigate the small RNA transcriptome in Cunninghamia lanceolata. Total RNAs of seeds and calli were extracted using RNAiso-mate for plant tissue and RNAiso plus (Takara, Dalian, Liaoning, China), whereas total RNAs of seedlings, adult leaves and stems were isolated with the Concert Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA), and were then treated with RNase-free DNase I (Promega, Madison, WI, USA). Equal amount of total RNAs from the 5 different samples were mixed to form a single RNA pool. Twenty micrograms of total RNAs from the pool were used and 16 to 30-nt sRNAs were purified using Novex 15% TBE-¬Urea gel (Invitrogen). Two adaptors were sequentially ligated to the 5' and 3' ends of purified sRNAs. The ligation products were further purified from Novex 10% TBE-Urea gel. Reverse transcriptase SuperScript II (Invitrogen) and high-fidelity DNA polymerase Phusion (New England Biolabs, Ipswich, MA, USA) were used in the following RT-PCR reaction. The amplification products were cut from Novex 6% TBE-Urea gel. The purified DNA fragments were used for sequencing on an Illumina 1G Genome Analyzer at the Beijing Genomics Institute, Shenzhen, China.
Project description:MicroRNAs (miRNAs) play a important part in post-transcriptional gene regulation and have been shown to control many genes involved in various biological and metabolic processes. There have been extensive studies to discover miRNAs and analyze their functions in model plant species, such as Arabidopsis and rice and other plants. However, the number of miRNAs discovered in grape is relatively low and little is known about miRNAs responded gibberellin during fruit germination. In this study, a small RNA library from gibberellin grape fruits was sequenced by the high throughput sequencing technology. A total of 16,033,273 reads were obtained. 812,099 total reads representing 1726 unique sRNAs matched to known grape miRNAs. Further analysis confirmed a total of 149 conserved grapevine miRNA (Vv-miRNA) belonging to 27 Vv-miRNA families were validated, and 74 novel potential grapevine-specific miRNAs and 23 corresponding novel miRNAs* were discovered. Twenty-seven (36.5%) of the novel miRNAs exhibited differential QRT-PCR expression profiles in different development gibberellin-treated grapevine berries that could further confirm their existence in grapevine. QRT-PCR analysis on transcript abundance of 27 conserved miRNA family and the new candidate miRNAs revealed that most of them were differentially regulated by the gibberellin, with most conserved miRNA family and 26 miRNAs being specifically induced by gibberellin exposure. All novel sequences had not been earlier described in other plant species. In addition, 117 target genes for 29 novel miRNAs were successfully predicted. Our results indicated that miRNA-mediated gene expression regulation is present in gibberellin-treated grape berries. This study led to the confirmation of 101 known miRNAs and the discovery of 74 novel miRNAs in grapevine. Identification of miRNAs resulted in significant enrichment of the gibberellin of grapevine miRNAs and provided insights into miRNA regulation of genes expressed in grape berries. GSM604831 is the control for the gibberellin-treated sample. The mixture samples of young berries (one week after flowering) large berries (five week after flowering after flowering), and old berries (nine week after flowering) treated with gibberellin, respectively, were generated by deep sequencing, in triplicate, using Illumina 1G Genome Analyzer.
Project description:Small RNAs of 20 to 25 nucleotides in length maintain genome integrity and control gene expression in a multitude of developmental and physiological processes. Despite RNA silencing has been primarily studied in model plants, the advent of high-throughput sequencing technologies has enabled profiling of the small RNA component of more than 40 plant species. Here, use deep sequencing and molecular methods to report the first inventory of small RNAs in olive (Olea europaea). Small RNAs of 24 nts dominate the small RNA transcriptome and atypically accumulate to levels never seen in other plant species, suggesting an active role of heterochromatin silencing in the maintenance and integrity of its large genome. By contrast, small RNAs of 20 to 22 nts were poorly represented in the population at levels lower than those found in most plant species tested. A total of 14 known miRNA families were identified in two libraries prepared from growing and dormant lateral buds. We found that some known miRNAs showed tissue- and/or developmental-specific expression. Also, seven novel, olive-specific miRNA candidates were found in our sequenced set of which 1 were supported by their star strands. Potential precursors for these miRNA candidates with intramolecular folding capacities were found in the olive EST database. Target mRNAs of conserved miRNAs and new olive-specific miRNA were computationally predicted among the olive EST collection and experimentally validated through endonucleolytic cleavage assays.
Project description:We identified twin small non-coding RNAs regulating tricarboxylic acid (TCA) cycle activity in Neisseria meningitidis. Expression of TCA cycle enzymes was elevated in sRNA deletion mutants. Direct interaction between sRNAs and the ribosomal entry sites on target mRNAs was demonstrated. Expression of the sRNAs was down-regulated in cells grown in poor medium with glucose as the sole carbon source but not without lrp, indicating that sRNA expression is controlled by the stringent response. N. meningitidis, over-expressing the sRNAs replicated in blood, but not in human cerebrospinal fluid. In addition, Lrp synthesis was inhibited by direct interaction between the sRNA and the 5’ UTR of lrp. sRNAs control adaptation of N. meningitidis to variation in nutrient supply in different niches of its host.
Project description:We used deep sequencing to characterize 3 families of sRNAs (piRNAs, miRNAs, and tRFs) present in Sus scrofa gonads and focused on the sRNA fraction present in both male and female gonads. Of the sequences detected in the testes, 2.6% of piRNAs, 9% of miRNAs, and 10% of tRFs were also present in the ovaries. Notably, the majority of the shared piRNAs mapped to the introns of ribosomal RNAs and were derived from clustered loci. In addition, the most abundant miRNAs present in the ovaries and testes are conserved and are involved in many biological processes such as the regulation of homeobox genes, the control of cell proliferation, and carcinogenesis. Unexpectedly, we detected a novel sRNA type, the tRFs, which are 30–36-nt RNA fragments derived from tRNA molecules, in gonads Determination miRNA, piRNA and tRF expression in swine gonads
Project description:This project aims to identify novel protein partners of conserved trans-sRNAs in the α-proteobacterium Sinorhizobium meliloti. Trans-sRNAs (AbcR2, NfeR1 and EcpR1) were tagged at their 5’ ends with the MS2 aptamer. To identify the proteins associated with the trans-sRNAs we: i) expressed and purified the MS2 coat protein fused to maltose-binding protein (MS2-MBP); ii) purified MS2 tagged sRNAs conjugated with MS2-MBP via amylase column; and iii) subjected retained proteins to mass spectrometry analysis. To discard unspecific interactions we also analysed several control samples: i) samples expressing untagged trans-sRNAs; ii) samples expressing an MS2 tagged Rho-independent transcriptional terminator.
Project description:Small RNAs (sRNAs) are hypothesized to contribute to hybrid vigor because they maintain genome integrity, contribute to genetic diversity, and control gene expression. We used Illumina sequencing to assess how sRNA populations vary between two maize inbred lines (B73, Mo17) and their hybrid. We sampled sRNAs from the seedling shoot apex and the developing ear, two rapidly growing tissues that program the greater growth of maize hybrids. We found that parental differences in siRNAs primarily originate from repeat regions. Although the maize genome contains greater number and complexity of repeats compared to Arabidopsis or rice, we confirmed that like these simpler plant genomes, 24-nt siRNAs whose abundance differs between maize parents also show a trend of downregulation following hybridization. Surprisingly, hybrid vigor is fully maintained when 24-nt siRNAs are globally reduced by mutation of the RNA-dependent RNA polymerase2 (RDR2) encoded by modifier of paramutation1 (mop1). We also discovered that 21-22nt siRNAs derived from a number of distinct retrotransposon families differentially accumulate between B73 and Mo17 as well as their hybrid. Thus, maize possesses a novel source of genetic variation for regulating both transposons and genes at a genomic scale, which may contribute to its high degree of observed heterosis. sRNA libraries were derived from RNA isolated from the seedling shoot apex and developing ear tissues from B73, Mo17, B73xMo17 and Mo17xB73. The shoot apex was chosen because it is enriched for meristematic tissue where cell proliferation occurs, rates of organ initiation are determined, and organ size is specified. The developing ear was examined because it is enriched in meristematic tissue and is undergoing rapid growth, and also because the mature ear shows the highest degree of heterosis. Total RNA was isolated and separated on a 15% TBE-Urea polyacrylamide gel. Using a 10-bp ladder, the sRNA fraction representing 10-40-bp was excised. sRNA libraries were prepared according to Lu et al. (2007) or manufacturer's instructitions (Illumina). A combination of Perl scripts and FASTX toolkit scripts were used to remove adapters, collapse identical sequences and count reads per sequence. Supplementary processed data text files contain the distinct sRNA sequences for all of the genotypes analyzed in that experiment. Abundance (reads per million) was calculated for each distinct sequence by dividing the number of reads of distinct sRNA in a library by the total number of sRNA reads for that library and multiplying this by 1 million. Genome builds: B73 genome, maizesequence.org release 4a.53 (October, 2009); Mo17 whole genome shotgun clones.
Project description:High-throughput sequence analysis of small RNAs of TYLCV-tolerant (containing tolerance gene Ty-1) and susceptible varieties indicated that miRNA-like vsRNAs and secondary vsiRNAs are mainly contributed to hotpots. Interestingly, various characteristics of vsRNAs among each sample are consistent, but in different number of expression; Deep sequencing of degradome provided evidence for the function of vsRNAs-mediated viral transcripts slicing; And bisulfite sequencing PCR suggested the geminivirus DNA methylation induced by vsRNAs. Comparison of the expression quantity and trend of viral DNA, mRNA and vsRNA inferred that the quantity of vsRNAs is significantly corresponding to the expression level of viral mRNA. Nevertheless, vsRNAs had finite effect on inhibition of virus replication and expression. Here, we also speculated that by RDR catalysis of vsRNAs amplification, the tolerance gene Ty-1 may take an effective inhibition of viral transcription at the beginning of TYLCV infection. Examination of 6 different sRNA and 4 degradome library in 2 tomato material. The main result of our paper is distinguish the host plant sRNA and viral-derived sRNA from the .fa files (MMS_21dpi, 30dpi and TY1S_21dpi, 30dpi).