Distribution of Small RNAs Along Transposable Elements in Vitis vinifera During Somatic Embryogenesis
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ABSTRACT: In the present study we have generated a parallel analysis of the 5' RNA ends (PARE) referred to polyadenylated RNAs in somatic embryos from in vitro culture of immature anthers of Vitis vinifera. PARE analysis include also a shortRNA analysis of same material. We have submitted the sRNAseq to mirPROOF and miRCAT (srna_workbench software at http://srna-workbench.cmp.uea.ac.uk) in order to recognize known and putative novel miRNAs, respectively that could be present in somatic embryos. In addition, aRNAs and PARE libraries were integrated in order to identify those 5' RNA ends that could be explained by sRNAs (i.e. identify transcripts that could be cleaved by sRNAs). We have used publicly available annotations and we gained a deep insight into the gene families and the transcriptional regulation mediated by miRNAs in V. vinifera somatic embryos. Gene expression is finely regulated to specific paralogues in gene families such as the NADPH-dependent diflavin oxidoreductase, Phosphoserine aminotransferase, Ethylene-responsive transcription factors, glutathione S-transferase par C, just to name a few. We have indeed characterized at least 4,000 mRNA targets. Size fractionated small RNA from total RNA extracts of somatic Embryos cv. "Brachetto" were ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Illumina high throughput pyrosequencing. The kit used is TrueSeq Small RNA kit, Illumina. Please see www.illumina.com for details of the sequencing technology. PARE libraries were generated starting from Poly(A) fraction. The protocol used have been previously described in German et al., 2009, Nature protocols. Please see www.illumina.com for details of the sequencing technology.
Project description:In the present study we diagnosed cleaved transcripts in either healthy or virus infected plants, by sRNAome and metagenome of degraded mRNAs. We integrated the experimentally obtained data with publicly available annotations and we gained a deep insight into the parallel evolution of gene families and the transcriptional regulation mediated by miRNAs in Tomato. Gene expression is finely regulated to specific paralogues in gene families such as the squamosa-promoter binding protein-like, the Auxin Response Factor, the Class III homeodomain-leucine zipper, the Argonaute gene families, just to name a few. In some cases, different miRNA are expressed to target different copies of the same family. Size fractionated small RNA from total RNA extracts of Solanum lycopesicum cv. "Money Maker" were ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Illumina high throughput pyrosequencing. The kit used is TrueSeq Small RNA kit, Illumina. Please see www.illumina.com for details of the sequencing technology. PARE libraries were generated starting from Poly(A) fraction used for sRNA isolation. The protocol used have been previously described in German et al., 2009, Nature protocols. Please see www.illumina.com for details of the sequencing technology.
Project description:Size fractionated small RNA from total RNA extracts of Vitis vinifera leaves were ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Illumina high throughput pyrosequencing. The kit used is TrueSeq Small RNA kit Please see www.illumina.com for details of the sequencing technology.
Project description:Vitis vinifera endogenous small RNAs Size fractionated small RNA from total RNA extracts of Vitis vinifera leaves, inflorescences, tendrils and small berries were ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Solexa/Illumina high throughput pyrosequencing. Please see www.illumina.com for details of the sequencing technology.
Project description:Vitis vinifera RNA degradome Isolated polyadenylated RNA from total RNA extracts of Vitis vinifera leaves, were ligated to 5'-adapter that include san MmeI recognition site. The ligated products were purified again, reverse transcribed and cleaved with MmeI. The 5' fragments were purified from gel and to a 3'- dsDNA adapter and PCR amplified. After PCR amplification the sample was subjected to Solexa/Illumina high throughput pyrosequencing. Please see www.illumina.com for details of the sequencing technology.
Project description:To investigate the roles of sRNAs in keeping embryo dormancy or germination in Larix leptolepis, we deciphered the endogenous "sRNAome" in dormant and germinated embryos. High-throughput sequencing of the sRNA libraries showed that dormant embryos exhibited a length bias towards 24-nt, while germinated embryos showed a bias towards a 21-nt and/or 22-nt length. Both of proportions for miRNAs to the non-redundant and redundant sRNAs were higher in germinated embryos than those in dormant embryos, while the ratio of unknown sRNAs was higher in dormant embryos than in germinated embryos. The proportion of 21-nt and 22-nt sRNAs increased in germinated embryos, which might attribute to the higher expression level of miRNAs. We identified a total of 160 conserved miRNAs from 39 families, 16 novel miRNAs, and 14 plausible miRNA candidates, of which novel and non-conserved known miRNAs might be the main contributors. These findings indicate that larch and possibly other gymnosperms have complex mechanisms of gene regulation involving sRNAs and miRNAs operating transcriptionally and post-transcriptionally during embryo dormancy and germination. One embryogenic cell line of Japanese larch (Larix leptolepis), designated as D878, with a high embryo maturation capacity was used in this study. Embryogenic callus were induced from immature embryos of larch on induction medium, followed by sub-culture, and culture on ABA-containing mature medium in a dark environment at 25 2 C. After cultured 45 days in mature medium, embryogenic calli developed into mature somatic embryos. In our study, the samples were harvested at day 57, one sample was collected after mature embryos continued to stay for 12 days on ABA-containing medium, and the other one was harvested after cultured for 12 days on ABA-removing medium. All samples were snap-frozen in liquid nitrogen, and stored in liquid nitrogen until RNA extraction.
Project description:Purpose: identify sites in endogenous mRNAs that are cut by KSHV SOX; Method: parallel analysis of RNA ends (PARE, following Zhai et al., 2014); Results: SOX cuts at discrete locations in mRNAs human Xrn1 was knocked down in HEK293T cells by shRNAs or siRNAs to stabilize degradation fragments with free 5' ends; GFP-SOX or GFP were transfected for ~24 hrs; total RNA samples were collected and subjected to PARE protocol (Zhai et al., 2014)
Project description:The narrow-specificity endoribonuclease RNase III and the 5’ exonuclease RNase J1 have been recently found to be not essential in the Gram-positive model organism, Bacillus subtilis. In this study, we performed a global analysis of internal 5’ ends that are generated or acted upon by these enzymes. An RNA-Seq protocol known as PARE (Parallel Analysis of RNA Ends) was used to capture 5’ monophosphorylated RNA ends in ribonuclease wild-type and mutant strains. Comparison of PARE peaks in strains with RNase III present or absent showed that, in addition to its well-known role in ribosomal (rRNA) processing, many coding sequences and intergenic regions were direct targets of RNase III. A set of regular RNA-seq experiments were performed to investigate RNA profiles in these strains and used to account for the changes in RNA abundance indirectly caused by the loss of RNase III in PARE. The PARE analysis also revealed an accumulation of 3’-proximal peaks that correlated with the absence of RNase J1, confirming the importance of RNase J1 in degrading RNA fragments that contain the transcription terminator structure. In addition, an endonuclease cleavage just two nucleotides downstream of the 16S rRNA 3’ end was discovered with PARE analysis. This latter observation begins to answer, at least for B. subtilis, a long-standing question on the exonucleolytic vs. endonucleolytic nature of 16S rRNA maturation
Project description:Parallel Analysis of RNA Ends (PARE) sequencing reads were generated to validate putative microRNAs and identify cleavage sites in Sorghum bicolor and Setaria viridis.
Project description:Small non-coding RNAs (sncRNAs) are emerging as key regulators of embryogenesis. To investigate the roles of sRNAs in regulating synchronism of somatic embryogenesis in Larix leptolepis, we deciphered the endogenous "sRNAome" in synchronous and desynchronous embryos. The 24-nt class sRNAs were overrepresented in both synchronous embryos and desynchronous embryos, accounting for 85.29% and 44.79%. A total of 29 miRNAs were upregulated in synchronous embryos, whereas 59 miRNAs were upregulated in desynchronous embryos. We describe the emerging theme for sncRNAs function: inhibiting the precocious expression, thus regulating the synchronism of somatic embryogenesis. These findings indicate that larch and possibly other gymnosperms have complex mechanisms of gene regulation involving sRNAs and miRNAs operating transcriptionally and post-transcriptionally during the regulation of synchronism.
Project description:To investigate the roles of sRNAs in keeping embryo dormancy or germination in Larix leptolepis, we deciphered the endogenous "sRNAome" in dormant and germinated embryos. High-throughput sequencing of the sRNA libraries showed that dormant embryos exhibited a length bias towards 24-nt, while germinated embryos showed a bias towards a 21-nt and/or 22-nt length. Both of proportions for miRNAs to the non-redundant and redundant sRNAs were higher in germinated embryos than those in dormant embryos, while the ratio of unknown sRNAs was higher in dormant embryos than in germinated embryos. The proportion of 21-nt and 22-nt sRNAs increased in germinated embryos, which might attribute to the higher expression level of miRNAs. We identified a total of 160 conserved miRNAs from 39 families, 16 novel miRNAs, and 14 plausible miRNA candidates, of which novel and non-conserved known miRNAs might be the main contributors. These findings indicate that larch and possibly other gymnosperms have complex mechanisms of gene regulation involving sRNAs and miRNAs operating transcriptionally and post-transcriptionally during embryo dormancy and germination.