Arabidopsis thaliana
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA309713 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR3116240.fastq.gz | Fastqsanger.gz | |||
| SRR3116250.fastq.gz | Fastqsanger.gz | |||
| SRR3116260.fastq.gz | Fastqsanger.gz | |||
| SRR3116270.fastq.gz | Fastqsanger.gz | |||
| SRR3116280.fastq.gz | Fastqsanger.gz |
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Project description:GC-rich coding sequences reduce transposon-like sRNA-mediated transgene silencing in Arabidopsis and maize
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Project description:GC-rich coding sequences reduce transposon-like sRNA-mediated transgene silencing in Arabidopsis and maize
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Project description:The molecular basis of transgene susceptibility to silencing is poorly characterized in plants, thus we evaluated several transgene design parameters as means to reduce heritable sRNA-mediated transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposable element (TE)-like silencing of canola-biased PUFA transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three Bacillus thuringiensis (Bt) crystal proteins tested the impact of CDSs recoding using different codon bias tables. Transgenes with the higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.
2017-10-30 | GSE103037 | GEO
Project description:The molecular basis of transgene susceptibility to silencing is poorly characterized in plants, thus we evaluated several transgene design parameters as means to reduce heritable sRNA-mediated transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposable element (TE)-like silencing of canola-biased PUFA transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three Bacillus thuringiensis (Bt) crystal proteins tested the impact of CDSs recoding using different codon bias tables. Transgenes with the higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.
2017-10-30 | GSE76406 | GEO
Project description:The piRNA pathway controls transposon expression in animal germ cells, thereby ensuring genome stability over generations. piRNAs are maternally deposited and required for proper transposon silencing in adult offspring. However, a long-standing question in the field is the precise function of maternally deposited piRNAs and its associated factors during embryogenesis. Here, we probe the spatio-temporal expression patterns of several piRNA pathway components during early stages of development. Amongst those, factors required for transcriptional gene silencing (TGS) showed ubiquitous abundance in somatic and pole cells throughout the first half of embryogenesis. We further analysed the transcriptomes of various embryo stages and correlated these with the presence of selected chromatin marks. We found that a number of transposon families show bursts of transcription during early embryonic stages. Transposons heavily targeted by maternally deposited piRNAs accumulated repressive chromatin marks following their spike in expression. Furthermore, depletion of maternally deposited Piwi protein in early embryos resulted in increased expression of transposons targeted by inherited piRNAs and was accompanied by a strong loss of repressive chromatin marks at coding sequences. Overall, our data suggests a pivotal role for the piRNA pathway in transposon defence during Drosophila embryogenesis in somatic cells.
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Project description:We aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology was preferred over small-RNA sequencing (sRNA-seq) since the latter underrepresented miRNAs with a guanine-cytosine (GC) content of at least 75% (p = 0.0002, n = 2). miRNA abundances measured on the microarray were ranked for consistency and uniformity using nine normalization approaches. The eleven most stable sequences included miRNAs of moderate, but also extreme GC content (45%–65%: miR-320d, miR-425-5p, miR-185-5p, miR-486-5p; 80%–95%: miR-1915-3p, miR-3656-5p, miR-3665-5p, miR-3960-5p, miR-4488-5p, miR-4497 and miR-4787-5p). In contrast, the seven extremely GC-rich miRNAs were not found in the two plasma miRNomes screened by sRNA-seq. Stem-loop RT-qPCR was employed for stability verification in 32 plasma samples of healthy male Caucasians (age range: 18–55 years). In general, inter-individual variance of miRNA abundance was low or very low as indicated by coefficient of variation (CV) values of 0.6%–8.2%. miR-3665 and miR-1915-3p outperformed in this analysis (CVs: 0.6 and 2.4%, respectively). The eight most stable sequences included four extremely GC-rich miRNAs (miR-1915-3p, miR-3665, miR-4787-5p and miR-4497). The best-performing duo normalization factor (NF) for the condition of human plasma, miR-320d and miR-4787-5p, also included a GC-extreme miRNA. In summary, the identification of extremely guanine-cytosine-rich plasma normalizers will help to increase accuracy of PCR-based miRNA quantification, thus raise the potential that miRNAs become markers for psychological stress reactions or early and precise diagnosis of clinical phenotypes. The novel miRNAs might also be useful for orthologous contexts considering their conservation in related animal genomes.
2022-07-25 | GSE209568 | GEO
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2017-02-10 | GSE80466 | GEO
Project description:Next-generation Illumina sequencing technology was used to analyze small RNA associated with post-transcriptional gene silencing induced by intron-spliced hairpin RNA (ihpRNA) in Arabidopsis. The experimental induction of RNA silencing in plants often involves expression of transgenes encoding inverted repeat (IR) sequences to produce abundant dsRNAs that are processed into small RNAs (sRNAs). These sRNAs are key mediators of post-transcriptional gene silencing (PTGS) and determine the specificity of the inhibition of gene expression. Despite its broad utility as a research tool, IR-PTGS is only a partially understood mechanism of RNA silencing in plants. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE SYNTHASE (CHS) gene fragments. The levels of PTGS were dependent on the orientation and position of the fragment in the IR construct. To investigate these differences, we characterized the sRNA profiles by Illumina sequencing of seven libraries generated from transgenic families showing different levels of IR-PTGS. Mapping of sRNA sequences to their corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation. Analyses of these patterns and peaks revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. Our observations provide new guidelines for designing constructs to increase the efficiency of IR-PTGS. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes.
2012-06-26 | GSE38922 | GEO
Project description:Next-generation Illumina sequencing technology was used to analyze small RNA associated with post-transcriptional gene silencing induced by intron-spliced hairpin RNA (ihpRNA) in Arabidopsis. The experimental induction of RNA silencing in plants often involves expression of transgenes encoding inverted repeat (IR) sequences to produce abundant dsRNAs that are processed into small RNAs (sRNAs). These sRNAs are key mediators of post-transcriptional gene silencing (PTGS) and determine the specificity of the inhibition of gene expression. Despite its broad utility as a research tool, IR-PTGS is only a partially understood mechanism of RNA silencing in plants. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE SYNTHASE (CHS) gene fragments. The levels of PTGS were dependent on the orientation and position of the fragment in the IR construct. To investigate these differences, we characterized the sRNA profiles by Illumina sequencing of seven libraries generated from transgenic families showing different levels of IR-PTGS. Mapping of sRNA sequences to their corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation. Analyses of these patterns and peaks revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. Our observations provide new guidelines for designing constructs to increase the efficiency of IR-PTGS. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes. Sequencing of small RNA from Arabidopsis plants transformed with ihpRNA constructs. Seven small RNA libraries were sequenced: Lib 0, made from non-transgenic Arabidopsis plants, and Libs 1-6, made from plants transformed with different configurations of ihpRNA.
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