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Series GSE178565 Query DataSets for GSE178565
Status Public on Mar 31, 2022
Title Mismatch hpRNA prevents intrinsic self-silencing and enhances RNAi stability
Organism Arabidopsis thaliana
Experiment type Non-coding RNA profiling by high throughput sequencing
Summary Hairpin RNA (hpRNA) transgenes, with a perfect inverted-repeat (IR) DNA, have been the most successful RNA interference (RNAi) method in plants. Here we show that hpRNA transgenes were invariably methylated in the IR DNA and the adjacent promoter, causing transcriptional self-silencing and preventing the full potential of RNAi. Nucleotide substitutions in the sense sequence, which disrupts the perfect IR DNA structure, were sufficient to prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi. Substituting all cytosine (C) with thymine (T) nucleotides, in a G:U hpRNA design, prevented DNA methylation and self-silencing but still allowed for the formation of perfect hpRNA due to G:U wobble base-pairing. The G:U design induces effective RNAi in 90-96% of transgenic lines, compared to 57-65% for the traditional hpRNA design. Furthermore, while a traditional hpRNA transgene showed increasing DNA methylation and self-silencing from cotyledons to true leaves, the G:U transgenes avoided this developmental progression of self-silencing and induced RNAi throughout plant growth. The G:U and traditional hpRNA transgenes generated small interfering RNA (siRNA) with different 5’ phosphorylation, which resembled the endogenous tasiRNA and miRNA, respectively. Furthermore, our results suggest that siRNAs from the two transgene designs function differently to induce target DNA methylation, one (from traditional hpRNA) through the canonical RdDM pathway and the other (G:U hpRNA) a non-canonical pathway. Our study not only revealed a methylation-resistant RNAi transgene design but also provided new mechanistic insights into small RNA biogenesis and function in plants
Overall design sRNA sequencing to compare different hpRNA structures for small RNA processing.
Contributor(s) Wang M, Zhang D, Zhong C, Smith N, Defeyter R, Greaves I, Swain S, Zhang R
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Submission date Jun 21, 2021
Last update date Mar 31, 2022
Contact name Ian K Greaves
Phone 61 6246 4828
Organization name csiro
Street address clunies ross street
City Acton
State/province act
ZIP/Postal code 2601
Country Australia
Platforms (2)
GPL19580 Illumina NextSeq 500 (Arabidopsis thaliana)
GPL21785 Illumina HiSeq 4000 (Arabidopsis thaliana)
Samples (20)
GSM5393881 hpEIN2[WT]-15
GSM5393882 hpEIN2[WT]-17
GSM5393883 hpEIN2[G:U]-12
BioProject PRJNA739666
SRA SRP324842

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Supplementary file Size Download File type/resource
GSE178565_size_distribution_of_sense_and_antisense_siRNAs_derived_from_the_dsRNA_stem.xlsx 18.8 Kb (ftp)(http) XLSX
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Raw data are available in SRA
Processed data are available on Series record

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