Mismatch hpRNA prevents intrinsic self-silencing and enhances RNAi stability
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA739666 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14870900.fastq.gz | Fastqsanger.gz | |||
| SRR14870901.fastq.gz | Fastqsanger.gz | |||
| SRR14870902.fastq.gz | Fastqsanger.gz | |||
| SRR14870903.fastq.gz | Fastqsanger.gz | |||
| SRR14870904.fastq.gz | Fastqsanger.gz |
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Project description:Hairpin RNA (hpRNA) transgenes are the most successful RNA interference (RNAi) method in plants. Here, we show that hpRNA transgenes are invariably methylated in the inverted-repeat (IR) DNA and the adjacent promoter, causing transcriptional self-silencing. Nucleotide substitutions in the sense sequence, disrupting the IR structure, prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi. Substituting all cytosine with thymine nucleotides, in a G:U hpRNA design, prevents self-silencing but still allows for the formation of 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. While a traditional hpRNA transgene shows increasing self-silencing from cotyledons to true leaves, its G:U counterpart avoids this and induce RNAi throughout plant growth. Furthermore, siRNAs from G:U and traditional hpRNA show different characteristics and appear to function via different pathways to induce target DNA methylation.
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