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Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence [RNA-Seq]

ABSTRACT: Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence [RNA-Seq]

PROVIDER: PRJNA1063169 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR27476740.fastq.gz	Fastqsanger.gz
	SRR27476741.fastq.gz	Fastqsanger.gz
	SRR27476742.fastq.gz	Fastqsanger.gz
	SRR27476743.fastq.gz	Fastqsanger.gz
	SRR27476744.fastq.gz	Fastqsanger.gz

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Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence II

Project description:Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript amplifying the silencing signal in plants. 22nt siRNAs are under-represented due to competition with DCL4, while only a small number of 22nt miRNAs exist due to the rare occurrence of an asymmetric bulge in the precursor miRNA stem. Here we report a strategy to produce abundant 22nt siRNAs and other desired siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges periodically into the antisense strand of hpRNA, we successfully shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. We showed that the asymmetric hpRNA constructs effectively silenced a β-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes targeting both GUS and EIN2 showed increased amount of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. Consistent with the production of secondary siRNAs, the 22nt asymmetric hpRNA constructs increased gene silencing phenotypes and enhanced virus resistance in plants compared to the traditional hpRNA constructs.

2024-06-27 | GSE243296 | GEO

Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence I

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Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence [RNA-Seq]

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Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence

Project description:Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence

| PRJNA1017608 | ENA

Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence I

Project description:Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence I

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Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence II

Project description:Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence II

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Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence

Project description:This SuperSeries is composed of the SubSeries listed below.

2024-06-27 | GSE243297 | GEO

RNAi-mediated rheostat for dynamic control of AAV-delivered transgenes

Project description:Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein AAV transgene expression is silenced using the clinically validated modality of chemically modified short interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.

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The bulges control in pri-miRNA processing in the position and strand-dependent manner

Project description:The microRNA (miRNA) biogenesis is responsible for the production of miRNAs that play critical roles in gene expression and numerous human diseases. The adequate biogenesis of miRNAs is largely determined by the efficiency and fidelity of primary microRNA (pri-miRNA) processing by Microprocessor. Here, we investigated the roles of a secondary RNA element, an RNA bulge, in pri-miRNA processing. We discovered that the 3p-strand bulges in positions 7-9 from the Microprocessor cleavage sites (midB_7-9) contributes to determining the cleavage sites of Microprocessor, the 5p- and 3p-strand bugles in positions 10-12 (midB_10-12) blocked the unproductive cleavage, and the 3p-strand bulges in positions 6-7 (seedB) inhibited the productive cleavage of Microprocessor. The 5p-strand midB_10-12 was found enriched and conserved in many pri-miRNAs of humans and other organisms. In addition, by analyzing the published Microprocessor-RNA structure and doing mutagenesis, we identified several amino acid residues of Microprocessor that explains a structure basis for the processing inhibition caused by seedB. The revealed functions of bulges in our study improves our understanding of the pri-miRNA processing by Microprocessor and implies their roles in regulating miRNA expression.

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RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway.

Project description:RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.

| S-EPMC1679793 | biostudies-literature

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