Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana Nicotiana tabacum
PROVIDER: GSE243297 | GEO | 2024/06/27
REPOSITORIES: GEO
Similar Datasets
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
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 | GSE243255 | GEO
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 | GSE252890 | GEO
Project description:Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence
| PRJNA1017608 | ENA
Project description:Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence I
| PRJNA1017482 | ENA
Project description:Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence II
| PRJNA1017611 | ENA
Project description:Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence [RNA-Seq]
| PRJNA1063169 | ENA
Project description:Induction of cytokines by small interfering RNA (siRNA) polyplexes has been a significant concern of researchers attempting to minimize the toxicity of this promising therapy. Although cationic carriers of siRNA are known to increase cytokine levels, few systematic studies have been done to determine what properties of the carrier are important to modulate cytokines. Because branched histidine-lysine (HK) peptides are effective carriers of siRNA and their sequence can be readily modified, we selected this class of carrier to determine which sequences of the peptide were important for cytokine induction. With the use of peripheral blood mononuclear cells (PBMCs), the HK peptide with a higher number of histidines (H3K(+H)4b) in complex with siRNA induced lower levels of cytokines compared with other HK (e.g., H2K4b, H3K4b, H3K(+N)4b) siRNA nanoplexes. Notably, these peptides' siRNA polyplexes showed a similar pattern of cytokine induction when injected intravenously in a mouse model, i.e., the HK with higher content of histidines induced cytokines the least. As indicated by the pH-sensitive dye within acidic endosomes, the greater pH-buffering capacity of H3K(+H)4b compared with other HK peptides may explain why cytokine levels were reduced. In addition to buffering capacity, the size of HK polyplexes markedly influenced cytokine production.
| S-EPMC3519993 | biostudies-literature
Project description:Plant microRNAs (miRNAs) are predominantly 21 nucleotides (nt) long but non-canonical lengths of 22 and 20 nt are commonly observed in diverse plant species. While miRNAs longer than 21 nt can be attributed to the neglect of unpaired bases within asymmetric bulges by the ruler function of dicer-like 1 (DCL1), how 20-nt miRNA is generated remains obscure. Analysis of small RNA data revealed that 20-nt miRNA can be divided into 3 main groups featured by atypical 3' overhangs or shorter duplex regions. Asymmetric bulges or mismatches at specific positions are commonly observed within each group and were shown to be crucial for 20-nt miRNA formation. Analysis of DCL1 cleavage sites on 20-nt miRNA precursors suggests that these determinants might alter precursor structure or trigger 3'-end decay of mature miRNA. The results herein advance our understanding of miRNA biogenesis and demonstrate that the effect of asymmetric bulges on miRNA length could be position-dependent.
| S-EPMC4615586 | biostudies-literature
Project description:Various sequences in the mammalian genomes are unstable. One class of sequence arrangement is long inverted repeats, which are known to be unstable in bacteria and yeast. While in mammals some evidence suggests that short inverted repeats (<10 bp long) may show instability, nothing is known about the stability of long inverted repeats. Here we describe two unrelated multicopy transgenes in the mouse (loci 109 and OX1-5), each of which contains a long inverted repeat that shows substantial mitotic instability. This instability also occurs in the germline so that mutant transgenes appear within pedigrees at a high frequency. The mutation processes acting at these two inverted repeats are complex and can involve insertion or deletion, and can result in stabilization of the transgene. At transgene 109 mutational events range from very small rearrangements at the centre of the inverted repeat to complete transgene deletion. In addition we show that the rates of mutation at the inverted repeat of transgene OX1-5 can vary between the male and female germlines and between inbred strains of mice, suggesting the possibility of a genetic analysis to identify loci that modulate inverted repeat instability.
| S-EPMC450015 | biostudies-other