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Specific silencing of pathogenic mRNA by a novel compact RNA-targeting tool TaqTth-hpRNA [Amplicon sequencing]

ABSTRACT: Pathogenic allele silencing is a promising treatment for genetic hereditary diseases. However, the concern about the specificity of present RNA-knockdown strategies has limited their in vivo applications. Here a TaqTth-hpRNA system consisting of a small, chimeric protein (TaqTth) and hairpin-RNA probe (hpRNA) is provided. The TaqTth-hpRNA showed a high-specific knockdown against targeted mRNA with minimal flanking sequence-motif requirement and less cell viability damage, then was applied to mutant APPswe mRNA silencing without altering the wild-type APP mRNA in Alzheimer’s disease. Notably, the combination of the TaqTth and human apolipoprotein E 2 (APOE2) overexpression encoded in a single AAV vector is available due to the compact size of TaqTth, and showed stronger inhibition of pathologies in the mouse model. Altogether, we provided the basis for a small RNA-targeting tool with high selectivity, minimal flanking sequence-motif requirement, less cell viability damage and potentially being an alternative in therapeutic applications.

ORGANISM(S): Escherichia coli BL21

PROVIDER: GSE269593 | GEO | 2024/06/20

REPOSITORIES: GEO

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Specific silencing of pathogenic mRNA by a novel compact RNA-targeting tool TaqTth-hpRNA

Project description:Pathogenic allele silencing is a promising treatment for genetic hereditary diseases. However, the concern about the specificity of present RNA-knockdown strategies has limited their in vivo applications. Here a TaqTth-hpRNA system consisting of a small, chimeric protein (TaqTth) and hairpin-RNA probe (hpRNA) is provided. The TaqTth-hpRNA showed a high-specific knockdown against targeted mRNA with minimal flanking sequence-motif requirement and less cell viability damage, then was applied to mutant APPswe mRNA silencing without altering the wild-type APP mRNA in Alzheimer’s disease. Notably, the combination of the TaqTth and human apolipoprotein E 2 (APOE2) overexpression encoded in a single AAV vector is available due to the compact size of TaqTth, and showed stronger inhibition of pathologies in the mouse model. Altogether, we provided the basis for a small RNA-targeting tool with high selectivity, minimal flanking sequence-motif requirement, less cell viability damage and potentially being an alternative in therapeutic applications.

2024-06-30 | GSE236392 | GEO

Specific silencing of pathogenic mRNA by a novel compact RNA-targeting tool TaqTth-hpRNA [RNA-seq]

2024-06-20 | GSE269592 | GEO

Mismatch hpRNA prevents intrinsic self-silencing and enhances RNAi stability

Project description: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

2022-03-31 | GSE178565 | GEO

Specific silencing of pathogenic mRNA by a novel compact RNA-targeting tool TaqTth-hpRNA

Project description:Specific silencing of pathogenic mRNA by a novel compact RNA-targeting tool TaqTth-hpRNA

| PRJNA990747 | ENA

Mismatch hpRNA prevents intrinsic self-silencing and enhances RNAi stability

Project description:Mismatch hpRNA prevents intrinsic self-silencing and enhances RNAi stability

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Gene expression in HeLa cells expressing GFP-IRF3 (non-targeting control and clonal knockouts of DDX58, MAVS, ATP13A1, CAPN15, TADA2B, MED16, and MED24) following Sendai virus (SeV) infection or hpRNA transfection

Project description:IRF3 translocates from the cytoplasm to the nucleus upon recognition of a cytoplasmic RNA stimulus by RIG-I or MDA5. Using an optical pooled CRISPR knockout screen in HeLa-Cas9 cells, we identified ATP13A1, CAPN15, TADA2B, MED16, and MED24 as novel genes affecting the translocation of IRF3 upon Sendai virus infection. We generated isogenic clonal knockout HeLa-Cas9 lines and confirmed that loss of these five novel genes results in either decreased (ATP13A1 and CAPN15) or increased (TADA2B, MED16, and MED24) translocation of IRF3 upon Sendai virus or synthetic hpRNA stimulation.

2023-03-05 | GSE179288 | GEO

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

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

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

2024-06-27 | GSE252890 | GEO

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

2024-06-27 | GSE243296 | GEO

The methyltransferase METTL9 mediates pervasive 1-methylhistidine modification in mammalian proteomes

Project description:Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3- methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase (MTase) that mediates the formation of the majority of 1MH present in mouse and human proteomes. The minimal requirement for METTL9-catalyzed methylation is a His-x-His (HxH) motif, where "x" is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins. Here, we show that the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I are methylated at HxH motif using MALDI-TOF MS.

2020-11-23 | PXD022067 | Pride

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