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TaqTth-hpRNA: a novel compact RNA-targeting tool for specific silencing of pathogenic mRNA.

ABSTRACT: Pathogenic allele silencing is a promising treatment for genetic hereditary diseases. Here, we develop an RNA-cleaving tool, TaqTth-hpRNA, consisting of a small, chimeric TaqTth, and a hairpin RNA guiding probe. With a minimal flanking sequence-motif requirement, in vitro and in vivo studies show TaqTth-hpRNA cleaves RNA efficiently and specifically. In an Alzheimer's disease model, we demonstrate silencing of mutant APP^swe mRNA without altering the wild-type APP mRNA. Notably, due to the compact size of TaqTth, we are able to combine with APOE2 overexpression in a single AAV vector, which results in stronger inhibition of pathologies.

SUBMITTER: Xu C

PROVIDER: S-EPMC11229350 | biostudies-literature | 2024 Jul

REPOSITORIES: biostudies-literature

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TaqTth-hpRNA: a novel compact RNA-targeting tool for specific silencing of pathogenic mRNA.

Xu Chong C Cao Jiyanuo J Qiang Huanran H Liu Yu Y Wu Jialin J Luo Qiudan Q Wan Meng M Wang Yujie Y Wang Peiliang P Cheng Qian Q Zhou Guohua G Sima Jian J Guo Yongjian Y Xu Shu S

Genome biology 20240707 1

Pathogenic allele silencing is a promising treatment for genetic hereditary diseases. Here, we develop an RNA-cleaving tool, TaqTth-hpRNA, consisting of a small, chimeric TaqTth, and a hairpin RNA guiding probe. With a minimal flanking sequence-motif requirement, in vitro and in vivo studies show TaqTth-hpRNA cleaves RNA efficiently and specifically. In an Alzheimer's disease model, we demonstrate silencing of mutant APP<sup>swe</sup> mRNA without altering the wild-type APP mRNA. Notably, due to ...[more]

PMID: 38972974

Similar Datasets

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

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

2024-06-20 | GSE269593 | 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

RNA-dependent RNA polymerase 6 is required for efficient hpRNA-induced gene silencing in plants.

Project description:In plants, transgenes with inverted repeats are used to induce efficient RNA silencing, which is also frequently induced by highly transcribed sense transgenes. RNA silencing induced by sense transgenes is dependent on RNA-dependent RNA polymerase 6 (RDR6), which converts single-stranded (ss) RNA into double-stranded (ds) RNA. By contrast, it has been proposed that RNA silencing induced by self-complementary hairpin RNA (hpRNA) does not require RDR6, because the hpRNA can directly fold back on itself to form dsRNA. However, it is unclear whether RDR6 plays a role in hpRNA-induced RNA silencing by amplifying dsRNA to spread RNA silencing within the plant. To address the efficiency of hpRNA-induced RNA silencing in the presence or absence of RDR6, Wild type (WT, Col-0) and rdr6-11 Arabidopsis thaliana lines expressing green fluorescent protein (GFP) were generated and transformed with a GFP-RNA interference (RNAi) construct. Whereas most GFP-RNAi-transformed WT lines exhibited almost complete silencing of GFP expression in the T1 generation, various levels of GFP expression remained among the GFP-RNAi-transformed rdr6-11 lines. Homozygous expression of GFP-RNAi in the T3 generation was not sufficient to induce complete GFP silencing in several rdr6-11 lines. Our results indicate that RDR6 is required for efficient hpRNA-induced RNA silencing in plants.

| S-EPMC3887914 | biostudies-literature

A compact regulatory RNA element in mouse Hsp70 mRNA.

Project description:Hsp70 (70 kDa heat shock protein) performs molecular chaperone functions by assisting the folding of newly synthesized and misfolded proteins, thereby counteracting various cell stresses and preventing multiple diseases, including neurodegenerative disorders and cancers. It is well established that, immediately after heat shock, Hsp70 gene expression is mediated by a canonical mechanism of cap-dependent translation. However, the molecular mechanism of Hsp70 expression during heat shock remains elusive. Intriguingly, the 5' end of Hsp70 messenger RNA (mRNA) appears to form a compact structure with the potential to regulate protein expression in a cap-independent manner. Here, we determined the minimal length of the mHsp70 5'-terminal mRNA sequence that is required for RNA folding into a highly compact structure. This span of this RNA element was mapped and the secondary structure characterized by chemical probing, resulting in a secondary structural model that includes multiple stable stems, including one containing the canonical start codon. All of these components, including a short stretch of the 5' open reading frame (ORF), were shown to be vital for RNA folding. This work provides a structural basis for future investigations on the role of translational regulatory structures in the 5' untranslated region and ORF sequences of Hsp70 during heat shock.

| S-EPMC10840451 | biostudies-literature

Nucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes to enhance RNAi stability in plants.

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.

| S-EPMC9263138 | biostudies-literature

Gene silencing by RNA interference in Sarcoptes scabiei: a molecular tool to identify novel therapeutic targets.

Project description:Scabies is one of the most common and widespread parasitic skin infections globally, affecting a large range of mammals including humans, yet the molecular biology of Sarcoptes scabiei is astonishingly understudied. Research has been hampered primarily due to the difficulty of sampling or culturing these obligatory parasitic mites. A further and major impediment to identify and functionally analyse potential therapeutic targets from the recently emerging molecular databases is the lack of appropriate molecular tools.We performed standard BLAST based searches of the existing S. scabiei genome databases using sequences of genes described to be involved in RNA interference in Drosophila and the mite model organism Tetranychus urticae. Experimenting with the S. scabiei mu-class glutathione S-transferase (SsGST-mu1) as a candidate gene we explored the feasibility of gene knockdown in S. scabiei by double-stranded RNA-interference (dsRNAi).We provide here an analysis of the existing S. scabiei draft genomes, confirming the presence of a double stranded RNA (dsRNA) - mediated silencing machinery. We report for the first time experimental gene silencing by RNA interference (RNAi) in S. scabiei. Non-invasive immersion of S. scabiei in dsRNA encoding an S. scabiei glutathione S-transferase mu-class 1 enzyme (SsGST-mu1) resulted in a 35% reduction in the transcription of the target gene compared to controls.A series of experiments identified the optimal conditions allowing systemic experimental RNAi without detrimental side effects on mite viability. This technique can now be used to address the key questions on the fundamental aspects of mite biology and pathogenesis, and to assess the potential therapeutic benefits of silencing S. scabiei target genes.

| S-EPMC5466799 | biostudies-literature

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA.

Project description:MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex.

| S-EPMC4703055 | biostudies-literature

Novel Broad-Spectrum Antiviral Inhibitors Targeting Host Factors Essential for Replication of Pathogenic RNA Viruses.

Project description:Recent RNA virus outbreaks such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV) have caused worldwide health emergencies highlighting the urgent need for new antiviral strategies. Targeting host cell pathways supporting viral replication is an attractive approach for development of antiviral compounds, especially with new, unexplored viruses where knowledge of virus biology is limited. Here, we present a strategy to identify host-targeted small molecule inhibitors using an image-based phenotypic antiviral screening assay followed by extensive target identification efforts revealing altered cellular pathways upon antiviral compound treatment. The newly discovered antiviral compounds showed broad-range antiviral activity against pathogenic RNA viruses such as SARS-CoV-2, EBOV and Crimean-Congo hemorrhagic fever virus (CCHFV). Target identification of the antiviral compounds by thermal protein profiling revealed major effects on proteostasis pathways and disturbance in interactions between cellular HSP70 complex and viral proteins, illustrating the supportive role of HSP70 on many RNA viruses across virus families. Collectively, this strategy identifies new small molecule inhibitors with broad antiviral activity against pathogenic RNA viruses, but also uncovers novel virus biology urgently needed for design of new antiviral therapies.

| S-EPMC7762994 | biostudies-literature

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