RAN translation of the expanded CAG repeats in the SCA3 disease context

Jazurek-Ciesiolka, Magdalena; Ciesiolka, Adam; Komur, Alicja A.; Urbanek-Trzeciak, Martyna Olga; Fiszer, Agnieszka

doi:10.1101/2020.05.07.082354

Cited by 4 publications

(4 citation statements)

References 99 publications

(179 reference statements)

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“…Again, these observations require confirmation in a larger set of cell lines, e.g., for investigation of processes that might be related to the phenomenon of neuronal vulnerability to mutations causing polyQ diseases [46]. However, these results support the idea that presented approach can be applied in studies aimed at the investigation of RNA biology of mutant transcripts leading to a better understanding of the pathogenesis of polyQ disorders [4,5,[47][48][49]. Additionally, our approach could be also used for analyses of non-neuronal cells, as these cells were reported to be implicated in the pathogenesis of both SCA3 and HD [4,50,51].…”

Section: Discussionmentioning

confidence: 80%

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models

et al. 2023

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Background The majority of genes in the human genome is present in two copies but the expression levels of both alleles is not equal. Allelic imbalance is an aspect of gene expression relevant not only in the context of genetic variation, but also to understand the pathophysiology of genes implicated in genetic disorders, in particular, dominant genetic diseases where patients possess one normal and one mutant allele. Polyglutamine (polyQ) diseases are caused by the expansion of CAG trinucleotide tracts within specific genes. Spinocerebellar ataxia type 3 (SCA3) and Huntington’s disease (HD) patients harbor one normal and one mutant allele that differ in the length of CAG tracts. However, assessing the expression level of individual alleles is challenging due to the presence of abundant CAG repeats in the human transcriptome, which make difficult the design of allele-specific methods, as well as of therapeutic strategies to selectively engage CAG sequences in mutant transcripts. Results To precisely quantify expression in an allele-specific manner, we used SNP variants that are linked to either normal or CAG expanded alleles of the ataxin-3 (ATXN3) and huntingtin (HTT) genes in selected patient-derived cell lines. We applied a SNP-based quantitative droplet digital PCR (ddPCR) protocol for precise determination of the levels of transcripts in cellular and mouse models. For HD, we showed that the process of cell differentiation can affect the ratio between endogenous alleles of HTT mRNA. Additionally, we reported changes in the absolute number of the ATXN3 and HTT transcripts per cell during neuronal differentiation. We also implemented our assay to reliably monitor, in an allele-specific manner, the silencing efficiency of mRNA-targeting therapeutic approaches for HD. Finally, using the humanized Hu128/21 HD mouse model, we showed that the ratio of normal and mutant HTT transgene expression in brain slightly changes with the age of mice. Conclusions Using allele-specific ddPCR assays, we observed differences in allele expression levels in the context of SCA3 and HD. Our allele-selective approach is a reliable and quantitative method to analyze low abundant transcripts and is performed with high accuracy and reproducibility. Therefore, the use of this approach can significantly improve understanding of allele-related mechanisms, e.g., related with mRNA processing that may be affected in polyQ diseases.

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Section: Discussionmentioning

confidence: 80%

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models

et al. 2023

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“…This bears the advantage to increase the stability of the resulting polyQ tract [53], which has not changed in our model since the first measurements of the fragment length in 2015. A disadvantage of the interrupted repeat, however, is that our mice could miss the feature of potential RNA toxicity, that is less prominent in interrupted repeats and can also contribute to disease progression [54,55]. Further, it is not possible to assess the impact of repeat associated non-ATG (RAN) translation, which might also play a role in SCA3, but could be extenuated in our 304Q KI mice.…”

Section: Discussionmentioning

confidence: 99%

A Novel SCA3 Knock-in Mouse Model Mimics the Human SCA3 Disease Phenotype Including Neuropathological, Behavioral, and Transcriptional Abnormalities Especially in Oligodendrocytes

et al. 2021

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Spinocerebellar ataxia type 3 is the most common autosomal dominant inherited ataxia worldwide, caused by a CAG repeat expansion in the Ataxin-3 gene resulting in a polyglutamine (polyQ)-expansion in the corresponding protein. The disease is characterized by neuropathological, phenotypical, and specific transcriptional changes in affected brain regions. So far, there is no mouse model available representing all the different aspects of the disease, yet highly needed for a better understanding of the disease pathomechanisms. Here, we characterized a novel Ataxin-3 knock-in mouse model, expressing a heterozygous or homozygous expansion of 304 CAACAGs in the murine Ataxin-3 locus using biochemical, behavioral, and transcriptomic approaches. We compared neuropathological, and behavioral features of the new knock-in model with the in SCA3 research mostly used YAC84Q mouse model. Further, we compared transcriptional changes found in cerebellar samples of the SCA3 knock-in mice and post-mortem human SCA3 patients. The novel knock-in mouse is characterized by the expression of a polyQ-expansion in the murine Ataxin-3 protein, leading to aggregate formation, especially in brain regions known to be vulnerable in SCA3 patients, and impairment of Purkinje cells. Along these neuropathological changes, the mice showed a reduction in body weight accompanied by gait and balance instability. Transcriptomic analysis of cerebellar tissue revealed age-dependent differential expression, enriched for genes attributed to myelinating oligodendrocytes. Comparing these changes with those found in cerebellar tissue of SCA3 patients, we discovered an overlap of differentially expressed genes pointing towards similar gene expression perturbances in several genes linked to myelin sheaths and myelinating oligodendrocytes.

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“…6-fold lower than that for Δ133p53 isoform synthesized from AUG3 with mRNA(AUG3CUA4). The second product most likely reflected translation initiation from a non-standard initiation codon [ 34 , 35 ]. Thus, even with the mRNA bearing only one initiation codon AUG4, translation in vitro from this codon was so ineffective that another non-standard initiation codon took over.…”

Section: Resultsmentioning

confidence: 99%

Translation of human Δ133p53 mRNA and its targeting by antisense oligonucleotides complementary to the 5′-terminal region of this mRNA

et al. 2021

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The p53 protein is expressed as at least twelve protein isoforms. Within intron 4 of the human TP53 gene, a P2 transcription initiation site is located and this transcript encodes two p53 isoforms: Δ133p53 and Δ160p53. Here, the secondary structure of the 5′-terminal region of P2-initiated mRNA was characterized by means of the SHAPE and Pb2+-induced cleavage methods and for the first time, a secondary structure model of this region was proposed. Surprisingly, only Δ133p53 isoform was synthetized in vitro from the P2-initiated p53 mRNA while translation from both initiation codons occurred after the transfection of vector-encoded model mRNA to HCT116 cells. Interestingly, translation performed in the presence of the cap analogue suggested that the cap-independent process contributes to the translation of P2-initiated p53 mRNA. Subsequently, several antisense oligonucleotides targeting the 5′-terminal region of P2-initiated p53 mRNA were designed. The selected oligomers were applied in in vitro translation assays as well as in cell lines and their impact on the Δ133p53 synthesis and on cell viability was investigated. The results show that these oligomers are attractive tools in the modulation of the translation of P2-initiated p53 mRNA through attacking the 5′ terminus of the transcript. Since cell proliferation is also reduced by antisense oligomers that lower the level of Δ133p53, this demonstrates an involvement of this isoform in tumorigenesis.

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RAN translation of the expanded CAG repeats in the SCA3 disease context

Cited by 4 publications

References 99 publications

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models

Allele-specific quantitation of ATXN3 and HTT transcripts in polyQ disease models

A Novel SCA3 Knock-in Mouse Model Mimics the Human SCA3 Disease Phenotype Including Neuropathological, Behavioral, and Transcriptional Abnormalities Especially in Oligodendrocytes

Translation of human Δ133p53 mRNA and its targeting by antisense oligonucleotides complementary to the 5′-terminal region of this mRNA

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