Pseudouridine-mediated stop codon readthrough in <i>S. cerevisiae</i> is sequence context–independent

Adachi, Hironori; Yu, Yi‐Tao

doi:10.1261/rna.076042.120

Cited by 30 publications

(15 citation statements)

References 35 publications

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“…(c) Polyadenylation sites annotated by the Poly(A)Site 2.0 atlas35 . GM12878 specific data from: (d) PolII ChiP-seq sites47 ; (e) CAGE sites for the positive strand48 ; (f) Three replicate DNase-HS tracks49 . (g) Read coverage from full-length nanopore 5′ RACE sequencing.…”

mentioning

confidence: 99%

Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing

Mulroney

Wulf

Schildkraut

et al. 2021

RNA

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Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional methods the 5′ and 3′ ends of poly(A) RNA cannot be identified unambiguously. This is due in part to the architecture of the nanopore/enzyme-motor complex, and in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoform scaffolds among ~4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5′ m7G caps, we exchanged the biological cap for a modified cap attached to a 45-nucleotide oligomer. This oligomer adaptation method improved 5′ end sequencing and ensured correct identification of the 5′ m7G capped ends. Second, among these 5′-capped nanopore reads, we screened for ionic current signatures consistent with a 3′ polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds, most of which (257,721) aligned to protein-coding genes. Of these, 4,876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data confirmed the validity of these high-confidence RNA scaffolds.

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mentioning

confidence: 99%

Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing

Mulroney

Wulf

Schildkraut

et al. 2021

RNA

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“…Base miscalls relative to canonical training data in our ADGRE1 aligned reads ( Fig. 8g) strongly suggest an unannotated pseudouridine in the stop codon, which is known to cause translation read through 37 . Importantly, this miscall was absent from in vitro transcribed nanopore direct RNA sequence data [11][12][13]15 .…”

Section: A Dhesion G Protein-coupled Receptor E1 ( Adgre1)mentioning

confidence: 93%

“…The * (red background) is the canonical stop codon for ADGRE1. A pseudouridine at the first nucleotide of that stop codon can promote ribosome read through in other genes 37 . ( b ) High confidence mRNA scaffolds aligned to the unannotated isoform.…”

Section: Adaptation Ofmentioning

confidence: 99%

Identification of high confidence human poly(A) RNA isoform scaffolds using nanopore sequencing

Mulroney

Wulf²,

Schildkraut³

et al. 2020

Preprint

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Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional methods the 5′ and 3′ ends of poly(A) RNA cannot be identified unambiguously. This is due in part to the architecture of the nanopore/enzyme-motor complex, and in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoform scaffolds among ∼4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5′ m7G caps, we exchanged the biological cap for a modified cap attached to a 45-nucleotide oligomer. This oligomer adaptation method improved 5′ end sequencing and ensured correct identification of the 5′ m7G capped ends. Second, among these 5′-capped nanopore reads, we screened for ionic current signatures consistent with a 3′ polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds, most of which (257,721) aligned to protein-coding genes. Of these, 4,876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data confirmed the validity of these high-confidence RNA scaffolds.

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“…The authors were also able to identify the amino acids incorporated at the pseudouridylated PTC codons: primarily tyrosine and phenylalanine at ΨGA codons and serine and threonine at ΨAA and ΨAG codons. The Yu lab recently showed that the readthrough effect promoted by pseudouridylation of PTCs is independent of the target sequence, suggesting that this technology is theoretically applicable to all nonsense mutations [ 202 ]. Importantly, it appears that a single U-to-Ψ conversion at the PTC not only promotes PTC-readthrough during translation but also suppresses NMD.…”

Section: Therapeutic Potential Of Rna Modificationsmentioning

confidence: 99%

From Antisense RNA to RNA Modification: Therapeutic Potential of RNA-Based Technologies

Adachi

Hengesbach

et al. 2021

Biomedicines

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Therapeutic oligonucleotides interact with a target RNA via Watson-Crick complementarity, affecting RNA-processing reactions such as mRNA degradation, pre-mRNA splicing, or mRNA translation. Since they were proposed decades ago, several have been approved for clinical use to correct genetic mutations. Three types of mechanisms of action (MoA) have emerged: RNase H-dependent degradation of mRNA directed by short chimeric antisense oligonucleotides (gapmers), correction of splicing defects via splice-modulation oligonucleotides, and interference of gene expression via short interfering RNAs (siRNAs). These antisense-based mechanisms can tackle several genetic disorders in a gene-specific manner, primarily by gene downregulation (gapmers and siRNAs) or splicing defects correction (exon-skipping oligos). Still, the challenge remains for the repair at the single-nucleotide level. The emerging field of epitranscriptomics and RNA modifications shows the enormous possibilities for recoding the transcriptome and repairing genetic mutations with high specificity while harnessing endogenously expressed RNA processing machinery. Some of these techniques have been proposed as alternatives to CRISPR-based technologies, where the exogenous gene-editing machinery needs to be delivered and expressed in the human cells to generate permanent (DNA) changes with unknown consequences. Here, we review the current FDA-approved antisense MoA (emphasizing some enabling technologies that contributed to their success) and three novel modalities based on post-transcriptional RNA modifications with therapeutic potential, including ADAR (Adenosine deaminases acting on RNA)-mediated RNA editing, targeted pseudouridylation, and 2′-O-methylation.

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Pseudouridine-mediated stop codon readthrough in S. cerevisiae is sequence context–independent

Cited by 30 publications

References 35 publications

Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing

Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing

Identification of high confidence human poly(A) RNA isoform scaffolds using nanopore sequencing

From Antisense RNA to RNA Modification: Therapeutic Potential of RNA-Based Technologies

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