Induction of cryptic pre-mRNA splice-switching by antisense oligonucleotides

Abstract Antisense oligomers (AOs) are increasingly being used for modulating RNA splicing in live cells, both for research and for therapeutic purposes. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven such examples of AO-induced cryptic splice site activation – five new examples from our own experiments and three from reports published by others. We modelled the predicted effects that AO binding would have on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was a disruption to the exon definition signal within the exon’s excluded segment.

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Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites

Molecular and Cellular Biology ◽

10.1128/mcb.4.5.966-972.1984 ◽

1984 ◽

Vol 4 (5) ◽

pp. 966-972

Author(s):

C Montell ◽

E F Fisher ◽

M H Caruthers ◽

A J Berk

Keyword(s):

Splice Site ◽

Rna Splicing ◽

Early Phase ◽

Late Phase ◽

Productive Infection ◽

Splice Sites ◽

Mrna Synthesis ◽

Adenovirus E1b ◽

Cryptic Splice Sites ◽

Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

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Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites.

Molecular and Cellular Biology ◽

10.1128/mcb.4.5.966 ◽

1984 ◽

Vol 4 (5) ◽

pp. 966-972 ◽

Cited By ~ 23

Author(s):

C Montell ◽

E F Fisher ◽

M H Caruthers ◽

A J Berk

Keyword(s):

Splice Site ◽

Rna Splicing ◽

Early Phase ◽

Late Phase ◽

Productive Infection ◽

Splice Sites ◽

Mrna Synthesis ◽

Adenovirus E1b ◽

Cryptic Splice Sites ◽

Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

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Usage of Cryptic Splice Sites in Citrullinemia Fibroblasts Suggests Role of Polyadenylation in Splice-Site Selection during Terminal Exon Definition

DNA and Cell Biology ◽

10.1089/dna.1998.17.717 ◽

1998 ◽

Vol 17 (8) ◽

pp. 717-725 ◽

Cited By ~ 3

Author(s):

TING-FEN TSAI ◽

MEI-JANE WU ◽

TSUNG-SHENG SU

Keyword(s):

Splice Site ◽

Site Selection ◽

Splice Sites ◽

Terminal Exon ◽

Splice Site Selection ◽

Exon Definition ◽

Cryptic Splice Sites

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The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing

Nature Communications ◽

10.1038/s41467-021-23505-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Evi Goulielmaki ◽

Maria Tsekrekou ◽

Nikos Batsiotos ◽

Mariana Ascensão-Ferreira ◽

Eleftheria Ledaki ◽

...

Keyword(s):

Dna Damage ◽

Rna Splicing ◽

Excision Repair ◽

Intron Retention ◽

Dna Lesions ◽

Splicing Factor ◽

Loop Formation ◽

Rna Targets ◽

Underlying Mechanisms

AbstractRNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.

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Multiple cryptic splice sites can be activated by IDS point mutations generating misspliced transcripts

Journal of Molecular Medicine ◽

10.1007/s00109-006-0057-1 ◽

2006 ◽

Vol 84 (8) ◽

pp. 692-700 ◽

Cited By ~ 15

Author(s):

Susanna Lualdi ◽

Maria G. Pittis ◽

Stefano Regis ◽

Rossella Parini ◽

Anna E. Allegri ◽

...

Keyword(s):

Point Mutations ◽

Splice Sites ◽

Cryptic Splice Sites

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Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation

10.1101/2021.03.04.433849 ◽

2021 ◽

Author(s):

Xiaoxuan Su ◽

Wenxiao Ma ◽

Boyang Cheng ◽

Qian Wang ◽

Zefeng Guo ◽

...

Keyword(s):

Nucleic Acid ◽

Antisense Oligonucleotides ◽

Target Recognition ◽

Vero Cells ◽

Rnase L ◽

Rna Targets ◽

Infection Models ◽

Antisense Sequence ◽

Innate Antiviral Response ◽

Chimeric Oligonucleotides

AbstractThere is an urgent need for effective antiviral drugs to alleviate the current COVID-19 pandemic. Here, we rationally designed and developed chimeric antisense oligonucleotides to degrade envelope and spike RNAs of SARS-CoV-2. Each oligonucleotide comprises a 3’ antisense sequence for target recognition and a 5’-phosphorylated 2’-5’ poly(A)4 for guided ribonuclease L (RNase L) activation. Since RNase L can potently cleave single strand RNA during innate antiviral response, the improved degradation efficiency of chimeric oligonucleotides was twice as much as classic antisense oligonucleotides in Vero cells, for both SARS-CoV-2 RNA targets. In pseudovirus infection models, one of chimeric oligonucleotides targeting spike RNA achieved potent and broad-spectrum inhibition of both SARS-CoV-2 and its recently reported N501Y and/or ΔH69/ΔV70 mutants. These results showed that the constructed chimeric oligonucleotides could efficiently degrade pathogenic RNA of SARS-CoV-2 facilitated by immune activation, showing promising potentials as antiviral nucleic acid drugs for COVID-19.

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Activation of cryptic splice sites in three patients with chronic granulomatous disease

Molecular Genetics & Genomic Medicine ◽

10.1002/mgg3.854 ◽

2019 ◽

Vol 7 (9) ◽

Author(s):

Martin de Boer ◽

Karin van Leeuwen ◽

Mathias Hauri‐Hohl ◽

Dirk Roos

Keyword(s):

Chronic Granulomatous Disease ◽

Granulomatous Disease ◽

Splice Sites ◽

Cryptic Splice Sites

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A Secondary Structure That Contains the 5′ and 3′ Splice Sites Suppresses Splicing of Duck Hepatitis B Virus Pregenomic RNA

Journal of Virology ◽

10.1128/jvi.76.20.10195-10202.2002 ◽

2002 ◽

Vol 76 (20) ◽

pp. 10195-10202 ◽

Cited By ~ 21

Author(s):

Daniel D. Loeb ◽

Amanda A. Mack ◽

Ru Tian

Keyword(s):

Hepatitis B Virus ◽

Secondary Structure ◽

Hepatitis B ◽

Rna Splicing ◽

Splice Sites ◽

Base Pairing ◽

Duck Hepatitis B Virus ◽

Duck Hepatitis ◽

B Virus ◽

Cytoplasmic Accumulation

ABSTRACT Pregenomic RNA (pgRNA) plays two major roles in the hepadnavirus life cycle. It is the mRNA for two proteins required for DNA replication, C and P, and it is the template for reverse transcription. pgRNA is a terminally redundant transcript whose synthesis does not involve RNA splicing. For duck hepatitis B virus (DHBV), a spliced RNA is derived from pgRNA by removal of a single intron. The mechanism for the simultaneous cytoplasmic accumulation of unspliced (pgRNA) and spliced RNA was not known. We found that mutations within two regions of the DHBV genome reduced the level of pgRNA while increasing the level of spliced RNA. One region is near the 5′ end of pgRNA (region A), while the second is near the middle of pgRNA (region B). Inspection of the DHBV nucleotide sequence indicated that region A could base pair with region B. The 5′ and 3′ splice sites of the intron of the spliced RNA are within regions A and B, respectively. Substitutions that disrupted the predicted base pairing reduced the accumulation of pgRNA and increased the accumulation of spliced RNA. Restoration of base pairing, albeit mutant in sequence, resulted in restoration of pgRNA accumulation with a decrease in the level of spliced RNA. Our data are consistent with a model in which splicing of the pgRNA is suppressed by a secondary structure between regions A and B that occludes the splicing machinery from modifying pgRNA.

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