scholarly journals Cryptic U2-dependent Pre-mRNASplice Site Usage Induced by Splice Switching Antisense Oligonucleotides

2021 ◽  
Author(s):  
Kristin Ham ◽  
Niall Keegan ◽  
Craig McIntosh ◽  
May Aung-Htut ◽  
Khine Zaw ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Splice Site ◽  
Rna Splicing ◽  
Antisense Oligonucleotides ◽  
Live Cells ◽  
Cryptic Splice Site ◽  
Exon Definition ◽  
Rna Targets ◽  
Intended Effect

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.

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

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kristin A. Ham ◽  
Niall P. Keegan ◽  
Craig S. McIntosh ◽  
May T. Aung-Htut ◽  
Khine Zaw ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Antisense Oligonucleotides ◽  
Live Cells ◽  
Splice Sites ◽  
Cryptic Splice Site ◽  
Exon Definition ◽  
Rna Targets ◽  
Cryptic Splice Sites ◽  
Six Genes ◽  
Intended Effect

AbstractAntisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. 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 AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding 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 disruption of the exon definition signal within the exon’s excluded segment.

scholarly journals A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient

2021 ◽  
Vol 22 (24) ◽  
pp. 13248
Author(s):  
John G. Conboy
Keyword(s):  
Secondary Structure ◽  
Splice Site ◽  
Intron Retention ◽  
Donor Site ◽  
Von Willebrand Disease ◽  
Splice Donor Site ◽  
Cryptic Splice Site ◽  
Splice Donor ◽  
Nucleotide Mutation ◽  
Von Willebrand

A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.

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

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.

scholarly journals Common variant of human NEDD4L activates a cryptic splice site to form a frameshifted transcript

2002 ◽  
Vol 47 (12) ◽  
pp. 0665-0676 ◽  
Author(s):  
D. M. Dunn ◽  
T. Ishigami ◽  
J. Pankow ◽  
A. von Niederhausern ◽  
J. Alder ◽  
...  
Keyword(s):  
Splice Site ◽  
Common Variant ◽  
Cryptic Splice Site

Activation of a cryptic splice-site in intron 24 leads to the formation of apolipoprotein B-27.6

1997 ◽  
Vol 133 (2) ◽  
pp. 163-170 ◽  
Author(s):  
Attilla Nemeth-Slany ◽  
Phillipa Talmud ◽  
Scott M Grundy ◽  
Shailendra B Patel
Keyword(s):  
Splice Site ◽  
Apolipoprotein B ◽  
Cryptic Splice Site

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

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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