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

Antisense 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.

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

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.

The minor and major spliceosome interact to regulate alternative splicing around minor introns

Mutations in minor spliceosome components are linked to diseases such as Roifman syndrome, Lowry-Wood syndrome, and early-onset cerebellar ataxia (EOCA). Here we report that besides increased minor intron retention, Roifman syndrome and EOCA can also be characterized by elevated alternative splicing (AS) around minor introns. Consistent with the idea that the assembly/activity of the minor spliceosome informs AS in minor intron-containing genes (MIGs), inhibition of all minor spliceosome snRNAs led to upregulated AS. Notably, alternatively spliced MIG isoforms were bound to polysomes in the U11-null dorsal telencephalon, which suggested that aberrant MIG protein expression could contribute to disease pathogenesis. In agreement, expression of an aberrant isoform of the MIG Dctn3 by in utero electroporation, affected radial glial cell divisions. Finally, we show that AS around minor introns is executed by the major spliceosome and is regulated by U11-59K of the minor spliceosome, which forms exon-bridging interactions with proteins of the major spliceosome. Overall, we extend the exon-definition model to MIGs and postulate that disruptions of exon-bridging interactions might contribute to disease severity and pathogenesis.

Alteration of Exon Definition Causes Amelogenesis Imperfecta

Amelogenesis imperfecta (AI) is a collection of genetic disorders affecting the quality and/or quantity of tooth enamel. More than 20 genes are, so far, known to be responsible for this condition. In this study, we recruited 3 Turkish families with hypomaturation AI. Whole-exome sequence analyses identified disease-causing mutations in each proband, and these mutations cosegregated with the AI phenotype in all recruited members of each family. The AI-causing mutations in family 1 were a novel AMELX mutation [NM_182680.1:c.143T>C, p.(Leu48Ser)] in the proband and a novel homozygous MMP20 mutation [NM_004771.3:c.616G>A, p.(Asp206Asn)] in the mother of the proband. Previously reported compound heterozygous MMP20 mutations [NM_004771.3:c.103A>C, p.(Arg35=) and c.389C>T, p.(Thr130Ile)] caused the AI in family 2 and family 3. Minigene splicing analyses revealed that the AMELX missense mutation increased exonic definition of exon 4 and the MMP20 synonymous mutation decreased exonic definition of exon 1. These mutations would trigger an alteration of exon usage during RNA splicing, causing the enamel malformations. These results broaden our understanding of molecular genetic pathology of tooth enamel formation.

Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides

Splice-switching antisense oligonucleotides (ASOs), which bind specific RNA-target sequences and modulate pre-mRNA splicing by sterically blocking the binding of splicing factors to the pre-mRNA, are a promising therapeutic modality to treat a range of genetic diseases. ASOs are typically 15–25 nt long and considered to be highly specific towards their intended target sequence, typically elements that control exon definition and/or splice-site recognition. However, whether or not splice-modulating ASOs also induce hybridization-dependent mis-splicing of unintended targets has not been systematically studied. Here, we tested the in vitro effects of splice-modulating ASOs on 108 potential off-targets predicted on the basis of sequence complementarity, and identified 17 mis-splicing events for one of the ASOs tested. Based on analysis of data from two overlapping ASO sequences, we conclude that off-target effects are difficult to predict, and the choice of ASO chemistry influences the extent of off-target activity. The off-target events caused by the uniformly modified ASOs tested in this study were significantly reduced with mixed-chemistry ASOs of the same sequence. Furthermore, using shorter ASOs, combining two ASOs, and delivering ASOs by free uptake also reduced off-target activity. Finally, ASOs with strategically placed mismatches can be used to reduce unwanted off-target splicing events.

Exon definition facilitates reliable control of alternative splicing in the RON proto-oncogene

ABSTRACTAlternative splicing is a key step in eukaryotic gene expression that allows the production of multiple protein isoforms from the same gene. Even though splicing is perturbed in many diseases, we currently lack insights into regulatory mechanisms promoting its precision and efficiency. We analyse high-throughput mutagenesis data obtained for an alternatively spliced exon in the proto-oncogene RON and determine the functional units that control this splicing event. Using mathematical modeling of distinct splicing mechanisms, we show that alternative splicing is based in RON on a so-called ‘exon definition’ mechanism. Here, the recognition of the adjacent exons by the spliceosome is required for removal of an intron. We use our model to analyze the differences between the exon and intron definition scenarios and find that exon definition is crucial to prevent the accumulation of deleterious, partially spliced retention products during alternative splicing regulation. Furthermore, it modularizes splicing control, as multiple regulatory inputs are integrated into a common net input, irrespective of the location and nature of the corresponding cis-regulatory elements in the pre-mRNA. Our analysis suggests that exon definition promotes robust and reliable splicing outcomes in RON splicing.SIGNIFICANCEDuring mRNA maturation, pieces of the pre-mRNA (introns) are removed during splicing, and remaining parts (exons) are joined together. In alternative splicing, certain exons are either included or excluded, resulting in different splice products. Inclusion of RON alternative exon 11 leads to a functional receptor tyrosine kinase, while skipping results in a constitutively active receptor that promotes epithelial-to-mesenchymal transition and contributes to tumour invasiveness. Intron retention results in to deleterious isoforms that cannot be translated properly. Using kinetic modeling, we investigate the combinatorial regulation of this important splicing decision, and find that the experimental data supports a so-called exon definition mechanism. We show that this mechanism enhances the precision of alternative splicing regulation and prevents the retention of introns in the mature mRNA.