Modification of splicing with antisense oligonucleotides in the Insulin receptor exon 11 and Apolipoprotein B exon 26.
Abstract
Background - The alternatively spliced insulin receptor (IR) exon 11 (36
nucleotides) and the constitutively spliced Apolipoprotein B (APOB) exon 26 (7572
nucleotides) are examples of the shortest and longest exons in the genome.
Aim - The aim of this study was to investigate the regulation of splicing of these 2
exons in cell culture using 2′-O-methyl RNA antisense oligonucleotides (ASOs) and
peptide nucleic acid (PNA)-peptide hybrid ASOs.
Methods - ASOs were designed to target key sequences involved in the splicing of
the IR exon 11 and exonic splicing silencer sequences (ESS) in APOB exon 26.
HepG2 cells were reverse-transfected with the ASOs for 48 hours, mRNA
harvested and RT-PCR was performed to amplify the IR isoform and APOB cDNAs
which were separated by PAGE and quantified.
Results
Insulin receptor exon 11 - 2′-O-methyl RNA ASOs targeted to two intronic sites,
the 3′ half of exon 11 and spanning the entire exon caused significant exon
skipping.
PNA-peptide hybrids predicted to increase exon 11 splicing, paradoxically caused
exon skipping. PNA-peptide hybrids with 3′ tails caused exon 11 skipping more
effectively than hybrids with 5′ tails.
Apolipoprotein B exon 26 - Only combinations of 2′-O-methyl RNA ASOs
targeting multiple ESSs in APOB exon 26 caused a small proportion of aberrant
splicing. This consisted of complete exon 26 skipping and the selection of a
downstream cryptic 3′ splice site in preference to the native 3′ splice site.
Discussion - Exclusion of the IR exon 11 can be induced by targeting a
combination of intronic or exonic sequences. PNA-peptide hybrid ASOs were
unable to increase exon 11 splicing. The aberrant splicing of large constitutive
exons such as APOB exon 26 can be induced by targeting multiple ESS sites along
its course.
Authors
Srirangalingam, UmasuthanCollections
- Theses [3831]