Abstract
BackgroundWe previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins.In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures.ResultsWe show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition.ConclusionOur in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.
Highlights
We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo
Splicing interference by 5' tails in bifunctional oligonucleotides We have shown previously that a bifunctional oligonucleotide with a 5' overhang that contains binding sites for the hnRNP A1/A2 proteins strongly inhibits splicing when the oligonucleotide hybridizes immediately upstream of the 5' splice site of Bcl-xL
Our previous work demonstrated that an oligonucleotide carrying a 5' overhang bound by hnRNP A1/A2 proteins and a portion complementary to the sequence immediately upstream of the Bcl-xL 5' splice site compromised the splicing to the Bcl-xL site and improved splicing to the upstream Bcl-xS 5' splice site in vitro and in vivo [17]
Summary
We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. Alternative pre-messenger RNA splicing involves the differential use of splice sites, a process that represents a powerful and versatile way to control protein function. Striking examples highlighting the importance of alternative splicing are found in many systems including programmed cell death or apoptosis. Bcl-x is alternatively spliced to produce Bcl-xL and Bcl-xS, two proteins with antagonistic function in apoptosis [4]. In cancers and cancer cell lines, the expression of the anti-apoptotic protein Bcl-xL is increased and overexpression of Bcl-xL is associated with decreased apoptosis, increased risk of metastasis, resistance to chemotherapeutic drugs and poor clinical outcome [5,6,7,8,9]. The pro-apoptotic isoform Bcl-xS can sensitize cells to chemotherapeutic agents [9,10,11,12,13]
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