Determining the impact of alternative splicing events on transcriptome dynamics

Emmanuelle Wilhelm,Arndt Benecke,Brendan Bell,François-Xavier Pellay

doi:10.1186/1756-0500-1-94

Abstract

BackgroundThe complete sequencing of the human genome and its subsequent analysis revealed a predominant role for alternative splicing in the generation of proteome diversity. Splice switching oligonucleotides (SSOs) are a powerful and specific tool to experimentally control alternative splicing of endogenous messenger RNAs in living cells. SSOs also have therapeutic potential to treat diseases that are caused by aberrant splicing. The assignment of biological roles to alternative splicing events of currently unknown function promises to provide a largely untapped source of potential new therapeutic targets. Here we have developed a protocol that combines high sensitivity microarrays with the transfection of SSOs to monitor global changes in gene expression downstream of alternate, endogenous splice events.ResultsWhen applied to a well-characterized splicing event in the Bcl-x gene, the application of high sensitivity microarrays revealed a link between the induction of the Bcl-xS isoform and the repression of genes involved in protein synthesis.ConclusionThe strategy introduced herein provides a useful approach to define the biological impact of any given alternative splicing event on global gene expression patterns. Furthermore, our data provide the first link between Bcl-xS expression and the repression of ribosomal protein gene expression.

Highlights

The complete sequencing of the human genome and its subsequent analysis revealed a predominant role for alternative splicing in the generation of proteome diversity
We chose as a model system the Bcl-x gene to study downstream effects of inducing the endogenous Bcl-xS splice variant because: 1) splice-switching oligonucleotides were first developed and tested to enforce splice site selection of the endogenous Bcl-x gene [11,12], 2) the biological consequences of inducing the pro-apoptotic Bcl-xS isoform are reasonably well-characterized, and 3) the Bcl-x gene is a potential target for SSObased anti-cancer therapies
Lipofectamine was selected as the transfection reagent as it has previously been demonstrated to provide efficient delivery of 2'-O-methyl (2'-O-Me) phosphorothioate oligonucleotides into the nucleus of HeLa cells [14]

Summary

Introduction

The complete sequencing of the human genome and its subsequent analysis revealed a predominant role for alternative splicing in the generation of proteome diversity. Technical advances in the application of antisense RNA oligonucleotides to manipulate alternative splicing either in cultured cells for functional genomic studies, or potentially in patients for therapeutic purposes, are opening unprecedented avenues to characterize the function of new alternative splicing events and eventually to exploit these functions clinically. These splice switching oligonucleotides (SSOs) can be used to induce loss of function or, when modified to bind cellular proteins, to induce gain of function by directing splicing regulatory factors to target a given splicing event [6,8,9]

Methods

Results

Conclusion