Determining the impact of alternative splicing events on transcriptome dynamics
© Wilhelm et al; licensee BioMed Central Ltd. 2008
Received: 15 May 2008
Accepted: 24 October 2008
Published: 24 October 2008
The 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.
When 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.
The 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.
The completion of the human genome sequence [1, 2] and subsequent analysis of the annotated genome  have revealed that the process of alternative splicing plays a key role in the generation of proteome diversity. Alternative splicing decisions control many biological processes ranging from sex determination in fruit flies to programmed cell death in human cells . Recent estimates suggest that at least 74% of human genes undergo alternative splicing . Moreover, a rapidly growing body of evidence shows that defects in alternative splicing of mRNA are an important cause of human disease [6–8]. Strikingly, although alternative splicing is pervasive within human genes, the functions of the vast majority of alternative splicing events remain unknown .
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].
The use of genome-wide microarrays in conjunction with SSO is an underutilized but potentially powerful method to determine the functions of alternative splicing events. Here we have recorded transcriptome dynamics during SSO-mediated induction of a well-characterized Bcl-xS splice variant using high-sensitivity microarrays. The results reveal a new link between Bcl-xS expression and the repression of ribosomal protein gene expression.
Cell culture conditions, molecular biology techniques and an extensive description of SSO-coupled transcriptome analysis are provided [see Additional file 1].
Treatment of cells with Bcl-xS-inducing SSOs can either sensitize cells to apoptotic stimuli, or induce apoptosis outright depending on the cell type and the levels of expression of Bcl-x [11–13]. In order to correlate changes in gene expression induced by Bcl-xS expression with the phenotypic outcome it was important to measure the rates of apoptosis in our experimental system. To analyze the impact of shifting the expression of endogenous Bcl-x from the long to the short isoform on apoptosis, the percentage of apoptotic cells was analyzed by flow cytometry using monoclonal antibodies that detect caspase cleaved cytokeratin-18, or by measuring Sub G1 DNA content by staining with propidium iodide. HeLa cells treated with Bcl-x SSO did not show a statistically significant increase in levels of apoptosis, in line with the previous results of Mercatante et al. in HeLa cells . Therefore although Bcl-xS mRNA expression is strongly induced in HeLa cells, this change is not accompanied by cell death through apoptosis.
Here we have combined s plice-s witching o ligonucleotides (SSOs) with high-sensitivity genome-wide microarrays to reveal the impact of shifting the expression of the endogenous Bcl-xL splice variant towards the Bcl-xS splice variant. To our knowledge, only a single study attempted to address the impact of SSO on gene expression patterns using microarrays . In that study, no statistically significant impact of Bcl-xS SSO on specific cellular functions or pathways was uncovered. Here we demonstrate a statistically significant impact of Bcl-xS SSOs on the repression of ribosomal protein-encoding genes and genes that code for nucleic acid binding proteins. Several factors could contribute to the different outcome of our study when compared to the earlier study . For example, different cell lines can give different transcriptome footprints, and we have employed HeLa whereas Mercatante et al. employed MCF7 and PC3 cells. Moreover the time of SSO treatment in our study was 18 hours versus 36 hours in that of Mercatante et al., a difference that could influence the number of primary versus secondary effects of the SSO. Here we have combined for the first time SSO with second-generation microarrays that possess substantially increased sensitivity and genome coverage and thus provide improved detection of statistically significant changes in specific gene ontologies. The capacity to detect specific cellular pathways that are impacted upon SSO represents an advance that can provide new biological insights into the function of alternative splicing events. Our study has focused on the transcriptome signature induced by Bcl-x SSO in the HeLa cell model system, however the methodology we describe should also be applicable to any alternative splicing event in any cell line that responds efficiently to SSO transfection. Genome-wide technologies have revealed the pervasive role of alternative splicing in proteome diversity, yet the functions of the vast majority of splice variants remain unknown . The merging of SSO and second-generation microarray technology that we present here provides a powerful and broadly applicable methodology to decipher the biological functions that are controlled by alternative splicing events.
The transcriptome footprint caused by the induction of endogenous Bcl-xS results in a dramatic suppression of genes involved in protein synthesis, particularly ribosomal protein encoding genes (Figure 3C). Since we do not observe a strong apoptotic response in cells treated with Bcl-x SSO (Figure 2C), we conclude that the measured decreases in the expression of genes related to protein synthesis cannot alone induce apoptosis. Bcl-xS has been characterized as a pro-apoptotic factor that is localized largely to the mitochondria . An impact of Bcl-xS-inducing SSO on the suppression of genes that participate in protein synthesis was therefore an unanticipated outcome. Boone-Unge and colleagues  have recently reported a biological link between the inhibition of protein translation and Bcl-xS using a completely unrelated approach. Their data showed that the translational inhibitor emitine causes a significant increase in the cellular levels of Bcl-xS mRNA. It is therefore conceivable that a feedback loop between Bcl-xS expression and translational inhibition exists. Further work will be required to confirm the hypothesis of such a feedback mechanism.
In summary, we report here a protocol that enables the measurement of the impact of endogenous alternative splicing events on transcriptome dynamics. When applied to the induction of Bcl-xS mRNA by splice-switching oligonucleotides we demonstrate a statistically significant suppression of ribosomal protein encoding genes. The approach applied here should accelerate efforts to define the biological roles of the vast numbers of uncharacterized alternative splice variants within the human genome. Finally, the approach we describe can also be applied to the analysis of the mechanism of action of clinically useful SSOs.
F.X.P. is recipient of a doctoral fellowship from the Nausicaa Combat Sa Leucémie association. A.B.'s group received funds from the European Hematology Association – José Carreras Foundation, and the French Ministry of Research through the "Complexité du Vivant – Action STICS-Santé" program. Work in B.B's laboratory was funded through a Discovery grant from Natural Sciences and Engineering Research Council of Canada. B.B. holds the Canada Research Chair in Genomic Regulation.
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