Determining the impact of alternative splicing events on transcriptome dynamics

Background 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. Findings 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. Conclusions 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. Biological triplicates, 2 conditions: control oligonucleotide, Bcl-x oligonucleotide

研究背景
人类基因组全测序及其后续分析揭示了可变剪接（alternative splicing）在蛋白质组多样性产生过程中的主导作用。剪接切换寡核苷酸（Splice Switching Oligonucleotides，SSOs）是一种高效且特异性强的工具，可在活细胞中实验性调控内源信使RNA（messenger RNA）的可变剪接。此类寡核苷酸还具备治疗异常剪接相关性疾病的潜力。为当前功能未明的可变剪接事件明确其生物学功能，有望发掘大量尚未被开发的新型治疗靶点。本研究开发了一种将高灵敏度微阵列与SSO转染相结合的实验方案，用于监测内源可变剪接事件下游的全局基因表达变化。

研究结果
当将该方案应用于Bcl-x基因中一个已被充分表征的剪接事件时，高灵敏度微阵列分析揭示了Bcl-xS剪接变体的诱导与蛋白质合成相关基因的表达抑制之间存在关联。

研究结论
本研究提出的策略可为明确任意特定可变剪接事件对全局基因表达模式的生物学影响提供实用方法。此外，本研究数据首次揭示了Bcl-xS表达与核糖体蛋白基因表达抑制之间的关联。

生物学重复三次，共2组实验条件：对照寡核苷酸组、Bcl-x寡核苷酸组