Stoilov Peter, Lin Chia-Ho, Damoiseaux Robert, Nikolic Julia, Black Douglas L
Department of Microbiology, Immunology, and Molecular Genetics and Howard Hughes Medical Institute, University of California, Los Angeles, CA 90095-1662, USA.
Proc Natl Acad Sci U S A. 2008 Aug 12;105(32):11218-23. doi: 10.1073/pnas.0801661105. Epub 2008 Aug 4.
Alternative splicing has emerged as a promising therapeutic target in a number of human disorders. However, the discovery of compounds that target the splicing reaction has been hindered by the lack of suitable high-throughput screening assays. Conversely, the effects of known drugs on the splicing reaction are mostly unclear and not routinely assessed. We have developed a two-color fluorescent reporter for cellular assays of exon inclusion that can accommodate nearly any cassette exon and minimizes interfering effects from changes in transcription and translation. We used microtubule-associated protein tau (MAPT) exon 10, whose missplicing causes frontotemporal dementia, to test the reporter in screening libraries of known bioactive compounds. These screens yielded several compounds that alter the splicing of the exon, both in the reporter and in the endogenous MAPT mRNA. One compound, digoxin, has long been used in the treatment of heart failure, but was not known to modulate splicing. The positive compounds target different signal transduction pathways, and microarray analysis shows that each compound affects the splicing of a different set of exons in addition to MAPT exon 10. Our results identify currently prescribed cardiotonic steroids as modulators of alternative splicing and demonstrate the feasibility of screening for drugs that alter exon inclusion.
可变剪接已成为多种人类疾病中一个有前景的治疗靶点。然而，由于缺乏合适的高通量筛选检测方法，阻碍了靶向剪接反应的化合物的发现。相反，已知药物对剪接反应的影响大多不清楚，也未进行常规评估。我们开发了一种用于外显子包含细胞检测的双色荧光报告系统，它几乎可以容纳任何盒式外显子，并将转录和翻译变化产生的干扰效应降至最低。我们使用微管相关蛋白tau（MAPT）的外显子10（其错配剪接会导致额颞叶痴呆）在已知生物活性化合物的筛选文库中测试该报告系统。这些筛选产生了几种可改变报告系统及内源性MAPT mRNA中外显子剪接的化合物。一种化合物地高辛长期以来用于治疗心力衰竭，但此前未知其可调节剪接。阳性化合物靶向不同的信号转导途径，微阵列分析表明，除了MAPT外显子10外，每种化合物还会影响不同外显子组的剪接。我们的结果确定了目前处方的强心甾类化合物为可变剪接的调节剂，并证明了筛选改变外显子包含的药物的可行性。
