Department of Neurology and Suzhou Clinical Research Center of Neurological Disease, The Second Affiliated Hospital of Soochow University, Suzhou 215004, China.
Institute of Neuroscience, Soochow University, 199 Renai Road, Suzhou, Jiangsu 215123, China.
Nucleic Acids Res. 2022 Jan 25;50(2):731-749. doi: 10.1093/nar/gkab1280.
Intronic splicing enhancers and silencers (ISEs and ISSs) are two groups of splicing-regulatory elements (SREs) that play critical roles in determining splice-site selection, particularly for alternatively spliced introns or exons. SREs are often short motifs; their mutation or dysregulation of their cognate proteins frequently causes aberrant splicing and results in disease. To date, however, knowledge about SRE sequences and how they regulate splicing remains limited. Here, using an SMN2 minigene, we generated a complete pentamer-sequence library that comprises all possible combinations of 5 nucleotides in intron 7, at a fixed site downstream of the 5' splice site. We systematically analyzed the effects of all 1023 mutant pentamers on exon 7 splicing, in comparison to the wild-type minigene, in HEK293 cells. Our data show that the majority of pentamers significantly affect exon 7 splicing: 584 of them are stimulatory and 230 are inhibitory. To identify actual SREs, we utilized a motif set enrichment analysis (MSEA), from which we identified groups of stimulatory and inhibitory SRE motifs. We experimentally validated several strong SREs in SMN1/2 and other minigene settings. Our results provide a valuable resource for understanding how short RNA sequences regulate splicing. Many novel SREs can be explored further to elucidate their mechanism of action.
内含子剪接增强子和沉默子（ISEs 和 ISSs）是两组剪接调控元件（SREs），它们在决定剪接位点选择方面起着关键作用，特别是对于选择性剪接的内含子或外显子。SREs 通常是短的基序；它们的突变或与其同源蛋白的失调经常导致异常剪接，并导致疾病。然而，到目前为止，关于 SRE 序列及其如何调节剪接的知识仍然有限。在这里，我们使用 SMN2 迷你基因，生成了一个完整的五聚体序列文库，其中包含 7 号内含子中 5 个核苷酸的所有可能组合，位于 5'剪接位点下游的固定位置。我们系统地分析了所有 1023 个突变五聚体相对于野生型迷你基因在 HEK293 细胞中外显子 7 剪接的影响。我们的数据表明，大多数五聚体显著影响外显子 7 的剪接：其中 584 个是刺激的，230 个是抑制的。为了识别实际的 SREs，我们利用了 motif set enrichment analysis（MSEA），从中我们确定了刺激和抑制 SRE 基序的群组。我们在 SMN1/2 和其他迷你基因中实验验证了几个强 SREs。我们的结果为理解短 RNA 序列如何调节剪接提供了有价值的资源。可以进一步探索许多新的 SREs，以阐明它们的作用机制。
