PDF(Pubmed)
Abstract：
Alternative splicing (AS) generates multiple RNA isoforms and increases the complexities of transcriptomes and proteomes. However, it remains unclear how RNA structures contribute to AS regulation. Here, we systematically search transcriptomes for secondary structures with concealed branch sites (BSs) in the alternatively spliced introns and predict thousands of them from six organisms, of which many are evolutionarily conserved. Intriguingly, a highly conserved stem-loop structure with concealed BSs is found in animal SF3B3 genes and colocalizes with a downstream poison exon (PE). Destabilization of this structure allows increased usage of the BSs and results in enhanced PE inclusion in human and Drosophila cells, leading to decreased expression of SF3B3. This structure is experimentally validated using an in-cell SHAPE-MaP assay. Through RNA interference screens of 28 RNA-binding proteins, we find that this stem-loop structure is sensitive to U2 factors. Furthermore, we find that SF3B3 also facilitates DNA repair and protects genome stability by enhancing interaction between ERCC6/CSB and arrested RNA polymerase II. Importantly, both Drosophila and human cells with the secondary structure mutated by genome editing exhibit altered DNA repair in vivo. This study provides a novel and common mechanism for AS regulation of PEs and reveals a physiological function of SF3B3 in DNA repair.
摘要：
可变剪接（AS）产生多种RNA同种型，并增加转录组和蛋白质组的复杂性。然而,目前尚不清楚RNA结构如何促进AS调节。这里,我们系统地搜索具有可变剪接内含子中隐藏分支位点（BS）的二级结构的转录组，并从六个生物体中预测数千个，其中许多是进化保守的。有趣的是，在动物SF3B3基因中发现了具有隐藏BS的高度保守的茎环结构，并与下游毒物外显子（PE）共定位。这种结构的不稳定允许增加BS的使用，并导致在人和果蝇细胞中增强的PE包含。导致SF3B3表达降低。该结构使用细胞内SHAPE-MaP测定进行实验验证。通过28种RNA结合蛋白的RNA干扰筛选，我们发现这种茎环结构对U2因子敏感。此外，我们发现SF3B3还通过增强ERCC6/CSB与被阻滞的RNA聚合酶II之间的相互作用来促进DNA修复并保护基因组稳定性.重要的是,果蝇和具有通过基因组编辑突变的二级结构的人类细胞在体内表现出改变的DNA修复。本研究为PE的AS调控提供了新的共同机制，揭示了SF3B3在DNA修复中的生理功能。
