Functional Role of Transcriptional Factor TBX5 in Pre-mRNA Splicing and Holt-Oram Syndrome via Association with SC35
Functional Role of Transcriptional Factor TBX5 in Pre-mRNA Splicing and Holt-Oram Syndrome via Association with SC35
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DOI：
10.1074/jbc.m109.041368
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发表时间：
2009-09-18
影响因子：
4.8
通讯作者：
Wang, Qing Kenneth
中科院分区：
文献类型：
--
作者：
Fan, Chun;Chen, Qiuyun;Wang, Qing Kenneth
TBX5 是心脏发生和肢体发育所需的 T 盒转录因子。 TBX5 突变会导致 Holt-Oram 综合征，其特征是先天性心脏缺陷和上肢变形。在这里，我们建立了 TBX5 在前 mRNA 剪接中的新功能，并证明该功能与 Holt-Oram 综合征的发病机制相关，为该疾病提供了一种新的致病机制。蛋白质组学与亲和纯化相结合，将剪接因子 SC35 鉴定为候选 TBX5 相关蛋白。免疫共沉淀和谷胱甘肽 S-转移酶 Pulldown 测定证实了 TBX5 和 SC35 之间的复合物形成。 TBX5 可以与 RNA 同聚物（多核糖核苷酸）和 5' 剪接位点结合，从而取代 SC35 与相同 RNA 的结合。 TBX5 的过表达提高了前 mRNA 剪接的效率并调节可变剪接位点选择。然而，TBX5和SC35的共表达会拮抗彼此对剪接的积极作用。最严重的 TBX5 突变 G80R 具有心脏表型的完全外显率，强烈影响前 mRNA 剪接，而心脏表型不完全外显的其他突变（包括 R237Q）不会改变 TBX5 的剪接活性。这项研究将 TBX5 确立为第一个心脏基因和第一个在转录激活和前 mRNA 剪接中具有双重作用的人类疾病基因。
TBX5 is a T-box transcriptional factor required for cardio-genesis and limb development. TBX5 mutations cause Holt-Oram syndrome characterized by congenital heart defects and upper limb deformations. Here we establish a novel function for TBX5 in pre-mRNA splicing, and we show that this function is relevant to the pathogenesis of Holt-Oram syndrome, providing a novel pathogenic mechanism for the disease. Proteomics in combination with affinity purification identifies splicing factor SC35 as a candidate TBX5-associating protein. Co-immunoprecipitation and glutathione S-transferase pulldown assays confirm the complex formation between TBX5 and SC35. TBX5 can bind to RNA homopolymers (polyribonucleotides) and to the 5'-splice site, which overrides the binding of SC35 to the same RNA. Overexpression of TBX5 increases the efficiency of pre-mRNA splicing and regulates alternative splice site selection. However, co-expression of TBX5 and SC35 antagonizes each other's positive effect on splicing. The most severe TBX5 mutation, G80R, with complete penetrance of the cardiac phenotype, strongly affects pre-mRNA splicing, whereas other mutations with incomplete penetrance of the cardiac phenotype, including R237Q, do not alter the splicing activity of TBX5. This study establishes TBX5 as the first cardiac gene and the first human disease gene with dual roles in both transcriptional activation and pre-mRNA splicing.