一种新的干扰素基因刺激因子剪接异构体STING(sv)的结构和功能

Structure and Function of an Alternative Splicing Isoform of Stimulator of Interferon Genes STING(sv)

干扰素基因刺激因子（Stimulator of interferon genes,STING）是病毒感染后机体抗病毒固有免疫反应过程中的重要蛋白分子。为了寻找STING新的剪接异构体,我们抽提了人类胚胎肾（Human embryonic kidney,HEK）293细胞RNA,用全长STING的mRNA（NM-198282.82）序列设计引物,作cDNA末端快速放大（RACE）及RTPCR,生物信息学方法比较新序列,亚克隆STING野生型（wt）及STING剪接异构体（sv）至真核表达载体pEGFPC1和pcDNA 3.1中。质粒EGFP-STING（wt）和EGFP-STING（sv）转染HEK 293细胞,用抗EGFP抗体作Western blot分析;质粒pcDNA-STING（wt）和pcDNA-STING（sv）转染HEK 293细胞,作荧光素酶功能分析以检测新剪接异构体对病毒诱导的干扰素β启动子荧光素酶活性的影响。我们发现了STING 3′端新的剪接异构体,与野生型相比,它缺失了第7外显子,导致阅读框架移位,相应蛋白质的C端30个氨基酸不同于野生型。Western blot出现相对分子质量（Mr）为58×103的STING（sv）蛋白强阳性条带。STING（sv）荧光素酶功能分析显示,该剪接异构体能明显抑制病毒诱导的干扰素β启动子活性。STING（sv）在多数组织和不同细胞系中都能表达。STING（sv）的发现为STING这一重要分子的功能调节提供了新的线索,它可能是病毒感染通路中干扰素的显性负性抑制剂,提示其为干扰素产生的精细调节成分,在人类病毒感染性疾病中的病理意义值得探究。

Stimulator of interferon genes （STING） is an important protein of the innate immune response, and protects against viral infections. To search for an alternative splicing isoform of STING, we undertook rapid amplification of cDNA ends （RACE） and RT-PCR with RNA extracted from human embryonic kid- ney （HEK）293 cells and primers designed according to the mRNA sequence of full-length STING（NM- 198282.82）. The new sequence was compared using a bioinformatics method. Then, a newly discovered, alternative splicing isoform of STING, named ＂STING（sv）＂, and STING（wt） were subcloned into the eu- karyotic expression vector pEGFP-C1 and pcDNA 3.1. Whole-cell extracts were analyzed by western blot- ting and then probed with monoclonal antibody against enhanced green fluorescent protein （EGFP） after transfection of EGFP-STING（wt） and EGFP-STING（wt） plasmids in HEK293 cells, pcDNA-STING（wt） and pcDNA-STING（wt） were transfected in HEK293 cells, and the luciferase assay carried out. Compared with STING（wt）, STING（sv） lacks exon 7 so that shift in the reading frame may produce a protein with a different C-terminal in amino acids 1-30. Western blotting confirmed an expected strong band at 58 × 10^3 kD. The functional luciferase assay showed that STING（sv） inhibited the activity of the interferon （IFN）- 13 promoter. STING（sv） can be expressed in multiple tissues and distinct cell lines. Our discovery of a new, alternative splicing isoform of STING provides new insights into the functional regulation of STING. STING（sv） could be a dominant negative inhibitor for the activity of the IFN-β promoter in the virus-infec- tion pathway. Hence, STING（sv） could participate in the ＂fine tuning＂ of the virus-induced activation of IFN. Therefore, exploring the role of STING（sv） in the pathogenesis of human diseases could be very worthwhile.