干扰素调节因子剪切的剪接异构体IRF-3c的结构及功能

Structure and function of a truncated spliced isoform of interferon regulatory factor IRF-3c

目的干扰素调节因子(IRF)3是病毒感染后调节干扰素基因转录重要的转录因子,寻找IRF-3新的剪接异构体,研究其结构及功能.方法抽提人类细胞RNA,用IRF-3已发表序列设计引物,作cDNA末端快速放大(RACE)及逆转录聚合酶链反应,生物信息学方法比较新序列,亚克隆IRF-3c至pcDNA3.1-flag,转染人胚胎肾上皮293细胞,用抗flag抗体作Western blot分析,用萤虫素酶功能分析的方法观测IRF-3c对病毒诱导的干扰素β启动子萤虫素酶活性的影响.结果发现了一种新的IRF3剪接异构体IRF-3c,IRF-3c用了紧接第六外显子后的180个第六内含子的碱基,新引入的最初第2、3、4个新碱基为一终止密码子,导致IRF-3c蛋白多肽终止于第327位.Western blot出现预期的相对分子质量为44 000的IRF-3c的蛋白强阳性条带.新的引用外显子序列可在小鼠的ETS表达库中找到同源序列,提示这种新的剪接体在生物演进中有其保守功能,IRF-3c萤虫素酶功能分析显示,该剪接体能抑制病毒诱导的干扰素β启动子活性至对照组的40%～50%.结论新的剪接体的发现为IRF-3这一重要分子的功能调节提供了新的线索,它是病毒感染通路中干扰素的显负性抑制剂,提示其功能为干扰素产生的精细调节成分,可防止干扰素的过度产生,其特异性序列可用于分析它们在人类疾病中的病理意义.

Objective Interferon regulatory factors 3 （IRF 3） is a key transcription factor to regulate gene expression of interferon after virus infection. This study aims to look for new spliced iso forms of IRF 3 and to investigate their structures and functions. Methods RNA extracts from human embryonic kidney 293 cells were amplified by RACE and RT PCR. New sequences were compared with published sequences of IRF 3 and murine EST database using bioinformatics method. A new se quence, IRF-3c, was subeloned into pcDNA3. 1-flag. The IRF-3c/pcDNA3.1-flag plasmid was transfected in HEK 293 cells. Whole cell extract was analysed by Western blot and then probed with monoclonal Flag antibody, Luciferase assay was carried out by co transfection with reporter plasmid, pGL2B with interferon β promoter, and IRF-3c cDNA expression plasmid. At 16 hours posttransfection, cells were infected with Sendal virus for 8 hours. Cells were collected and assayed for luciferase activity. Results A novel spliced isoform of IRF-3, named IRF-3c was discovered. The new isoform is almost the same as IRF 3. except for the utilization of the 180 bp bases in intron 6 adjacent to exon 6. The first 2,3 and 4 bases are a stop codon, which may produce a protein with a truncated C-terminal stoped at amino acids 327. Western blot analysis confirmed an expected 44 kDa strong band. The new inserted bases can be found in murine EST database, suggesting a conservative function in evolution. The functional luciferase assay showed that IRF 3c inhibited the IFNβ promoter activity toaround 40%-50% as that of control after Sendal virus infection. Conclusions The discovery of a new isoform of IRF-3 provides a new insight into the functional regulation of IRF-3 family. It is a dominant-negative inhibitor for interferon β3 promoter activity in the virus infection pathway, provides a mechanism for the fine-tuning of the virus-induced activation of the interferon response, and prevents interferon β from its overexpression and its toxic effects. It is worthwhile to explore the role of IRF-3c in the pathogenesis of human diseases using IRF-3c＇s specific sequence.