摘要：
哺乳动物中腺苷三磷酸结合盒转运蛋白(ATP-binding cassette transporter,ABCT)可通过可变剪切产生多种转录本,其中含有提前终止密码子(premature terminal codon,PTC)的转录本还可与无义介导的mRNA降解通路(nonsense-mediated mRNA decay,NMD)作用来调节蛋白的相关功能,但这些现象尚未在低等生物的ABCT研究中发现.该文以单细胞原生动物——嗜热四膜虫为对象,利用转录组数据发现ABCC10基因存在可变剪切,并产生两条转录本(SV1和SV2),其中SV2在第五个内含子处发生内含子保留事件,这段长49bp的序列使SV2发生移码并产生PTC.在构建NMD通路中关键因子UPF1基因的嗜热四膜虫敲降株的基础上,利用实时荧光定量PCR方法检测SV2的转录情况.结果显示:含有PTC的转录本SV2在UPF1敲降株中的转录水平相对于野生型显著增加,说明SV2可被NMD通路降解.这与高等动物中某些ABCC蛋白通过可变剪切引入含PTC转录本,并能被NMD降解的方式一致,推测该方式在真核生物中十分保守,并在真核生物的共同祖先(the last eukaryotic common ancestor)中就已形成.%ATP-binding cassette transporters (ABCT) could generate multiple transcripts through alternative splicing (AS) in mammalian. Some AS introduced PTC (premature terminal codon)-containing isoforms of ABCT couple with NMD (nonsense-mediated mRNA decay) to regulate relevant functions. However, there are no similar reports in lower organisms. This paper focuses on the unicellular protozoa Tetrahymena thermophila, based on the RNA-seq data of Tetrahymena thermophila, identified two alternative splicing variants of gene ABCC10 (SV1 and SV2). The SV2 contained an intron retention event at the fifth intron, and this 49 bp intron resulted in shift-frame and introduced PTC. Then, a knock-down Tetrahymena strain of gene UPF1 which is a key factor of NMD was constructed, and the expression levels of SV2 were performed using a real-time quantitative PCR. The results showed the expression levels of SV2 were up-regulated significantly in knock-down strain, indicating that SV2 was targeted by NMD, which is consistent to the mechanism which the AS introduced PTC-containing isoforms of ABCC proteins can be targeted by NMD in mammalian. Thus, we infer that this mechanism is highly evolutionary conserved in eukaryotes and was already functional in the last eukaryotic common ancestor.
