Ding Wenyong, Lin Lin, Ren Feng, Zou Hanfa, Duan Ziyuan, Dai Jianwu
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China.
J Genet Genomics. 2009 Aug;36(8):475-82. doi: 10.1016/S1673-8527(08)60137-X.
In the alternative splicing, intron retention, of histamine H(3) receptors in rats and mice, the short transcript isoforms that are excised alternatively spliced introns are easily detected in a very low level in rats and are undetectable in mice using the regular PCR protocol. The retained introns have common 5' splice site and different 3' splice sites. The detailed mechanism for the special alternative splicing remains largely unclear. In this study, we developed a minigene splicing system to recapitulate natural alternative splicing of the receptors and investigated the effects of 5' and 3' splice sites on intron retention in HeLa cells. Mutating weak 5' and 3' splice sites of the alternatively spliced introns toward the canonical consensus sequences promoted the splicing of the corresponding introns in rat and mouse minigenes. The effect of splice site strength was context-dependent and much more significant for the 3' splice site of the longer alternative intron than for the 3' splice site of the shorter alternative intron and the common 5' splice sites; it was also more significant in the rat minigene than in the mouse minigene. Mutating the 3' splice site of the longer alternative intron resulted in almost complete splicing of the intron and made the corresponding isoform to become the nearly exclusive transcript in the rat minigene.
在大鼠和小鼠组胺H(3)受体的可变剪接（内含子保留）中，通过常规PCR方法，切除可变剪接内含子的短转录本异构体在大鼠中仅能以极低水平被检测到，而在小鼠中则无法检测到。保留的内含子具有共同的5'剪接位点和不同的3'剪接位点。这种特殊可变剪接的详细机制在很大程度上仍不清楚。在本研究中，我们开发了一个微型基因剪接系统来重现受体的自然可变剪接，并研究了5'和3'剪接位点对HeLa细胞内含子保留的影响。将可变剪接内含子的弱5'和3'剪接位点突变为经典的共有序列，促进了大鼠和小鼠微型基因中相应内含子的剪接。剪接位点强度的影响取决于上下文，对于较长可变内含子的3'剪接位点而言，其影响比对较短可变内含子的3'剪接位点和共同的5'剪接位点更为显著；在大鼠微型基因中的影响也比在小鼠微型基因中更为显著。突变较长可变内含子的3'剪接位点导致该内含子几乎完全剪接，并使相应的异构体在大鼠微型基因中成为几乎唯一的转录本。
