Lin Jin-Huan, Wu Hao, Zou Wen-Bin, Masson Emmanuelle, Fichou Yann, Le Gac Gerald, Cooper David N, Férec Claude, Liao Zhuan, Chen Jian-Min
Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai, China.
Shanghai Institute of Pancreatic Diseases, Shanghai, China.
Front Genet. 2021 Aug 5;12:701652. doi: 10.3389/fgene.2021.701652. eCollection 2021.
Combining data derived from a meta-analysis of human disease-associated 5' splice site GT>GC (i.e., +2T>C) variants and a cell culture-based full-length gene splicing assay (FLGSA) of forward engineered +2T>C substitutions, we recently estimated that ∼15-18% of +2T>C variants can generate up to 84% wild-type transcripts relative to their wild-type counterparts. Herein, we analyzed the splicing outcomes of 20 +2T>C variants that generate some wild-type transcripts in two minigene assays. We found a high discordance rate in terms of the generation of wild-type transcripts, not only between FLGSA and the minigene assays but also between the different minigene assays. In the pET01 context, all 20 wild-type minigene constructs generated the expected wild-type transcripts; of the 20 corresponding variant minigene constructs, 14 (70%) generated wild-type transcripts. In the pSPL3 context, only 18 of the 20 wild-type minigene constructs generated the expected wild-type transcripts whereas 8 of the 18 (44%) corresponding variant minigene constructs generated wild-type transcripts. Thus, in the context of a particular type of variant, we raise awareness of the limitations of minigene splicing assays and emphasize the importance of sequence context in regulating splicing. Whether or not our findings apply to other types of splice-altering variant remains to be investigated.
结合从人类疾病相关的5'剪接位点GT>GC（即+2T>C）变体的荟萃分析中获得的数据，以及对正向工程化的+2T>C替代进行的基于细胞培养的全长基因剪接分析（FLGSA），我们最近估计，相对于野生型对应物，约15 - 18%的+2T>C变体可产生高达84%的野生型转录本。在此，我们分析了20个+2T>C变体在两种小基因分析中产生一些野生型转录本的剪接结果。我们发现，不仅在FLGSA和小基因分析之间，而且在不同的小基因分析之间，野生型转录本的产生率存在高度不一致。在pET01背景下，所有20个野生型小基因构建体都产生了预期的野生型转录本；在20个相应的变体小基因构建体中，有14个（70%）产生了野生型转录本。在pSPL3背景下，20个野生型小基因构建体中只有18个产生了预期的野生型转录本，而18个相应的变体小基因构建体中有8个（44%）产生了野生型转录本。因此，在特定类型变体的背景下，我们提高了对小基因剪接分析局限性的认识，并强调了序列背景在调节剪接中的重要性。我们的发现是否适用于其他类型的剪接改变变体仍有待研究。
