Division of Applied Chemistry, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan.
Division of Applied Chemistry, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, 2-16-1 Tokiwadai, Ube 755-8611, Japan; Research Center for Thermotolerant Microbial Resources, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8315, Japan; Yamaguchi University Biomedical Engineering Center, 2-16-1 Tokiwadai, Ube 755-8611, Japan.
Biochim Biophys Acta Gene Regul Mech. 2022 Jan;1865(1):194784. doi: 10.1016/j.bbagrm.2021.194784. Epub 2022 Jan 4.
Gene expression in eukaryotes is enhanced by the presence of introns in a process known as intron-mediated enhancement (IME), but its mechanism remains unclear. In Saccharomyces cerevisiae, sequences at the 5'-splice sites (SS) and branch point sites (BPS) are highly conserved compared with other higher eukaryotes. Here, the minimum intron sequence essential for IME was investigated using various short introns and a yeast codon-optimized luciferase gene as an IME model. Mutations at the 5'-SS conserved sequence and branch point in the QCR10 intron caused splicing deficiency with either a complete loss or a marked decrease in IME. By contrast, however, the 3'-AG to tG mutant was spliced and retained IME function. Moreover, heterologous introns, which did not show IME in S. cerevisiae, gained splicing competency and IME ability by substitutions to the S. cerevisiae-type 5'-SS and BPS sequences. Intriguingly, several deletion mutants between the 5'-SS and BPS in introns exhibited high levels of IME despite a loss in splicing competency. In most cases, further deletions or substitutions did not recover splicing competency and were found to decrease IME. However, a 16-nt variant consisting of the conserved 5'-SS and BPS sequences and 3'-CAG showed an IME level comparable with that of the wild-type intron. These results indicate that IME can be independent of splicing in S. cerevisiae while intron sequences at the 5'-SS and BPS play an essential role in IME.
真核生物的基因表达通过内含子的存在而增强，这个过程被称为内含子介导的增强（IME），但其机制尚不清楚。在酿酒酵母中，与其他高等真核生物相比，5'-剪接位点（SS）和分支点位点（BPS）的序列高度保守。在这里，使用各种短内含子和酵母密码子优化的荧光素酶基因作为 IME 模型，研究了 IME 所必需的最小内含子序列。QCR10 内含子 5'-SS 保守序列和分支点的突变导致剪接缺陷，无论是完全缺失还是显著减少 IME。相比之下，然而，3'-AG 到 tG 突变体被剪接并保留了 IME 功能。此外，在酿酒酵母中没有表现出 IME 的异源内含子通过取代酿酒酵母型 5'-SS 和 BPS 序列获得了剪接能力和 IME 能力。有趣的是，尽管丧失了剪接能力，但内含子 5'-SS 和 BPS 之间的几个缺失突变体仍表现出高水平的 IME。在大多数情况下，进一步的缺失或取代无法恢复剪接能力，并发现 IME 减少。然而，由保守的 5'-SS 和 BPS 序列和 3'-CAG 组成的 16-nt 变体表现出与野生型内含子相当的 IME 水平。这些结果表明，在酿酒酵母中 IME 可以独立于剪接，而 5'-SS 和 BPS 处的内含子序列在 IME 中发挥着重要作用。
