Fekete Erzsébet, Ág Norbert, Ág-Rácz Viktória, Márton Alexandra, Sándor Erzsébet, Scazzocchio Claudio, Flipphi Michel, Karaffa Levente
Department of Biochemical Engineering, Faculty of Science and Technology, University of Debrecen, Debrecen, Hungary.
Juhász-Nagy Pál Doctoral School of Biology and Environmental Sciences, University of Debrecen, Debrecen, Hungary.
Microbiol Spectr. 2025 Sep 2;13(9):e0292624. doi: 10.1128/spectrum.02926-24. Epub 2025 Aug 8.
[D1,2] stwintrons consist of nested U2 introns, where an internal intron splits the 5'-donor of an external intron between its first and second nucleotide. Almost all stwintrons described to date are of the [D1,2] type, suggesting unique means for their duplication. Sequence-similar [D1,2] stwintrons are typically integrated at new intron positions, specific for one species. Two hundred eighty-eight sequence-similar [D1,2] stwintrons were identified in the genomes of 14 . Occasional missplicing was apparent, where almost the entire stwintron was excised as one canonical intron. Near-identical sister stwintrons were identified in sp. MSU SB201401 and that share near-terminal inverted repeat elements of 10-nt length named 5'-NTIRE-10 and 3'-NTIRE-10, which are complementary as RNA. Complementary NTIRE-10 partners were also present in three species. These NTIRE-10s can form a near-terminal double-stranded RNA stem structure that brings in close proximity the terminal G's of the [D1,2] stwintron and of its alternative misspliced intron. The exact folding of the interior stwintron RNA appears irrelevant. Ten of the stwintrons with complementary NTIRE-10s are present in sp. MSU SB201401, implying that [D1,2] stwintron duplication occurs frequently in this species.IMPORTANCESpliceosomal introns are excised from pre-mRNAs by a ribonucleoprotein complex, the U2 spliceosome. Excision does not necessarily occur by one splicing reaction. We had identified and validated intronic sequences in fungi that consist of two nested U2 introns and called them spliceosomal twin introns (stwintrons). In this bioinformatics-based study, we identified and validated almost 300 [D1,2] stwintrons with sequence similarity in the genomes of 14 species in the order of . Thirty-seven of them feature small, fully complementary RNA inverted repeat elements. These elements form a near-terminal RNA stem structure, bringing the terminal G's of the stwintron in close proximity. We further demonstrated the existence of an alternative splicing pattern involving one excision event that removes the two constituent introns from the transcript together. This work contributes to the understanding of mechanisms of stwintron gain in fungi.
[D1,2] 型双内含子由嵌套的U2内含子组成，其中一个内部内含子在外部内含子的5'-供体的第一个和第二个核苷酸之间将其切开。迄今为止描述的几乎所有双内含子都是 [D1,2] 型，这表明它们具有独特的复制方式。序列相似的 [D1,2] 型双内含子通常整合到一个物种特有的新内含子位置。在14种生物的基因组中鉴定出288个序列相似的 [D1,2] 型双内含子。偶尔会出现明显的错配剪接，几乎整个双内含子作为一个典型内含子被切除。在稻瘟病菌株MSU SB201401和另一种菌株中鉴定出近乎相同的姐妹双内含子，它们共享长度为10个核苷酸的近末端反向重复元件，称为5'-NTIRE-10和3'-NTIRE-10，作为RNA时它们是互补的。互补的NTIRE-10对在三种稻瘟病菌株中也存在。这些NTIRE-10可以形成一个近末端双链RNA茎结构，使 [D1,2] 型双内含子及其可变错配剪接内含子的末端G紧密靠近。内部双内含子RNA的确切折叠似乎无关紧要。在稻瘟病菌株MSU SB201401中存在10个带有互补NTIRE-10的双内含子，这意味着 [D1,2] 型双内含子在该物种中频繁发生复制。
重要性
剪接体内含子通过核糖核蛋白复合物U2剪接体从前体mRNA中切除。切除不一定通过一次剪接反应发生。我们在真菌中鉴定并验证了由两个嵌套的U2内含子组成的内含子序列，并将它们称为剪接体双内含子（双内含子）。在这项基于生物信息学的研究中，我们在稻瘟菌目14个物种的基因组中鉴定并验证了近300个具有序列相似性的 [D1,2] 型双内含子。其中37个具有小的、完全互补的RNA反向重复元件。这些元件形成一个近末端RNA茎结构，使双内含子的末端G紧密靠近。我们进一步证明了存在一种可变剪接模式，涉及一个切除事件，该事件将两个组成内含子一起从转录本中去除。这项工作有助于理解真菌中双内含子获得的机制。
