Gao Jun-Li, Fan Yu-Jie, Wang Xiu-Ye, Zhang Yu, Pu Jia, Li Liang, Shao Wei, Zhan Shuai, Hao Jianjiang, Xu Yong-Zhen
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China;
Poochon Scientific, Frederick, Maryland 21704, USA.
Genes Dev. 2015 Apr 1;29(7):760-71. doi: 10.1101/gad.258863.115.
Unlike typical cis-splicing, trans-splicing joins exons from two separate transcripts to produce chimeric mRNA and has been detected in most eukaryotes. Trans-splicing in trypanosomes and nematodes has been characterized as a spliced leader RNA-facilitated reaction; in contrast, its mechanism in higher eukaryotes remains unclear. Here we investigate mod(mdg4), a classic trans-spliced gene in Drosophila, and report that two critical RNA sequences in the middle of the last 5' intron, TSA and TSB, promote trans-splicing of mod(mdg4). In TSA, a 13-nucleotide (nt) core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 small nuclear RNP (snRNP) through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB using the CRISPR/Cas9 system result in developmental defects in flies. Although it is not clear how the 5' intron finds the 3' introns, compensatory changes in U1 snRNA rescue trans-splicing of TSA mutants, demonstrating that U1 recruitment is critical to promote trans-splicing in vivo. Furthermore, TSA core-like motifs are found in many other trans-spliced Drosophila genes, including lola. These findings represent a novel mechanism of trans-splicing, in which RNA motifs in the 5' intron are sufficient to bring separate transcripts into close proximity to promote trans-splicing.
与典型的顺式剪接不同，反式剪接将来自两个独立转录本的外显子连接起来以产生嵌合mRNA，并且已在大多数真核生物中被检测到。锥虫和线虫中的反式剪接已被表征为一种剪接前导RNA促进的反应；相比之下，其在高等真核生物中的机制仍不清楚。在这里，我们研究了果蝇中一个经典的反式剪接基因mod(mdg4)，并报告了最后一个5'内含子中间的两个关键RNA序列TSA和TSB促进了mod(mdg4)的反式剪接。在TSA中，一个13个核苷酸（nt）的核心基序在果蝇物种中是保守的，对于反式剪接是必需且充分的，它通过与U1 snRNA的强碱基配对结合U1小核核糖核蛋白（snRNP）。在TSB中，一个保守的二级结构起到增强子的作用。使用CRISPR/Cas9系统删除TSA和TSB会导致果蝇出现发育缺陷。虽然尚不清楚5'内含子如何找到3'内含子，但U1 snRNA中的补偿性变化挽救了TSA突变体的反式剪接，表明U1的募集对于促进体内反式剪接至关重要。此外，在许多其他果蝇反式剪接基因中发现了类似TSA核心的基序，包括lola。这些发现代表了一种反式剪接的新机制，其中5'内含子中的RNA基序足以使独立的转录本紧密靠近以促进反式剪接。
