A conserved intronic U1 snRNP-binding sequence promotes trans-splicing in Drosophila.
A conserved intronic U1 snRNP-binding sequence promotes trans-splicing in Drosophila.
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保守的内含子 U1 snRNP 结合序列促进果蝇中的反式剪接
DOI：
10.1101/gad.258863.115
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发表时间：
2015-04-01
影响因子：
10.5
通讯作者：
Xu YZ
中科院分区：
文献类型：
--
作者：
Gao JL;Fan YJ;Wang XY;Zhang Y;Pu J;Li L;Shao W;Zhan S;Hao J;Xu YZ
Gao等研究了果蝇中经典的反式剪接基因mod（mdg 4），并报道了最后5′内含子中间的两个关键RNA序列TSA和TSB促进mod（mdg 4）的反式剪接。在TSA中，13-nt核心基序在果蝇物种中是保守的，并且对于反式剪接是必需的和足够的，其通过与U1 snRNA的强碱基配对结合U1 snRNP。在TSB中，保守的二级结构充当增强子。TSA和TSB的缺失导致果蝇的发育缺陷。与典型的顺式剪接不同，反式剪接将来自两个单独转录物的外显子连接以产生嵌合mRNA，并且已在大多数真核生物中检测到。在锥虫和线虫中，反式剪接被描述为一种剪接前导RNA促进的反应;相反，在高等真核生物中，其机制尚不清楚。我们研究了果蝇中一个经典的反式剪接基因mod（mdg 4），发现其5′端内含子中间的两个关键RNA序列TSA和TSB能够促进mod（mdg 4）的反式剪接。在TSA中，13个核苷酸（nt）的核心基序在果蝇物种中是保守的，并且对于反式剪接是必需的和足够的，其通过与U1 snRNA的强碱基配对结合U1小核RNP（snRNP）。在TSB中，保守的二级结构充当增强子。使用CRISPR/Cas9系统删除TSA和TSB会导致果蝇的发育缺陷。虽然还不清楚5′内含子如何找到3′内含子，但U1 snRNA的补偿性变化挽救了TSA突变体的反式剪接，表明U1募集对促进体内反式剪接至关重要。此外，TSA核心样基序在许多其他反式剪接的果蝇基因中发现，包括lola。这些发现代表了一种新的反式剪接机制，其中5′内含子中的RNA基序足以使单独的转录本靠近以促进反式剪接。
Gao et al. 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-nt core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 snRNP through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB result in developmental defects in flies. 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.
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