Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing-defective genes.
Splice-site mutations cause Rrp6-mediated nuclear retention of the unspliced RNAs and transcriptional down-regulation of the splicing-defective genes.
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DOI：
10.1371/journal.pone.0011540
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发表时间：
2010-07-12
期刊：
影响因子：
3.7
通讯作者：
Visa N
中科院分区：
文献类型：
--
作者：
Eberle AB;Hessle V;Helbig R;Dantoft W;Gimber N;Visa N
真核细胞已经发展出监视机制来防止异常转录本的表达。早期监视检查点作用于转录位点，防止携带加工缺陷的 mRNA 释放。酿酒酵母中的这个检查点需要外泌体亚基 Rrp6，但尚不清楚 Rrp6 是否也在高等真核生物的 mRNA 监视中发挥作用。我们开发了一种体内系统来研究果蝇核 mRNA 监测。我们已经生产了表达人类 β-珠蛋白基因的 S2 细胞，该基因的内含子 2 中的剪接位点发生突变（mut β-珠蛋白）。 mut β-珠蛋白基因编码的转录本通常在内含子 1 处剪接，但保留内含子 2。mut β-珠蛋白转录本的水平远低于从同一启动子转录的野生型 (wt) β-珠蛋白 mRNA 的水平。我们比较了 mut 和 wt β-珠蛋白基因的表达，以研究下调缺陷 mRNA 产生的机制。 wt 和 mut β-珠蛋白转录物均在 3' 处进行加工，但 mut β-珠蛋白转录物的切割效率低于 wt 转录物。此外，如 FISH 所示，mut β-珠蛋白转录物从转录位点释放的效率较低，并且通过 RNAi 耗尽 Rrp6 可以恢复这种缺陷。此外，测量 RNA 聚合酶 II 与转录基因关联的 ChIP 实验表明，mut β-珠蛋白基因的转录显着受损。我们还表明，mut β-珠蛋白基因显示 H3K4me3 水平降低。我们的结果表明，在昆虫细胞以及可能在所有后生动物中，至少有两种监视反应以共转录方式发挥作用。一种反应需要 Rrp6，并导致缺陷 mRNA 从转录位点无效释放。另一种反应在转录水平发挥作用，通过涉及组蛋白修饰的机制减少有缺陷的转录本的合成。
Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes. We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human β-globin gene with mutated splice sites in intron 2 (mut β-globin). The transcripts encoded by the mut β-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut β-globin transcripts are much lower than those of wild type (wt) ß-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt β-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut β-globin transcripts are processed at the 3′, but the mut β-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut β-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut β-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut β-globin gene shows reduced levels of H3K4me3. Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The other response acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications.
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