Genome-wide identification of alternative splice forms down-regulated by nonsense-mediated mRNA decay in Drosophila.
Genome-wide identification of alternative splice forms down-regulated by nonsense-mediated mRNA decay in Drosophila.
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DOI：
10.1371/journal.pgen.1000525
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发表时间：
2009-06
期刊：
影响因子：
4.5
通讯作者：
Brenner SE
中科院分区：
文献类型：
--
作者：
Hansen KD;Lareau LF;Blanchette M;Green RE;Meng Q;Rehwinkel J;Gallusser FL;Izaurralde E;Rio DC;Dudoit S;Brenner SE
选择性mRNA剪接为果蝇中数千个基因的表达增加了一层调控。并不是所有的选择性剪接都能产生功能性蛋白质;它也可以产生具有提前终止密码子的mRNA同种型，这些终止密码子通过无义介导的mRNA衰变（NMD）途径降解。选择性剪接和NMD的这种偶联提供了在哺乳动物中高度保守的基因调控机制。NMD在果蝇中也很活跃，但它对选择性剪接形式库的影响一直是未知的，它识别靶点的机制也是未知的。在这里，我们采用了一个定制的剪接敏感的微阵列，全球范围内测量替代mRNA加工和NMD对果蝇基因表达的影响。我们已经开发了一种新的算法来推断基于微阵列测量的基因的每个mRNA异构体的表达变化。这种方法是通用的解释剪接敏感的微阵列和高通量的序列数据。使用这种方法，我们已经确定了一个高置信度的45个基因，其中NMD对不同的替代亚型，包括许多RNA结合和核糖体蛋白质有不同的影响。偶联的选择性剪接和NMD降低这些基因的表达，这反过来可能对其他基因的表达产生下游影响。NMD影响的基因在翻译和有丝分裂中的作用是丰富的，这可能是先前观察到的NMD因子在细胞周期进展中的作用的基础。我们的研究结果具有普遍意义的理解NMD机制的苍蝇。最值得注意的是，我们发现NMD靶mRNA的3′非翻译区（UTR）明显长于相同基因的非靶亚型，这支持了3′ UTR长度在果蝇识别NMD靶中的作用。一个基因可以通过选择性剪接加工成多个mRNA。选择性剪接增加了基因组编码的蛋白质数量，但并非所有选择性mRNA都产生蛋白质。相反，一些被无义介导的mRNA衰变（NMD）降解，这是一种最初被确定为清除细胞中具有无义或终止密码子突变的mRNA的监视系统。引入早期终止密码子的选择性剪接将导致NMD，为细胞在基因转录后下调基因表达提供了一种方法。在本文中，我们已经开发了一种新的分析方法，研究选择性剪接和降解的果蝇基因芯片的联合作用。我们已经发现了一组严格定义的45个基因，它们可以被剪接成编码蛋白质的mRNA或被NMD降解的mRNA，从而下调整体基因表达。受影响的基因包括一些对细胞调控过程至关重要的基因，包括翻译、RNA剪接和细胞周期进程。我们的研究结果还有助于阐明NMD如何确定终止密码子是否过早，从而确定是否靶向mRNA进行降解。
Alternative mRNA splicing adds a layer of regulation to the expression of thousands of genes in Drosophila melanogaster. Not all alternative splicing results in functional protein; it can also yield mRNA isoforms with premature stop codons that are degraded by the nonsense-mediated mRNA decay (NMD) pathway. This coupling of alternative splicing and NMD provides a mechanism for gene regulation that is highly conserved in mammals. NMD is also active in Drosophila, but its effect on the repertoire of alternative splice forms has been unknown, as has the mechanism by which it recognizes targets. Here, we have employed a custom splicing-sensitive microarray to globally measure the effect of alternative mRNA processing and NMD on Drosophila gene expression. We have developed a new algorithm to infer the expression change of each mRNA isoform of a gene based on the microarray measurements. This method is of general utility for interpreting splicing-sensitive microarrays and high-throughput sequence data. Using this approach, we have identified a high-confidence set of 45 genes where NMD has a differential effect on distinct alternative isoforms, including numerous RNA–binding and ribosomal proteins. Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes. The NMD–affected genes are enriched for roles in translation and mitosis, perhaps underlying the previously observed role of NMD factors in cell cycle progression. Our results have general implications for understanding the NMD mechanism in fly. Most notably, we found that the NMD–target mRNAs had significantly longer 3′ untranslated regions (UTRs) than the nontarget isoforms of the same genes, supporting a role for 3′ UTR length in the recognition of NMD targets in fly. A gene can be processed into multiple mRNAs through alternative splicing. Alternative splicing increases the number of proteins encoded by the genome, but not all alternative mRNAs produce protein. Instead, some are degraded by nonsense-mediated mRNA decay (NMD), a surveillance system that was originally identified as a means of clearing the cell of mRNAs with nonsense, or stop codon, mutations. Alternative splicing that introduces early stop codons will lead to NMD, offering a way for the cell to down-regulate gene expression after a gene has been transcribed. In this paper, we have developed a new analysis method to study the combined effect of alternative splicing and degradation in the fruit fly Drosophila melanogaster using microarrays. We have found a stringently defined set of 45 genes that can be spliced either into an mRNA that encodes a protein or into an mRNA that is degraded by NMD, down-regulating the overall gene expression. The affected genes include a number that are central to the cell's regulatory processes, including translation, RNA splicing, and cell cycle progression. Our results also help shed light on how NMD determines whether a stop codon is premature, and thus whether to target an mRNA for degradation.
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作者：
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