Genome-Wide Identification of Alternative Splice Forms Down-Regulated by Nonsense-Mediated mRNA Decay in <em>Drosophila</em>

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.可变mRNA剪接（alternative mRNA splicing）为黑腹果蝇（Drosophila melanogaster）内数千个基因的表达增添了一层调控层级。并非所有可变剪接均可产生功能性蛋白质；部分可变剪接会生成携带提前终止密码子的mRNA剪接异构体（mRNA isoforms），这类异构体可通过无义介导的mRNA降解（nonsense-mediated mRNA decay, NMD）通路被降解。可变剪接与NMD的这种耦合机制，为基因调控提供了一种在哺乳动物中高度保守的调控路径。NMD在黑腹果蝇中同样具有活性，但其对可变剪接形式谱的影响，以及其识别靶标的分子机制，此前均未明确。本研究采用定制剪接敏感型微阵列，在全基因组范围内定量检测可变mRNA加工与NMD对黑腹果蝇基因表达的影响。我们开发了一种全新算法，可基于微阵列检测数据，精准推断单个基因各mRNA剪接异构体的表达变化量。该方法可广泛适用于解读剪接敏感型微阵列与高通量测序数据。通过该分析策略，我们鉴定得到一组包含45个基因的高置信度基因集，NMD对这些基因的不同可变剪接异构体存在差异化调控作用，其中涵盖大量RNA结合蛋白与核糖体蛋白。可变剪接与NMD的协同作用会降低这类基因的表达水平，进而可能对其他基因的表达产生下游调控效应。受NMD调控的基因在翻译与有丝分裂过程中显著富集，这或许为此前观测到的NMD因子参与细胞周期进程的现象提供了机制层面的解释。本研究结果对于解析果蝇体内的NMD机制具有普遍的参考价值。最值得关注的是，我们发现相较于同一基因的非靶标异构体，NMD靶标mRNA的3'非翻译区（3' untranslated regions, UTRs）长度显著更长，这一发现支持了3'UTR长度在果蝇NMD靶标识别过程中发挥关键作用的观点。