Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved AS-NMD pathways

Background: Alternative splicing (AS), which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing AS pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. Results: RNA immunoprecipitation in tandem (RIPiT) of exon junction complex (EJC) components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compared EJC RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our EJC RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identified thousands of previously unannotated splicing events; while many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis. Conclusions: Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the NMD-linked AS pathway predominates. EJC RIPiT-Seq also enabled identification of numerous conserved but previously unknown AS-NMD events. Overall design: We created and sequenced rRNA-depleted whole cell RNA-Seq libraries (84-93 million mate pairs each) wherein the captured fragments were of similar length (220-500 nts) to our previously published EJC RIPiT-Seq libraries (GEO: GSE115788). The new RNA-Seq libraries were generated from cultures (three biological replicates, two technical replicates each) that were (+) or were not (–) subjected to a one hour pre-treatment with harringtonine. Technical replicates were later combined.

背景：可变剪接（Alternative splicing, AS）可从单个基因产生多种mRNA同工型，对真核基因表达的调控至关重要。然而，传统RNA测序（RNA-Seq）无法测定竞争性可变剪接通路的通量，因为不同mRNA同工型的降解速率差异极大。事实上，部分半衰期极短的mRNA同工型（例如受翻译依赖型无义介导降解（translation-dependent nonsense-mediated decay, NMD）调控的可变剪接相关无义介导降解（AS-NMD）产物），即便在最全面的RNA-Seq分析中也可能被完全遗漏。结果：对外显子连接复合物（exon junction complex, EJC）组分进行串联RNA免疫共沉淀（RNA immunoprecipitation in tandem, RIPiT），可纯化尚未经历翻译的剪接后mRNA-蛋白质颗粒（pre-translational mRNPs），从而规避翻译依赖型mRNA降解的干扰。本研究将EJC RIPiT测序（RIPiT-Seq）与HEK293细胞的全细胞RNA-Seq数据进行了对比。正如预期，已知AS-NMD通路的通量远高于RNA-Seq所捕获的水平。我们通过EJC RIPiT-Seq还明确证实，剪接机器本身无法识别阅读框。本研究鉴定了数千个此前未被注释的剪接事件：其中多数可归因于“剪接噪声”，其余则为进化保守的事件，可产生新型AS-NMD同工型，可能参与蛋白质稳态的维持。其中部分事件发生在过表达与癌症不良预后相关的基因中。结论：对剪接后、翻译前的mRNPs中的RNA进行深度测序，可在不受差异化mRNA降解干扰的前提下鉴定并定量剪接事件。对于多数已知的AS-NMD靶标而言，与NMD相关的可变剪接通路占主导地位。EJC RIPiT-Seq还可鉴定出大量保守但此前未被发现的AS-NMD事件。整体实验设计：本研究构建并测序了核糖体RNA去除型全细胞RNA-Seq文库（每个文库包含8400万至9300万对双端测序读段），其捕获的片段长度（220~500 nt）与本团队此前发表的EJC RIPiT-Seq文库（GEO: GSE115788）一致。新的RNA-Seq文库来自经（+）或未经（–）长梗霉素（harringtonine）预处理1小时的细胞培养物，设置3次生物学重复，每个生物学重复包含2次技术重复，后续将技术重复的数据合并分析。