Extensive location bias of the GPCR-dependent translatome via site-selective activation of mTOR. Extensive location bias of the GPCR-dependent translatome via site-selective activation of mTOR

G protein-coupled receptors (GPCRs) modulate various physiological functions by re-wiring cellular gene expression in response to extracellular signals. Control of gene expression by GPCRs has been studied almost exclusively at the transcriptional level, neglecting an extensive amount of regulation that takes place translationally. Hence, little is known about the nature and mechanisms of gene-specific post-transcriptional regulation downstream of receptor activation. Here, we apply an unbiased multiomics approach to delineate an extensive translational regulatory program initiated by the prototypical beta2-adrenergic receptor (β2-AR) and provide mechanistic insights into how these processes are orchestrated. Using ribosome profiling (Ribo-seq), we identify nearly 120 novel gene targets of adrenergic receptor activity for which expression is exclusively regulated at the level of translation. We next show that all translational changes are induced selectively by endosomal β2-ARs and report that this proceeds through activation of the mammalian target of rapamycin (mTOR) pathway. Specifically, within the set of translational GPCR targets we discover significant enrichment of genes with 5' terminal oligopyrimidine (TOP) motifs, a gene class classically known to be translationally regulated by mTOR. We then demonstrate that endosomal β2-ARs are required for mTOR activation and subsequent mTOR-dependent TOP mRNA translation. This site-selective crosstalk between the pathways is observed in multiple cell models with native β2-ARs, across a range of endogenous and synthetic adrenergic agonists, and for other GPCRs with intracellular activity. Together, this comprehensive analysis of drug-induced translational regulation establishes a critical role for location-biased GPCR signaling in fine-tuning the cellular protein landscape. Overall design: To investigate transcription and translation responses downstream of β2-AR, we conducted parallel RNA-seq and Ribo-seq in HEK293 cells. Cells were incubated with DMSO or Dyngo (dynamin inhibitor) for 20 minutes, then stimluated with 1uM Isoproterenol. RNAseq samples were isolated through standard library preparation. Ribo-seq samples were prepared by isolating ribosome-protected-fragments and small RNA library preparation

G蛋白偶联受体（G protein-coupled receptors, GPCRs）可通过重塑细胞基因表达调控网络以响应胞外信号，进而调控多种生理功能。目前针对GPCRs介导的基因表达调控的研究几乎仅局限于转录层面，却忽略了大量发生在翻译层面的调控过程，因此人们对受体激活下游基因特异性转录后调控的本质与调控机制仍知之甚少。
本研究采用无偏多组学策略，勾勒了经典型β2肾上腺素能受体（β2-adrenergic receptor, β2-AR）介导的广泛翻译调控程序，并阐明了这些过程的协同调控机制。
利用核糖体谱（ribosome profiling, Ribo-seq）技术，我们鉴定出近120个肾上腺素能受体活性相关的新型基因靶点，这些靶点的表达仅在翻译层面受到调控。后续研究发现，所有翻译层面的表达变化均由内体定位的β2-AR选择性诱导，且该过程通过激活哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）通路得以实现。
具体而言，在GPCR翻译调控靶点集合中，我们发现携带5'端寡嘧啶（5' terminal oligopyrimidine, TOP）基序的基因显著富集，这类基因经典上被认为可通过mTOR通路实现翻译调控。我们进一步证实，内体定位的β2-AR是mTOR激活以及后续mTOR依赖型TOP mRNA翻译的必要条件。这种通路间的位点选择性串扰现象，在多种表达天然β2-AR的细胞模型、一系列内源性及合成型肾上腺素能激动剂处理的样本中，以及具有细胞内活性的其他GPCRs中均得到验证。
综上，这项针对药物诱导翻译调控的全面分析，阐明了定位偏向性GPCR信号通路在精细调控细胞蛋白质组景观中的关键作用。
整体实验设计：为探究β2-AR下游的转录与翻译响应，我们在HEK293细胞中并行开展了RNA测序（RNA-seq）与核糖体谱测序（Ribo-seq）实验。将细胞先用二甲基亚砜（DMSO）或动力蛋白抑制剂Dyngo预处理20分钟，随后用1μM异丙肾上腺素（Isoproterenol）进行刺激。RNA-seq样本通过标准文库制备流程完成分离与建库；Ribo-seq样本则通过分离核糖体保护片段并进行小RNA文库制备完成。