Co-translational targeting of transcripts to endosomes

Asymmetric localization and translation of mRNAs is used by single cells to sense their environment and integrate extrinsic cues with the appropriate cellular response. Here we investigate the extent to which endosomes impact subcellular patterning of transcripts and provide a platform for localized translation. Using image-based transcriptomics, indirect immunofluorescence, and RNAseq of isolated organelles, we discover mRNAs that associate with early endosomes in a translation-dependent and -independent manner. We explore this in more detail for the mRNA of a major endosomal tethering factor and fusogen, Early Endosomal Antigen 1, EEA1, which localises to early endosomes in a puromycin-sensitive manner. By reconstituting EEA1 knock-out cells with either the coding sequence or 3’UTR of EEA1, we show that the coding region is sufficient for endosomal localisation of mRNA. Finally, we use quantitative proteomics to discover proteins associated with EEA1 mRNA and identify CSRP1 as a factor that controls EEA1 translational efficiency. Our findings reveal that multiple transcripts associate with early endosomes in a translation-dependent manner and identify mRNA-binding proteins that may participate in controlling endosome-localised translation.

单细胞通过非对称的mRNA定位与翻译，感知其环境并整合外部信号与相应的细胞反应。本研究旨在探究内体对转录本亚细胞模式形成及局部翻译平台的影响。通过基于图像的转录组学、间接免疫荧光和分离器官的RNA测序技术，我们发现了在翻译依赖性和非依赖性方式下与早期内体关联的mRNA。我们进一步详细研究了主要内体连接因子和融合蛋白早期内体抗原1（EEA1）的mRNA，EEA1以嘌呤霉素敏感的方式定位于早期内体。通过重构EEA1敲除细胞，使用EEA1的编码序列或3'非翻译区（3'UTR），我们证实编码区足以实现mRNA在内体的定位。最终，我们运用定量蛋白质组学技术发现与EEA1 mRNA关联的蛋白质，并鉴定CSRP1为调控EEA1翻译效率的因素。我们的研究揭示了多个转录本以翻译依赖性方式与早期内体关联，并确定了可能参与调控内体局部翻译的mRNA结合蛋白。