Enzyme-mediated proximity labeling reveals the co-translational targeting of DLGAP5 mRNA to the centrosome during mitosis

Subcellular RNA localization is a conserved mechanism in eukaryotic cells and plays critical roles in diverse physiological processes including cell proliferation, differentiation, and embryo development. Nevertheless, the characterization of centrosome-localized mRNAs remains under-explored due to technical difficulties. In this study, we utilize APEX2-mediated proximity labeling to map centrosome-proximal transcriptome, identifying DLGAP5 mRNA as a novel centrosome-localized transcript during mitosis. Using a combination of drug perturbation, truncation, deletion, and mutagenesis, we demonstrate that microtubule binding of nascent MBD1 polypeptide is required for centrosomal transport of DLGAP5 mRNA. Our data also reveals that mRNA targeting efficiency is tightly linked to the coding sequence length. Thus, our study provides a transcriptomic resource for future investigation of centrosome-localized RNAs, and sheds light on mechanisms underlying mRNA centrosomal localization. Overall design: MERR APEX-seq libraries were generated from the following two cell lines: (1) APEX2-PCNT-EGFP (Centrosome); (2) APEX2-NES (Cytosol). For each cell line, three biological replicates were performed. Total RNAs directly extracted from the cells were set as Input. RNAs labeled by centrosome- or cytosol-localized APEX2 were set as Enrich. RNAs enriched from negative controls omitting probes were set as Control.

RNA的亚细胞定位是真核细胞中的保守机制，在细胞增殖、分化、胚胎发育等多种生理过程中发挥关键作用。然而，受技术障碍制约，目前对中心体定位mRNA的表征仍有待深入探索。本研究利用APEX2介导的邻近标记技术绘制中心体邻近转录组图谱，鉴定出DLGAP5 mRNA为有丝分裂阶段一种新的中心体定位转录本。本研究结合药物扰动、截短、缺失与诱变实验，证实新生MBD1多肽与微管的结合是DLGAP5 mRNA完成中心体转运的必要条件。研究数据同时揭示，mRNA的靶向效率与编码序列长度紧密相关。综上，本研究为未来开展中心体定位RNA的相关研究提供了转录组学资源，并为阐明mRNA中心体定位的潜在机制提供了新视角。整体实验设计：本研究从以下两种细胞系中构建MERR APEX-seq文库：(1) APEX2-PCNT-EGFP（中心体组）；(2) APEX2-NES（细胞质组）。每种细胞系均设置3次生物学重复。直接从细胞中提取的总RNA设为Input样本；被中心体或细胞质定位的APEX2标记的RNA设为Enrich样本；从省略探针的阴性对照中富集得到的RNA设为Control样本。