Records of RNA locations in living yeast revealed through covalent marks

RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific locations, we developed a new strategy in which a uridine-adding enzyme is anchored to subcellular sites, where it directly and progressively marks RNAs with 3' terminal uridines. This Localized RNA Recording approach yields a record of RNA locations, is neither toxic nor limited by diffusion of chemical probes, and here is validated through identification of many mRNAs localized selectively to ER or mitochondria. We identify a broad dual localization pattern conserved from yeast to human cells, in which the same battery of mRNAs encounter both ER and mitochondria in both species, and include an mRNA encoding a key stress sensor. Subunits of many multiprotein complexes localize to both the ER and mitochondria indicating coordination of complex assembly. Non-coding RNAs in the course of RNA surveillance and processing encounter both organelles. By providing a record of RNA locations over time, the approach complements those that capture snapshots of instantaneous positions.RNA的动态变化与定位分布广泛存在于生命活动的全流程，覆盖胚胎发育至疾病发生的各个阶段。为实现特异性RNA定位，我们开发了一项全新策略：将尿苷添加酶（uridine-adding enzyme）锚定至亚细胞位点，使其在原位直接且逐步地对RNA进行3'端尿苷化标记。该定位RNA记录技术可留存RNA的位置信息，既无细胞毒性，也不受化学探针扩散的限制；本研究通过筛选特异性定位于内质网（Endoplasmic Reticulum, ER）或线粒体（mitochondria）的多种mRNA，验证了该方法的有效性。我们发现了一种从酵母到人类细胞均保守的广谱双重定位模式：在酵母与人类两个物种中，均有一组mRNA同时靶向内质网与线粒体，其中还包含编码关键应激感应蛋白的mRNA。多种多蛋白复合物的亚基同时定位于内质网与线粒体，这提示复合物的组装过程存在协同调控机制。在RNA监视与加工过程中的非编码RNA，同样会与这两类细胞器发生相互作用。该方法可随时间留存RNA的定位信息，能够弥补仅能捕获瞬时位置快照的技术短板。