Proximity-specific ribosome profiling reveals the logic of localized mitochondrial translation

Localized translation broadly enables spatiotemporal control of gene expression. Here we present LOCL-TL (LOV-domain-Controlled Ligase for Translation Localization), an optogenetic approach for monitoring translation with codon resolution at any defined subcellular location under physiological conditions. Application of LOCL-TL to mitochondrially-localized translation revealed that ~20% of human nuclear-encoded mitochondrial genes are translated on the outer mitochondrial membrane (OMM). Mitochondrially-translated messages form two classes distinguished by encoded protein length, recruitment mechanism, and cellular function. An evolutionarily ancient mechanism allows nascent chains to drive cotranslational recruitment of long proteins via an unanticipated bipartite targeting signal. Conversely, mRNAs of short proteins, especially eukaryotic origin electron transport chain (ETC) components, are specifically recruited by the OMM protein A-Kinase Anchoring Protein 1 (AKAP1), in a translation-independent manner that depends on mRNA splicing. AKAP1 loss lowers ETC levels. LOCL-TL thus reveals a hierarchical strategy that enables preferential translation of a subset of proteins on the OMM. Overall design: Ribosome profiling data were generated from cell lines engineered with LOCL-TL components. Two replicates were used for each group: 1) ER-LOV-BirA and 2) Mito-LOV-BirA. RNA-seq data were collected from cell lines with LOCL-TL components and LOV-BirA targeted to the mitochondria in replicates. RNA-seq data were also collected from cell lines with perturbations on AKAP1, LARP4, or negative control gRNA.

定位翻译可广泛实现基因表达的时空调控。本研究报道了LOCL-TL（LOV结构域控制的翻译定位连接酶，LOV-domain-Controlled Ligase for Translation Localization），这是一种光遗传学方法，能够在生理条件下于任意指定亚细胞位点以密码子分辨率监测翻译过程。

将LOCL-TL应用于线粒体定位的翻译研究后发现，约20%的人类核编码线粒体基因在线粒体外膜（outer mitochondrial membrane, OMM）上完成翻译。

线粒体翻译的转录本可分为两类，其分类依据为编码蛋白长度、招募机制与细胞功能。

一种进化上古老的机制可通过未被预见的双靶向信号，引导新生肽链完成长蛋白的共翻译招募。

与之相反，短蛋白的mRNA（尤其是真核起源的电子传递链（electron transport chain, ETC）组分）可通过线粒体外膜蛋白A激酶锚定蛋白1（A-Kinase Anchoring Protein 1, AKAP1）实现特异性招募，该过程以翻译非依赖的方式进行，且依赖mRNA剪接。

AKAP1缺失会降低电子传递链复合物的水平。

综上，LOCL-TL揭示了一种层级化策略，可实现线粒体外膜上特定蛋白子集的偏好性翻译。

实验设计：核糖体谱分析（ribosome profiling）数据来自经LOCL-TL元件工程改造的细胞系。每组设置2次生物学重复：1）ER-LOV-BirA；2）Mito-LOV-BirA。RNA测序（RNA-seq）数据采集自携带LOCL-TL元件且靶向线粒体的LOV-BirA细胞系，设置重复样本。同时收集了经AKAP1、LARP4扰动或阴性对照向导RNA（guide RNA, gRNA）处理的细胞系的RNA测序数据。