RNA proximity labeling in log phase Caulobacter crescentus NA1000

APEX2 RNA proximity labeling is a powerful method for determining localized RNAs in vivo. APEX2 RNA proximity labeling was adapted to bacterial cells, using an APEX2 fusion to the core scaffold of BR-bodies, RNase E. As a control, APEX2 was also fused to a variant of RNase E that lacks its C-terminal IDR and is unable to form BR-bodies, RNase E delta CTD. RNA proximity labeling was performed, and we observed a similar pattern of enriched RNAs to density centrifugation isolated BR-bodies (Al-Husini et al. Mol Cell 2020). Overall design: Duplicate cultures of RNase E-APEX2 and RNase E(delta CTD)-APEX2 strains were growth to mid-log phase, and incubated with alkyne phenol for 30 minutes. The cells were incubated with H2O2 for 1 minute, and then harvested for RNA extraction. An aliquot of the lysate of each biological replicate was saved for normalization, and the proximity labled RNA was isolated by attaching a biotin-azide via click chemistry followed by purificaiton on streptavidin resin. Lysate RNA and proximity labeled RNA samples were then prepared for RNA-seq by the IU CGM core facility.

APEX2 RNA邻近标记（APEX2 RNA proximity labeling）是一种用于体内定位RNA的高效研究手段。本研究将该技术适配至细菌细胞，通过将APEX2与BR小体（BR-bodies）的核心支架蛋白RNase E融合构建实验体系。作为对照，APEX2还与缺失C端内在无序区域（intrinsically disordered region, IDR）且无法形成BR小体的RNase E变体（RNase E ΔCTD）进行了融合。完成RNA邻近标记实验后，我们观测到的富集RNA模式与密度离心分离得到的BR小体（Al-Husini等, 《分子细胞》（Mol Cell）, 2020）高度一致。
整体实验设计如下：将RNase E-APEX2与RNase E(ΔCTD)-APEX2菌株的重复培养物培养至对数中期，使用炔基苯酚（alkyne phenol）孵育30分钟。随后用H₂O₂处理细胞1分钟，收集细胞用于RNA提取。留存每份生物学重复的裂解液等分样本用于归一化校正，通过点击化学将生物素叠氮（biotin-azide）连接至经邻近标记的RNA，随后利用链霉亲和素树脂（streptavidin resin）完成纯化。裂解液总RNA与经邻近标记的RNA样本均由IU CGM核心实验室完成RNA测序建库流程。