Hwang Hun-Way, Saito Yuhki, Park Christopher Y, Blachère Nathalie E, Tajima Yoko, Fak John J, Zucker-Scharff Ilana, Darnell Robert B
Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA; Department of Pathology, University of Pittsburgh, School of Medicine, 3550 Terrace Street, Pittsburgh, PA 15213, USA.
Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA.
Neuron. 2017 Sep 13;95(6):1334-1349.e5. doi: 10.1016/j.neuron.2017.08.024.
Alternative polyadenylation (APA) is increasingly recognized to regulate gene expression across different cell types, but obtaining APA maps from individual cell types typically requires prior purification, a stressful procedure that can itself alter cellular states. Here, we describe a new platform, cTag-PAPERCLIP, that generates APA profiles from single cell populations in intact tissues; cTag-PAPERCLIP requires no tissue dissociation and preserves transcripts in native states. Applying cTag-PAPERCLIP to profile four major cell types in the mouse brain revealed common APA preferences between excitatory and inhibitory neurons distinct from astrocytes and microglia, regulated in part by neuron-specific RNA-binding proteins NOVA2 and PTBP2. We further identified a role of APA in switching Araf protein isoforms during microglia activation, impacting production of downstream inflammatory cytokines. Our results demonstrate the broad applicability of cTag-PAPERCLIP and a previously undiscovered role of APA in contributing to protein diversity between different cell types and cellular states within the brain.
可变聚腺苷酸化（APA）在调节不同细胞类型的基因表达方面日益受到认可，但从单个细胞类型获取APA图谱通常需要事先纯化，这是一个压力较大的过程，其本身可能会改变细胞状态。在此，我们描述了一种新平台cTag-PAPERCLIP，它能从完整组织中的单细胞群体生成APA图谱；cTag-PAPERCLIP无需组织解离，并能保持转录本的天然状态。将cTag-PAPERCLIP应用于分析小鼠大脑中的四种主要细胞类型，发现兴奋性神经元和抑制性神经元之间存在与星形胶质细胞和小胶质细胞不同的常见APA偏好，这部分受神经元特异性RNA结合蛋白NOVA2和PTBP2的调节。我们进一步确定了APA在小胶质细胞激活过程中切换Araf蛋白异构体方面的作用，影响下游炎性细胞因子的产生。我们的结果证明了cTag-PAPERCLIP的广泛适用性，以及APA在促成大脑中不同细胞类型和细胞状态之间蛋白质多样性方面以前未被发现的作用。
