Carnegie Institution for Science, Department of Embryology, Baltimore, MD.
Horace Mann School, The Bronx, NY.
J Cell Biol. 2021 Jan 4;220(1). doi: 10.1083/jcb.202002129.
The nuclear lamina (NL) is a meshwork found beneath the inner nuclear membrane. The study of the NL is hindered by the insolubility of the meshwork and has driven the development of proximity ligation methods to identify the NL-associated/proximal proteins, RNA, and DNA. To simplify and improve temporal labeling, we fused APEX2 to the NL protein lamin-B1 to map proteins, RNA, and DNA. The identified NL-interacting/proximal RNAs show a long 3' UTR bias, a finding consistent with an observed bias toward longer 3' UTRs in genes deregulated in lamin-null cells. A C-rich motif was identified in these 3' UTR. Our APEX2-based proteomics identifies a C-rich motif binding regulatory protein that exhibits altered localization in lamin-null cells. Finally, we use APEX2 to map lamina-associated domains (LADs) during the cell cycle and uncover short, H3K27me3-rich variable LADs. Thus, the APEX2-based tools presented here permit identification of proteomes, transcriptomes, and genome elements associated with or proximal to the NL.
核层（NL）是位于内核膜下的网格状结构。由于 NL 网络的不溶性，对其进行研究受到阻碍，这促使人们开发了接近连接方法来鉴定 NL 相关/邻近的蛋白质、RNA 和 DNA。为了简化和改善时间标记，我们将 APEX2 融合到 NL 蛋白 lamin-B1 上，以绘制蛋白质、RNA 和 DNA 的图谱。鉴定出的 NL 相互作用/邻近的 RNA 显示出长 3' UTR 偏倚，这一发现与在 lamin 缺失细胞中失调的基因中观察到的较长 3' UTR 偏倚一致。在这些 3' UTR 中发现了一个富含 C 的基序。我们基于 APEX2 的蛋白质组学鉴定了一个富含 C 的基序结合调节蛋白，该蛋白在 lamin 缺失细胞中的定位发生改变。最后，我们使用 APEX2 在细胞周期中绘制与核层相关的结构域（LAD），并揭示了富含 H3K27me3 的短可变 LAD。因此，这里介绍的基于 APEX2 的工具可以鉴定与 NL 相关或邻近的蛋白质组、转录组和基因组元件。
