拓扑异构酶 I (TOP1) 是一种重要的酶，可松弛 DNA 以防止和消除转录过程中的扭转应力。然而，TOP1 活性调节的潜在机制仍然难以捉摸。使用增强交联和免疫沉淀 (eCLIP) 和紫外交联 RNA 免疫沉淀，然后在人结肠癌细胞中进行总 RNA 测序 (UV-RIP-seq)，以及 RNA 电泳迁移率变动分析 (EMSA)、生物层干涉测量 (BLI) ），并且在体外 RNA 结合测定中，我们将 TOP1 确定为 RNA 结合蛋白 (RBP)。我们证明 TOP1 在体外和细胞内直接结合 RNA，并且 TOP1 结合的大多数 RNA 都是 mRNA。使用 TOP1 RNA 结合突变体和拓扑异构酶裂解复合物测序 (TOP1cc-seq) 绘制 TOP1 催化活性图谱，我们发现当 RNA 聚合酶 II (RNAPII) 开始转录活性基因时，RNA 会对抗 TOP1 活性。我们通过采用 DNA 超螺旋测定和磁力镊子进一步证明了 RNA 在调节 TOP1 活性中的抑制作用。这些发现提供了对 RNA 和 TOP1 在调节 RNAPII 依赖性转录固有的 DNA 拓扑应激中的协调作用的见解。由 Elsevier Inc. 出版。

Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and in vitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA in vitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.Published by Elsevier Inc.