Simpson Querrey Institute for Epigenetics, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA; Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Section of Molecular Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Mol Cell. 2024 Sep 5;84(17):3192-3208.e11. doi: 10.1016/j.molcel.2024.07.032. Epub 2024 Aug 21.
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.
拓扑异构酶 I（TOP1）是一种必需的酶，它可以松弛 DNA，以防止和消散转录过程中的扭转应力。然而，TOP1 活性调节的机制仍然难以捉摸。我们使用增强交联和免疫沉淀（eCLIP）以及紫外线交联 RNA 免疫沉淀后总 RNA 测序（UV-RIP-seq）在人结肠癌细胞中进行研究，同时进行 RNA 电泳迁移率变动分析（EMSA）、生物层干涉测量法（BLI）和体外 RNA 结合测定，鉴定 TOP1 是一种 RNA 结合蛋白（RBP）。我们表明 TOP1 可以在体外和细胞中直接结合 RNA，并且 TOP1 结合的大多数 RNA 是 mRNA。我们使用 TOP1 RNA 结合突变体和拓扑异构酶切割复合物测序（TOP1cc-seq）来绘制 TOP1 催化活性图，揭示了 RNA 拮抗 TOP1 活性，因为 RNA 聚合酶 II（RNAPII）开始转录活性基因。我们进一步通过 DNA 超螺旋化测定和磁镊实验证明了 RNA 在调节 TOP1 活性方面的抑制作用。这些发现为 RNA 和 TOP1 在调节与 RNAPII 依赖转录相关的 DNA 拓扑应力方面的协调作用提供了深入的了解。
