Cancer cells are dependent upon an abundance of ribosomes to maintain rapid cell growth and proliferation. The rate-limiting step of ribosome biogenesis is ribosomal RNA (rRNA) synthesis by RNA polymerase I (Pol I). Therefore, a goal of the cancer therapeutic field is to develop and characterize Pol I inhibitors. Here, we elucidate the mechanism of Pol I inhibition by a first-in-class small-molecule BMH-21. To characterize the effects of BMH-21 on Pol I transcription, we leveraged high-resolution in vitro transcription assays and in vivo native elongating transcript sequencing (NET-seq). We find that Pol I transcription initiation, promoter escape, and elongation are all inhibited by BMH-21 in vitro. In particular, the transcription elongation phase is highly sensitive to BMH-21 treatment, as it causes a decrease in transcription elongation rate and an increase in paused Pols on the ribosomal DNA (rDNA) template. In vivo NET-seq experiments complement these findings by revealing a reduction in Pol I occupancy on the template and an increase in sequence-specific pausing upstream of G-rich rDNA sequences after BMH-21 treatment. Collectively, these data reveal the mechanism of action of BMH-21, which is a critical step forward in the development of this compound and its derivatives for clinical use.

中文翻译：

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小分子 BMH-21 在体内和体外直接抑制 RNA 聚合酶 I 的转录延伸和 DNA 占据。

癌细胞依赖于大量的核糖体来维持快速的细胞生长和增殖。核糖体生物合成的限速步骤是通过 RNA 聚合酶 I (Pol I) 合成核糖体 RNA (rRNA)。因此，癌症治疗领域的一个目标是开发和表征 Pol I 抑制剂。在这里，我们阐明了一流的小分子 BMH-21 抑制 Pol I 的机制。为了表征 BMH-21 对 Pol I 转录的影响，我们利用高分辨率体外转录测定和体内天然延伸转录测序 (NET-seq)。我们发现 Pol I 转录起始、启动子逃逸和延伸都在体外被 BMH-21 抑制。特别是，转录延伸阶段对 BMH-21 处理高度敏感，因为它导致转录延伸率降低和核糖体 DNA (rDNA) 模板上暂停的 Pols 增加。体内 NET-seq 实验通过揭示模板上 Pol I 占用率的减少和 BMH-21 处理后富含 G 的 rDNA 序列上游的序列特异性暂停增加来补充这些发现。总的来说，这些数据揭示了 BMH-21 的作用机制，这是开发该化合物及其衍生物用于临床的关键一步。