The small-molecule BMH-21 directly inhibits transcription elongation and DNA occupancy of RNA polymerase I in vivo and in vitro

Cancer cells require a high number of ribosomes to support their rapid growth and proliferation. The key step in ribosome biogenesis is the synthesis of ribosomal RNA (rRNA) by RNA polymerase I (Pol I). As a result, developing and characterizing inhibitors of Pol I is a significant goal in cancer therapy.

In this study, we investigate the mechanism of action of BMH-21, a first-in-class small-molecule Pol I inhibitor. Using high-resolution in vitro transcription assays and in vivo native elongating transcript sequencing (NET-seq), we characterize how BMH-21 affects Pol I transcription. Our results show that BMH-21 inhibits Pol I transcription initiation, promoter escape, and elongation. Notably, the elongation phase is particularly sensitive to BMH-21, which decreases the transcription elongation rate and increases the accumulation of paused Pol I on the ribosomal DNA (rDNA) template. NET-seq experiments in vivo further confirm these findings by showing reduced Pol I occupancy on the template and increased sequence-specific pausing upstream of G-rich rDNA sequences following BMH-21 treatment.

These findings elucidate the mechanism of action of BMH-21 and represent a crucial advance in the development of this compound and its derivatives for potential clinical application.