Uncovering the mechanisms of transcription elongation by eukaryotic RNA polymerases I, II, and III

Jacobs, Ruth Q.; Lucius, Aaron L.; Schneider, David A.

doi:10.1016/j.isci.2022.105306

Cited by 13 publications

(35 citation statements)

References 96 publications

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“…Three Saccharomyces cerevisiae (yeast) strains were generated with a C-terminal tag (TEV cleavage site, three HA repeats, and 10 histidine residues) on the second largest subunit of Pol I, II, and III. Yeast were grown at the Bioexpression and Fermentation Facility, Department of Biochemistry and Molecular Biology at the University of Georgia and purified as detailed previously [ 49 , 50 ]. The purified Pol samples were analyzed with SDS PAGE and mass spectrometry [ 50 ].…”

Section: Methodsmentioning

confidence: 99%

“…Yeast were grown at the Bioexpression and Fermentation Facility, Department of Biochemistry and Molecular Biology at the University of Georgia and purified as detailed previously [ 49 , 50 ]. The purified Pol samples were analyzed with SDS PAGE and mass spectrometry [ 50 ]. All subunits of Pol I (14-subunits), Pol II (12-subunits), and Pol III (17-subunits) were present with no cross-contamination of subunits across Pol I, II, and III preparations.…”

Section: Methodsmentioning

confidence: 99%

“…Multi-nucleotide addition reactions were executed as previously published [ 49 , 50 , 51 ] and detailed in the Supplemental Materials . Briefly, elongation complexes (ECs) were assembled with purified Pol I, II, or III, pre-annealed RNA:DNA t hybrid, 9-mer RNA annealed to 64-nt DNA template strand, and DNA nt (DNA non-template strand) [ 52 ].…”

Section: Methodsmentioning

confidence: 99%

“…Multi-nucleotide addition time courses, composed of nine data sets (11-mer–19-mer), were fit globally using a previously published method [ 49 , 50 , 51 ], Multi-start Evolutionary Nonlinear OpTimizeR (MENOTR) [ 53 ]. MENOTR is available on GitHub: , accessed on 22 September 2021.…”

Section: Methodsmentioning

confidence: 99%

“…EC stability experiments were executed as previously published [ 49 , 50 , 54 , 55 ]. Radiolabeled EC 10-mer containing either Pol I, II, and III were mixed with 10 μM RNase A (#LS002132; Worthington Biochemical, Lakewood, NJ, USA), 1 M KCl, and BMH-21 (1 μM) or vehicle at t = 0.…”

Section: Methodsmentioning

confidence: 99%

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RNA Polymerase I Is Uniquely Vulnerable to the Small-Molecule Inhibitor BMH-21

Jacobs

Fuller

Cooper

et al. 2022

Cancers

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Cancer cells require robust ribosome biogenesis to maintain rapid cell growth during tumorigenesis. Because RNA polymerase I (Pol I) transcription of the ribosomal DNA (rDNA) is the first and rate-limiting step of ribosome biogenesis, it has emerged as a promising anti-cancer target. Over the last decade, novel cancer therapeutics targeting Pol I have progressed to clinical trials. BMH-21 is a first-in-class small molecule that inhibits Pol I transcription and represses cancer cell growth. Several recent studies have uncovered key mechanisms by which BMH-21 inhibits ribosome biosynthesis but the selectivity of BMH-21 for Pol I has not been directly measured. Here, we quantify the effects of BMH-21 on Pol I, RNA polymerase II (Pol II), and RNA polymerase III (Pol III) in vitro using purified components. We found that BMH-21 directly impairs nucleotide addition by Pol I, with no or modest effect on Pols II and III, respectively. Additionally, we found that BMH-21 does not affect the stability of any of the Pols’ elongation complexes. These data demonstrate that BMH-21 directly exploits unique vulnerabilities of Pol I.

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Section: Methodsmentioning

confidence: 99%