Expression of RNA polymerase I catalytic core is influenced by RPA12
Brittany L. Ford,Ting Wei, Hester Liu, Catherine E. Scull, Saman M. Najmi, Stephanie Pitts, Wenjun Fan, David A. Schneider, Marikki Laiho
Abstract
RNA Polymerase I (Pol I) has recently been recognized as a cancer therapeutic target. The activity of this enzyme is essential for ribosome biogenesis and is universally activated in cancers. The enzymatic activity of this multi-subunit complex resides in its catalytic core composed of RPA194, RPA135, and RPA12, a subunit with functions in RNA cleavage, transcription initiation and elongation. Here we explore whether RPA12 influences the regulation of RPA194 in human cancer cells. We use a specific small-molecule Pol I inhibitor BMH-21 that inhibits transcription initiation, elongation and ultimately activates the degradation of Pol I catalytic subunit RPA194. We show that silencing RPA12 causes alterations in the expression and localization of Pol I subunits RPA194 and RPA135.
Introduction
RNA Polymerase I (Pol I) is a multi-subunit enzyme that operates in the nucleolus of the cell [1–3]. The key enzymatic activity of Pol I is to transcribe the ribosomal DNA (rDNA) into the 13 kb long 47S precursor ribosomal RNA (rRNA), which is the first step of the complex, resource and energy-consuming process of ribosome biogenesis [1, 2, 4]. Ribosome biogenesis is especially critical at times requiring extensive protein synthesis, such as cellular divisions during development and regeneration or for highly specialized functions within differentiated cells [5–7]. In this process transcription by Pol I is the rate limiting factor [1, 2, 8]. The magnitude of Pol I transcription and related metabolic activity in cells underscores the importance of this enzyme; up to 60% of all transcription in cells is by Pol I [4].
Material and methods
Cell culture and reagents
A375 melanoma (CRL-1619) cells were purchased from American Type Culture Collection. This cell line was authenticated using short tandem repeat analysis by Johns Hopkins Genetic Resources Core Facility. Mycoplasma testing was conducted periodically using qPCR with negative results. The cells were maintained in a humidified atmosphere comprising of 5% CO2 at 37˚C. A375 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 4.5 g/L glucose. Pol I inhibitor BMH-21 (12H-benzo[g]pyrido[2,1-b] quinazoline-4-carboxamide, N-[2(dimethylamino)ethyl]-12-oxo) used in this study was synthesized as described by Colis et al. and verified for purity using liquid chromatography/ mass spectrometry and 1H nuclear magnetic resonance [13]. Yeast cells were grown at 23°C on Yeast Peptone Dextrose (YEPD) agar plates supplemented with indicated concentrations of BMH-21.
Results
Effect of Pol I inhibitor BMH-21 on RPA12
RPA12, the small 13.9 kDa subunit specific for Pol I, is required for the cleavage of nascent rRNA and assists in polymerase backtracking and proofreading [20, 26, 27]. Based on detailed dynamic studies in vitro, RPA12.2, the yeast RPA12 homologue, affects nucleotide addition kinetics and elongation complex stability [19, 20]. We have earlier shown that BMH-21, a small-molecule Pol I inhibitor, destabilizes RPA194 [12]. However, the effect of BMH-21 on RPA12 has not been analyzed before. We first conducted a kinetic experiment to assess the impact of BMH-21 on RPA12 using immunofluorescence analysis. BMH-21 (1 μM) was applied to the cells for increasing periods of time, up to 180 minutes, followed by staining of the cells for RPA12 (Fig 1A). Pol I transcription stress by BMH-21 leads to nucleolar reorganization, is amply documented [12, 39], and was monitored by staining for fibrillarin (FBL), an RNA methyltransferase and dense fibrillar center protein required for rRNA processing. BMH-21 treatment caused rearrangement and condensation of the nucleolus at 30–60 minutes of treatment. Nucleolar caps became prominent at 180 min.
Discussion
RPA12 is bestowed with several key activities in Pol I transcription. These include nucleotide addition, RNA cleavage, enzyme backtracking and proofreading, and transcription termination [18, 20, 26, 27, 41]. Not surprisingly, RPA12.2 deletion has temperature-associated lethality in yeast. However, when grown at cold temperatures, or if only the C-terminus containing the TFIIS paralog domain is deleted, viability is sustained [24, 42]. We were unsuccessful in establishing long-term stable knockout of RPA12 using shRNA or sgRNAs (not shown) in mammalian cancer cells, while transient knockdown was achieved using both shRNA and siRNA.
Conclusion
Here we showed that the transient depletion of RPA12, a core component of the Pol I enzyme complex and an essential factor for RNA cleavage, led to a decreased steady-state protein expression of the catalytic subunit RPA194. Despite this, RPA12 is nonessential for continued rRNA synthesis and chromatin engagement of the polymerase in human cancer cells. However, long-term sustained depletion of RPA12 was not achieved. This study has the limitation that it was performed in only one cancer cell line. Additional studies will be needed to investigate the implications of RPA12 knockdown and the functional impact it has on Pol I enzyme composition, transcription, and termination. As yeast RPA12.2 conveys proofreading functions for the rRNA transcript and its depletion leads to high polymerase error rates, it is plausible that long-term ablation of its activity leads to rRNA transcription errors that compromise ribosome function and cell survival.
Acknowledgments
The authors would like to thank the Light Microscopy Unit at the University of Helsinki for the use of their facility and assistance with the processing of IF images.
Citation: Ford BL, Wei T, Liu H, Scull CE, Najmi SM, Pitts S, et al. (2023) Expression of RNA polymerase I catalytic core is influenced by RPA12. PLoS ONE 18(5): e0285660.
https://doi.org/10.1371/journal.pone.0285660
Editor: Arunava Roy, University of South Florida, UNITED STATES
Received: December 30, 2022; Accepted: April 27, 2023; Published: May 11, 2023
Copyright: © 2023 Ford et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This study was funded in part by the National Cancer Institute P30CA006973 to M.L. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: M.L. holds patents (Activators and therapeutic applications thereof; 8,680,107, 10,214,491, 2195316, 2889297, 2,691,227, 2,912,456), (Compounds which inhibit RNA polymerase, compositions including such compounds, and their use; 3119781, 11,001,581), (Screening Method to Identify Scleroderma Immune Responses with Anti-cancer Activity, and Induction of such Immune Responses for Cancer Therapy; 1,454,630) and patent applications (Combinatory treatment strategies of cancer based on RNA polymerase I inhibition; 16/335,737, 17853955.7). M.L., H.L. and W.F. hold patent applications (Compounds which inhibit RNA polymerase, compositions including such compounds, and their use 18/024,407; 18/024,417; 18/024,421). These patents and patent applications are managed by the Johns Hopkins University. These do not alter our adherence to PLOS ONE policies on sharing data and materials. The other authors declare that they have no conflicts of interest with the contents of this article.
Abbreviations: Pol I, RNA polymerase I; rRNA, ribosomal RNA; rDNA, ribosomal DNA; IF, Immunofluorescence; qPCR, quantitative polymerase chain reaction; Co-IP, co-immunoprecipitation; ChIP, chromatin immunoprecipitation
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0285660#abstract0
