Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Jan;12(1):210305.
doi: 10.1098/rsob.210305. Epub 2022 Jan 26.

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Carson J Bryant  1 , Mason A McCool  1 , Laura Abriola  2 , Yulia V Surovtseva  2 , Susan J Baserga  1   3   4
Affiliations

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Carson J Bryant et al. Open Biol. 2022 Jan.

Abstract

Studies of the regulation of nucleolar function are critical for ascertaining clearer insights into the basic biological underpinnings of ribosome biogenesis (RB), and for future development of therapeutics to treat cancer and ribosomopathies. A number of high-throughput primary assays based on morphological alterations of the nucleolus can indirectly identify hits affecting RB. However, there is a need for a more direct high-throughput assay for a nucleolar function to further evaluate hits. Previous reports have monitored nucleolar rRNA biogenesis using 5-ethynyl uridine (5-EU) in low-throughput. We report a miniaturized, high-throughput 5-EU assay that enables specific calculation of nucleolar rRNA biogenesis inhibition, based on co-staining of the nucleolar protein fibrillarin (FBL). The assay uses two siRNA controls: a negative non-targeting siRNA control and a positive siRNA control targeting RNA Polymerase 1 (RNAP1; POLR1A), and specifically quantifies median 5-EU signal within nucleoli. Maximum nuclear 5-EU signal can also be used to monitor the effects of putative small-molecule inhibitors of RNAP1, like BMH-21, or other treatment conditions that cause FBL dispersion. We validate the 5-EU assay on 68 predominately nucleolar hits from a high-throughput primary screen, showing that 58/68 hits significantly inhibit nucleolar rRNA biogenesis. Our new method establishes direct quantification of nucleolar function in high-throughput, facilitating closer study of RB in health and disease.

Keywords: RNA polymerase 1; high-throughput screening; nucleolus; pre-rRNA processing; pre-rRNA transcription; ribosome biogenesis.

PubMed Disclaimer

Conflict of interest statement

We declare we have no competing interests.

Figures

Figure 1.
Figure 1.
A high-throughput assay for nucleolar rRNA biogenesis using 5-ethynyl uridine (5-EU). (a) Schematic of the 5-EU assay protocol. MCF10A cells are reverse-transfected in 384-well imaging plates with control or unknown siRNAs for 72 h. Following target depletion, 5-EU is incorporated into nascent RNA transcripts for 1 h, with the majority of label incorporated into nascent pre-ribosomal RNA (pre-rRNA). Treated cells are fixed and stained for DNA (Hoechst 33342, DAPI channel) and the nucleolar protein FBL (Cy5 channel). 5-EU in nascent transcripts is conjugated to an azide fluorophore (AF488 azide, FITC channel) via a copper-catalysed click reaction. After fluorescent imaging, cell nuclei and nucleoli are segmented in silico with CellProfiler, and nucleolar-specific 5-EU signal is quantified for each nucleolus object identified. (b) RNAP1 inhibition specifically inhibits nucleolar 5-EU incorporation. No 5-EU, experiment without 1 h 5-EU incorporation. Treatment with a non-targeting siRNA (siNT) leads to a high 5-EU signal within the nucleolus and moderate nucleoplasmic background signal. Acute treatment with BMH-21 (siNT + BMH) or siRNA-mediated depletion of POLR1A (siPOLR1A) decreases nucleolar 5-EU signal, although nucleoplasmic background remains. DNA (Hoechst staining), FBL (staining), 5-EU (5-EU staining) and DNA/5-EU (combined Hoechst and EU staining). Scale bars, 10 µm. (c) Schematic of CellProfiler segmentation and nucleolar 5-EU quantification. Panels 1 and 2, raw images of DNA and FBL staining. Panels 3 and 4, nuclei or nucleoli segmented by CellProfiler from DNA or FBL staining, respectively. Rainbow colouring identifies object number. Panel 5, overlay of segmented nucleoli (green) on top of 5-EU staining (magenta). Panel 6, quantification of median nucleolar 5-EU signal for nucleoli in cells treated with siNT, siNT and BMH-21, or siPOLR1A. n = 24, 8 or 16 wells, respectively. Scale bars, 10 µm.
Figure 2.
Figure 2.
Optimization of the miniaturized 5-EU assay for nucleolar rRNA biogenesis. (a) Median (blue) or maximum (red) nuclear 5-EU signal for cells treated across a range of 5-EU concentrations and click reaction times, without or with BMH-21 treatment at 1 µM. n ≥ 20 000 cells per condition. (b) Control signal-to-background (S/B) ratios for treatment conditions in (a). Control S/B is calculated as the ratio of mean DMSO-treated nuclear 5-EU signal divided by mean BMH-21-treated nuclear 5-EU signal, for each combination of 5-EU concentration and click reaction time. Median nuclear 5-EU signal (blue), maximum nuclear 5-EU signal (red). (c) Control S/B and Z’ factor values for nuclei or nucleoli objects with only 5-EU visualization, 5-EU plus blocking with 10% (v/v) FBS/PBS, or 5-EU plus blocking and FBL co-staining. Median 5-EU signal (blue), maximum 5-EU signal (red). (d) Maximum nuclear 5-EU signal (red) or median nucleolar 5-EU signal (blue) for cells treated with siNT, siNOL11 or siPOLR1A. n ≥ 130 000 cells per siRNA. (e) Control S/B and Z’ factor values for cells (from (d)) treated with siNOL11 or siPOLR1A as the positive control. Control S/B is calculated as the ratio of mean siNT-treated 5-EU signal divided by mean siNOL11- or siPOLR1A-treated 5-EU signal. Maximum nuclear 5-EU signal (red), median nucleolar 5-EU signal (blue).
Figure 3.
Figure 3.
Validation of the 5-EU assay for nucleolar rRNA biogenesis on 68 known RB factors. (a). Outline of assay validation experiments. Sixty-eight proteins known to regulate RB subprocesses, including RNAP1 transcription and pre-rRNA processing, modification or stability, were selected for assay validation. The 5-EU assay was performed on cells depleted of these factors in biological triplicate, as described. (b). Representative images of FBL staining and 5-EU visualization for cells treated with siNT (negative control), siPOLR1A (positive control, orange), or a subset of siRNAs targeting known RB factors. (c) Nucleolar rRNA biogenesis percentage inhibition values for cells depleted of each known RB factor. Black dots, individual percentage inhibition values for one biological replicate. Solid bars, mean percentage inhibition (n = 3). Orange bar, POLR1A positive control (percentage inhibition = 100%). Blue bars, RB factors illustrated in (b). Letters to right indicate factors involved in RNAP1 transcription (T), pre-rRNA processing (P), or transcription repression (R).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Woolford JL, Baserga SJ Jr. 2013. Ribosome biogenesis in the yeast Saccharomyces cerevisiae. Genetics 195, 643-681. (10.1534/genetics.113.153197) - DOI - PMC - PubMed
    1. Aubert M, O'Donohue MF, Lebaron S, Gleizes PE. 2018. Pre-ribosomal RNA processing in human cells: from mechanisms to congenital diseases. Biomolecules 8, 123. (10.3390/biom8040123) - DOI - PMC - PubMed
    1. Correll CC, Bartek J, Dundr M. 2019. The nucleolus: a multiphase condensate balancing ribosome synthesis and translational capacity in health, aging and ribosomopathies. Cells 8, 869. (10.3390/cells8080869) - DOI - PMC - PubMed
    1. Lafontaine DLJ, Riback JA, Bascetin R, Brangwynne CP. 2020. The nucleolus as a multiphase liquid condensate. Nat. Rev. Mol. Cell Biol. 22, 165-182. - PubMed
    1. Pederson T. 2011. The nucleolus. Cold Spring Harb. Perspect Biol. 3, a000638. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources