期刊
OPEN BIOLOGY
卷 12, 期 1, 页码 -出版社
ROYAL SOC
DOI: 10.1098/rsob.210305
关键词
ribosome biogenesis; nucleolus; pre-rRNA transcription; pre-rRNA processing; RNA polymerase 1; high-throughput screening
资金
- National Institutes of Health (NIH) [1R35GM131687, 1F31DE030332, T32GM007223]
Studies of nucleolar function regulation are important for understanding the basic biology of ribosome biogenesis and developing therapeutics for cancer and ribosomopathies. Existing high-throughput assays indirectly identify drugs affecting ribosome biogenesis based on morphological changes in the nucleolus, but a more direct assay is needed. We report a high-throughput assay for nucleolar function that quantifies nucleolar rRNA biogenesis using 5-ethynyl uridine (5-EU) and nucleolar protein fibrillarin (FBL). This new method enables closer study of ribosome biogenesis in health and disease.
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.
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