The Ribosomal Gene Loci—The Power behind the Throne

Panov, Kostya I.; Hannan, Katherine M.; Hein, Nadine

doi:10.3390/genes12050763

Cited by 16 publications

(11 citation statements)

References 148 publications

(200 reference statements)

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“…Cells of all organisms manufacture mature ribosomes, the core machinery of protein translation, through a process known as ribosome biogenesis (RB) (reviewed in [1,2]). In eukaryotic cells, the first steps of RB occur in the nucleolus, a membraneless nuclear organelle discovered in the 1830s (reviewed in [3][4][5]), where RNA Polymerase 1 (RNAP1) transcribes the primary preribosomal RNA ( pre-rRNA) precursor (reviewed in [6][7][8]). Subsequently, a series of RNA processing and modification steps transpire, largely within the nucleolus, to create the mature cytoplasmic 18S, 5.8S, and 28S rRNA molecules in human cells [9,10].…”

Section: Introductionmentioning

confidence: 99%

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

et al. 2022

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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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Section: Introductionmentioning

confidence: 99%

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

et al. 2022

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“…In human cells, three of the four mature ribosomal RNAs (rRNAs), the 18S, 5.8S and 28S rRNAs, are synthesized in the nucleolus as components of the polycistronic 47S primary pre-rRNA precursor transcript from tandem ribosomal DNA (rDNA) repeats by RNA Polymerase 1 (RNAP1) ( 4 ). The 5S rRNA is separately transcribed in the nucleus by RNA Polymerase 3 (RNAP3) ( 5 , 6 ). A myriad of ribosome assembly factors (AFs) execute endo- and exonucleolytic pre-rRNA processing and modification events to liberate the mature rRNAs from the 47S transcript, forming the small 40S and large 60S ribosomal subunits ( 7–10 ).…”

Section: Introductionmentioning

confidence: 99%

Discovery of novel microRNA mimic repressors of ribosome biogenesis

Bryant,

McCool,

Rosado González

et al. 2024

Nucleic Acids Research

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While microRNAs and other non-coding RNAs are the next frontier of novel regulators of mammalian ribosome biogenesis (RB), a systematic exploration of microRNA-mediated RB regulation has not yet been undertaken. We carried out a high-content screen in MCF10A cells for changes in nucleolar number using a library of 2603 mature human microRNA mimics. Following a secondary screen for nucleolar rRNA biogenesis inhibition, we identified 72 novel microRNA negative regulators of RB after stringent hit calling. Hits included 27 well-conserved microRNAs present in MirGeneDB, and were enriched for mRNA targets encoding proteins with nucleolar localization or functions in cell cycle regulation. Rigorous selection and validation of a subset of 15 microRNA hits unexpectedly revealed that most of them caused dysregulated pre-rRNA processing, elucidating a novel role for microRNAs in RB regulation. Almost all hits impaired global protein synthesis and upregulated CDKN1A (p21) levels, while causing diverse effects on RNA Polymerase 1 (RNAP1) transcription and TP53 protein levels. We provide evidence that the MIR-28 siblings, hsa-miR-28-5p and hsa-miR-708-5p, potently target the ribosomal protein mRNA RPS28 via tandem primate-specific 3′ UTR binding sites, causing a severe pre-18S pre-rRNA processing defect. Our work illuminates novel microRNA attenuators of RB, forging a promising new path for microRNA mimic chemotherapeutics.

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“…In human cells, three of the four mature ribosomal RNAs (rRNAs), the 18S, 5.8S, and 28S rRNAs, are synthesized in the nucleolus as components of the polycistronic 47S primary pre-rRNA precursor transcript from tandem ribosomal DNA (rDNA) repeats by RNA Polymerase 1 (RNAP1) (4). The 5S rRNA is separately transcribed in the nucleus by RNA Polymerase 3 (RNAP3) (5,6). A myriad of ribosome assembly factors (AFs) execute endo-and exonucleolytic pre-rRNA processing and modification events to liberate the mature rRNAs from the 47S transcript, forming the small 40S and large 60S ribosomal subunits (7)(8)(9)(10).…”

Section: Graphical Abstract Introductionmentioning

confidence: 99%

Discovery of novel microRNA mimic repressors of ribosome biogenesis

Bryant

McCool

Rosado-Gonzalez

et al. 2023

Preprint

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While microRNAs and other non-coding RNAs are the next frontier of novel regulators of mammalian ribosome biogenesis (RB), a systematic exploration of microRNA-mediated RB regulation has not yet been undertaken. We carried out a high-content screen in MCF10A cells for changes in nucleolar number using a library of 2,603 mature human microRNA mimics. Following a secondary screen for nucleolar rRNA biogenesis inhibition, we identified 72 novel microRNA negative regulators of RB after stringent hit calling. Hits were enriched for mRNA targets encoding proteins with nucleolar localization or functions in cell cycle regulation. Rigorous selection and validation of a subset of 15 microRNA hits unexpectedly revealed that most of them caused dysregulated pre-rRNA processing, elucidating a novel role for microRNAs in RB regulation. Almost all 15 hits impaired global protein synthesis and upregulated CDKN1A (p21) levels, but caused diverse effects on RNA Polymerase 1 (RNAP1) transcription and TP53 protein levels. We defined RPS28 as a direct target of the MIR-28 siblings, hsa-miR-28-5p and hsa-miR-708-5p, which caused a severe pre-18S pre-rRNA processing defect, and uncovered SPRR3's novel role in promoting RNAP1 transcription. Our work illuminates novel microRNA attenuators of RB, forging a promising new path for microRNA mimic chemotherapeutics.

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The Ribosomal Gene Loci—The Power behind the Throne

Cited by 16 publications

References 148 publications

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Discovery of novel microRNA mimic repressors of ribosome biogenesis

Discovery of novel microRNA mimic repressors of ribosome biogenesis

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