A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Bryant, Carson J; McCool, Mason A.; Abriola, Laura; Surovtseva, Yulia V.; Baserga, Susan J.

doi:10.1098/rsob.210305

Cited by 26 publications

(57 citation statements)

References 117 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Strikingly, cells depleted of RSL24D1 or PeBoW components exhibited a strong reduction in nucleolar rRNA biogenesis relative to siNT and siSBDS negative control cells (Fig. 3C, D), consistent with previous results following depletion of Ribi factors required for both pre-rRNA processing and transcription (Bryant et al 2022). Taken together, these results indicate that RSL24D1 and PeBoW modulate rDNA transcription, linking LSU processing factors to RNAPI transcription regulation.…”

Section: Resultssupporting

confidence: 91%

“…3B). This result was further corroborated by in cellulo pulse labeling with the uridine analogue 5-ethynyl uridine (5-EU) and the analysis of its incorporation into nascent nucleolar rRNA through immunofluorescent staining as in (Bryant et al 2022). Strikingly, cells depleted of RSL24D1 or PeBoW components exhibited a strong reduction in nucleolar rRNA biogenesis relative to siNT and siSBDS negative control cells (Fig.…”

Section: Resultsmentioning

confidence: 78%

“… (D) Quantitation of the results in (C). Nucleolar rRNA biogenesis was quantified in MCF10A cells treated with the indicated siRNAs, where strongly reduced 5-EU signal corresponds to RNAPI inhibition (Bryant et al 2022). The negative control non-targeting siRNA, siNT, (n = 16 wells per plate) was set at 0% inhibition and the positive control siRPA194 (n = 16 wells per plate) was set at 100% inhibition.…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing

Buhagiar

McCool

Bryant

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

In eukaryotes, the nucleolus is the site of ribosome biosynthesis, an essential process in all cells. While human ribosome assembly is largely evolutionarily conserved, many of the regulatory details underlying its control and function have not yet been well-defined. The nucleolar protein RSL24D1 was originally identified as a factor important for ribosome biogenesis, and as an interactor with the PeBoW complex (PES1, BOP1, WDR12) in high-throughput affinity purifications. The PeBoW complex has been shown to be required for pre-28S rRNA processing. In this study, we show that RSL24D1 depletion impairs both pre-ribosomal RNA (pre-rRNA) transcription and mature 28S rRNA production, leading to decreased protein synthesis and p53 stabilization in mammalian cells. Surprisingly, each of the PeBoW complex members is also required for pre-rRNA transcription. We also demonstrate that RSL24D1 is physically complexed with RNA polymerase I, revealing a connection between large ribosomal subunit biogenesis and rDNA transcription. These results uncover the dual role of RSL24D1 and the PeBoW complex in multiple steps of ribosome biogenesis, and provide evidence implicating large subunit biogenesis factors in pre-rRNA transcription control.

show abstract

Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing

Buhagiar

McCool

Bryant

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…This result was further corroborated by in cellula pulse labeling with the uridine analog 5-ethynyl uridine (5-EU) and the analysis of its incorporation into nascent nucleolar rRNA through immunofluorescent staining as in (Bryant et al 2022). Strikingly, cells depleted of RSL24D1 or PeBoW components exhibited a strong reduction in nucleolar rRNA biogenesis relative to siNT and siSBDS negative control cells (Fig.…”

Section: Rsl24d1 and The Pebow Complex Are Required For Rnapi Promote...supporting

confidence: 52%

Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing

McCool

Buhagiar

Bryant

et al. 2022

RNA

View full text Add to dashboard Cite

In eukaryotes, the nucleolus is the site of ribosome biosynthesis, an essential process in all cells. While human ribosome assembly is largely evolutionarily conserved, many of the regulatory details underlying its control and function have not yet been well-defined. The nucleolar protein RSL24D1 was originally identified as a factor important for 60S ribosomal subunit biogenesis. In addition, the PeBoW (BOP1-PES1-WDR12) complex has been well-defined as required for pre-28S rRNA processing and cell proliferation. In this study, we show that RSL24D1 depletion impairs both pre-ribosomal RNA (pre-rRNA) transcription and mature 28S rRNA production, leading to decreased protein synthesis and p53 stabilization in human cells. Surprisingly, each of the PeBoW complex members is also required for pre-rRNA transcription. We demonstrate that RSL24D1 and WDR12 co-immunoprecipitate with the RNA polymerase I subunit, RPA194, and regulate its steady state levels. These results uncover the dual role of RSL24D1 and the PeBoW complex in multiple steps of ribosome biogenesis, and provide evidence implicating large ribosomal subunit biogenesis factors in pre-rRNA transcription control.

show abstract

“…We therefore examined whether alt-LAMA3 affects rDNA transcription as well. Using a miniaturized 5-ethynyl uridine (5-EU) assay for nucleolar rRNA transcription (Hayashi et al, 2018;Ogawa et al, 2021;Stamatopoulou et al, 2018;Bryant et al, 2022), we found a significant defect in pre-rRNA synthesis in alt-LAMA3 KO cells (Figures 3E and S3I). Utilizing a dual-luciferase reporter (Ghoshal et al, 2004;Sondalle et al, 2019), we observed a defect in RNA polymerase I (RNAPI) transcription in the absence of alt-LAMA3 ll Resource (Figure 3F).…”

Section: Llmentioning

confidence: 99%