Nuclear stabilization of p53 requires a functional nucleolar surveillance pathway

Hannan, Katherine M.; Soo, Priscilla; Wong, Mei; Lee, Justine K.; Hein, Nadine; Poh, Perlita; Wysoke, Kira D.; Williams, Tobias D.; Montellese, Christian; Smith, Lorey; Al-Obaidi, Sheren; Núñez-Villacís, Lorena; Pavy, Megan; He, Jin-Shu; Parsons, Kate M; Loring, Karagh E.; Morrison, Tess; Diesch, Jeannine; Burgio, Gaétan; Ferreira, Rita; Feng, Zhiping; Gould, Cathryn M.; Madhamshettiwar, Piyush B.; Flygare, Johan; Gonda, Thomas J.; Simpson, Kaylene J.; Kutay, Ulrike; Pearson, Richard B.; Engel, Christoph; Watkins, Nicholas J.; Hannan, Ross D.; George, Amee J.

doi:10.1016/j.celrep.2022.111571

Cited by 27 publications

(37 citation statements)

References 69 publications

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“…2B). These results agree with a genome-wide RNAi screen in A549 cells to identify proteins whose depletion leads to TP53 stabilization, where APOBEC3A was a top hit (Data S1) (19). Taken together, these results indicate that acute depletion of APOBEC3A induces the nucleolar stress response which leads to inhibition of cell cycle progression and to reduced cell viability.…”

Section: Resultssupporting

confidence: 88%

“…No reuse allowed without permission. To connect APOBEC3A's potential role in ribosome biogenesis with the cell cycle, we tested for the induction of the nucleolar stress response (17)(18)(19) after APOBEC3A siRNA depletion. The nucleolar stress response results in a change in nucleolar morphology, increased levels of TP53 and its transcriptional target CDKN1A (62), and ultimately cell cycle arrest and apoptosis.…”

Section: Resultsmentioning

confidence: 99%

“…6G). While these categories were not enriched for established nucleolar regulators, RNA regulation and apoptosis are overlapping and tightly linked to the ribosome biogenesis pathway (1,(17)(18)(19). APOBEC3A potentially edits several pre-mRNA transcripts encoding proteins present in the nucleolus and with roles regulating RNA and the cell cycle.…”

Section: 712mentioning

confidence: 99%

“…The nucleolus is pleiotropic itself (13), as are various ribosome biogenesis factors with dual-functions in other cellular processes (14)(15)(16). A main connection to the nucleolus is cell cycle progression through the TP53-mediated nucleolar stress response (17)(18)(19). This stress response can result in nucleolar structure alterations, which is also an attribute of cancer cells; however, cancer cells exhibit increased number and size of nucleoli, correlating with greater ribosome production (20,21).…”

Section: Introductionmentioning

confidence: 99%

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The cytidine deaminase APOBEC3A is required for large ribosomal subunit biogenesis

McCool

Bryant

Abriola

et al. 2023

Preprint

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Cancer initiates as a consequence of genomic mutations, and its subsequent progression relies on increased production of ribosomes to maintain high levels of protein synthesis for unchecked cell growth. Recently, cytidine deaminases have been uncovered as sources of mutagenesis in cancer. To form more established connections between these two cancer driving processes, we interrogated the cytidine deaminase family of proteins for potential roles in human ribosome biogenesis. We identified and validated APOBEC3A and APOBEC4 as novel ribosome biogenesis factors through our laboratory's established screening platform for the discovery of regulators of nucleolar function in MCF10A cells. We show that APOBEC3A is required for cell cycle progression and global protein synthesis. More specifically, we highlight APOBEC3A's role within the processing and maturation steps that form the large subunit 5.8S and 28S ribosomal (r)RNAs. Through an innovative nuclear RNA sequencing methodology, we identify candidate APOBEC3A C-to-U editing sites on the pre-rRNA and pre-mRNAs for the first time. Our work reveals the exciting possibility that the pre-rRNA can be edited during its maturation. More broadly, we found an additional function of APOBEC3A in cancer pathology, expanding its relevance as a target for cancer therapeutics.

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Section: Resultssupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: 712mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The cytidine deaminase APOBEC3A is required for large ribosomal subunit biogenesis

McCool

Bryant

Abriola

et al. 2023

Preprint

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“…As patients with congenital ribosomopathies age, they are at a 2.5- to 8.5-fold increased risk for developing cancer [ 114 ]. In many cancer cells, the heterozygous deletion of an RP gene is often accompanied by a TP53 mutation [ 117 ]. These somatic mutations in RPs include uL18/RPL5, uL16/RPL10, uL5/RPL11, eL22/RPL22, and uL23/RPL23A.…”

Section: Disease-associated Ribosomal Heterogeneity and Specializationmentioning

confidence: 99%

Specialized Ribosomes in Health and Disease

Miller

MacDonald

Kellogg

et al. 2023

IJMS

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Ribosomal heterogeneity exists within cells and between different cell types, at specific developmental stages, and occurs in response to environmental stimuli. Mounting evidence supports the existence of specialized ribosomes, or specific changes to the ribosome that regulate the translation of a specific group of transcripts. These alterations have been shown to affect the affinity of ribosomes for certain mRNAs or change the cotranslational folding of nascent polypeptides at the exit tunnel. The identification of specialized ribosomes requires evidence of the incorporation of different ribosomal proteins or of modifications to rRNA and/or protein that lead(s) to physiologically relevant changes in translation. In this review, we summarize ribosomal heterogeneity and specialization in mammals and discuss their relevance to several human diseases.

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Purification of Mouse Embryonic Fibroblasts (MEFs)

Ferreira,

Hein

2023

Current Protocols

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Mouse embryonic fibroblasts (MEFs) are primary fibroblasts purified from mouse embryos at a defined time post‐fertilization. MEFs have versatile applications, including use as feeder cell layers or sources of untransformed primary cells for a variety of biological assays. MEFs are most commonly isolated between embryonic day (E)12.5 and E13.5 but can be isolated from embryos as early as E8.5 and as late as E15.5. The individual embryos are harvested by carefully removing uterine tissue, yolk sac, and placenta. The embryos are euthanized, and non‐mesenchymal tissues, such as the fetal liver and heart, are removed before tissue homogenization. The remaining fetal tissue is homogenized by mechanical mincing using a sterile blade, followed by enzymatic digestion and resuspension. During tissue dissociation, the duration of trypsin‐EDTA/DNase digestion and enzyme concentration are critical parameters to produce high‐quality MEFs with the highest rates of cell viability and proliferation potential. MEFs can be cryopreserved at passage (P) 0 if >80% confluent, passaged for further expansion before freezing down, or directly utilized for downstream applications, i.e., preparation as feeder cell layers. Primary MEFs possess a limited proliferation capacity of ∼20 cell divisions, beyond which the percentage of senescent cells rapidly increases; thus, cultures should only be expanded/passaged to a maximum of P5. Critical for cell viability during cryopreservation and thawing of MEFs is the slow decrease in temperature when freezing, the rapid increase when thawing, the use of a cryoprotective agent, and an optimal cell density. While it is critical to generate high‐quality MEFs to standardize and optimize preparation procedures and utilize fresh reagents, some variability in proliferation capacity and cell viability between MEF preparations remains. Thus, MEF preparation, culture, and cryopreservation procedures are continuously being optimized. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol: Purification, passaging, and expansion of MEFsSupporting Protocol: Cryopreservation and thawing of MEFs

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Nuclear stabilization of p53 requires a functional nucleolar surveillance pathway

Cited by 27 publications

References 69 publications

The cytidine deaminase APOBEC3A is required for large ribosomal subunit biogenesis

The cytidine deaminase APOBEC3A is required for large ribosomal subunit biogenesis

Specialized Ribosomes in Health and Disease

Purification of Mouse Embryonic Fibroblasts (MEFs)

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