The tumor suppressor HNRNPK induces p53-dependent nucleolar stress to drive ribosomopathies
Pedro Aguilar-Garrido, … , Sean M. Post, Miguel Gallardo
Pedro Aguilar-Garrido, … , Sean M. Post, Miguel Gallardo
Published May 8, 2025
Citation Information: J Clin Invest. 2025;135(12):e183697. https://doi.org/10.1172/JCI183697.
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Research Article Aging Hematology

The tumor suppressor HNRNPK induces p53-dependent nucleolar stress to drive ribosomopathies

Abstract

The nucleolus is a membraneless organelle and an excellent stress sensor. Any changes in its architecture or composition lead to nucleolar stress, resulting in cell cycle arrest and interruption of ribosomal activity, critical factors in aging and cancer. In this study, we identified and described the pivotal role of the RNA-binding protein HNRNPK in ribosome and nucleolar dynamics. We developed an in vitro model of endogenous HNRNPK overexpression and an in vivo mouse model of ubiquitous HNRNPK overexpression. These models showed disruptions in translation as the HNRNPK overexpression caused alterations in the nucleolar structure, resulting in p53-dependent nucleolar stress, cell cycle arrest, senescence, and bone marrow failure phenotype, similar to what is observed in patients with ribosomopathies. Together, our findings identify HNRNPK as a master regulator of ribosome biogenesis and nucleolar homeostasis through p53, providing what we believe to be a new perspective on the orchestration of nucleolar integrity, ribosome function and cellular senescence.

Authors

Pedro Aguilar-Garrido, María Velasco-Estévez, Miguel Ángel Navarro-Aguadero, Álvaro Otero-Sobrino, Marta Ibáñez-Navarro, Miguel Ángel Marugal, María Hernández-Sánchez, Prerna Malaney, Ashley Rodriguez, Oscar Benitez, Xiaroui Zhang, Marisa J.L. Aitken, Alejandra Ortiz-Ruiz, Diego Megías, Manuel Pérez, Gadea Mata, Jesús Gomez, Miguel Lafarga, Orlando Domínguez, Osvaldo Graña-Castro, Eduardo Caleiras, Pilar Ximénez-Embun, Marta Isasa, Paloma Jimena de Andres, Sandra Rodríguez-Perales, Raúl Torres-Ruiz, Enrique Revilla, Rosa María García-Martín, Daniel Azorín, Josune Zubicaray, Julián Sevilla, Oleksandra Sirozh, Vanesa Lafarga, Joaquín Martínez-López, Sean M. Post, Miguel Gallardo

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Figure 3

HNRNPK overexpression showed rRNA and ribosome protein reduction and drove cell cycle arrest and senescence.

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HNRNPK overexpression showed rRNA and ribosome protein reduction and dro...
(A) Left: Representative images of EU assay. Right: Fluorescence intensity values (dot plot, >1.000 cells of representative well; biological replicates analysis: P = 0.013). Scale bar: 100 μm. (B) HnrnpkSAM (n = 6) vs. empty vector (n = 3) MEF qRT-PCR results, with dot plot showing pre-rRNA 45S and mature rRNA transcripts 18S, 28S, and 5.8S (45S, P = 0.0095; 5.8S, P = 0.0397; 18S, P = NS; 28S, P = NS). (C) Northern blot membranes with ITS1 and ITS2 probes showing lower levels of 47S/45S rRNAs and aberrant levels of pre-RNA precursors such as 20S. (D) HnrnpkSAM (n ≥ 5) vs. empty vector (n ≥ 4) qRT-PCR results, with dot plot showing RPL (top) and RPS (bottom) gene expression (Rpl22, P = 0.0027; Rpl14, P = 0.0010; Rpl28, P = 0.0063; Rps9, P = 0.0020; Rps21, P = 0.0003; Rps16, P = 0.0007; Rps12, P = 0.0003; Rps3, P = 0.0007; Rplpo, P = 0.0095). (E) Western blot membrane of HNRNPK-overexpressing cells showing higher c-MYC and p53 expression. (F) Left: Representative cell cycle FCM histogram plots in HnrnpkSAM (n = 9) vs. empty vector (n = 9) MEFs. Right: Bar graph of percentage of G1, S and G2/M cells (P = 0.005) from FCM analysis. (G) Left: Bright-field microscope images of SA-β-galactosidase staining in HnrnpkSAM MEFs. Right: Dot plot of cells positive for SA-β-galactosidase staining in HnrnpkSAM (n = 8) vs. empty vector (n = 6) (P = 0.0007). Scale bar: 200 μm. (H) Western blot membrane of irradiated HnrnpkSAM cells showing an increase in the senescence markers p21 and p16. All graphs are shown as the median (A) or mean (B–E). Two-sided Student’s t test, with the exception of FCM cell cycle analysis (2-way ANOVA): *P < 0.05; **P < 0.01; ***P < 0.001. All experiments comprised at least n = 3 biological replicates and/or n = 3 technical replicates.