The tumor suppressor HNRNPK induces p53-dependent nucleolar stress to drive ribosomopathies
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
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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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 2

HNRNPK overexpression promotes nucleolar fragmentation and dysregulates nucleolar components.

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HNRNPK overexpression promotes nucleolar fragmentation and dysregulates ...
(A) Top row: Electron micrographs of control MEF (empty vector). Typical spindle-shaped MEF with a euchromatic nucleus and a prominent nucleolus (No) (empty vector, n = 2; HnrnpkSAM, n = 8) (left). Representative example of a normal nucleolus with several fibrillar centers (*) (middle). Detail of a nucleolus showing several typical fibrillar centers surrounded by an annular shell of dense fibrillar component intermingled with the granular component (GC) (right). Second row left: Electron micrographs of MEFs overexpressing HnrnpkSAM. Formation of multinucleolated MEFs. The nucleus shows multiple nucleoli and peripheral masses of heterochromatin (*) and nucleolar segregation and compaction of nucleolar components. SG, segregated nucleolar components. DFC, dense fibrillar component. Nucleolar fragments (f). Nucleolar caps (c). Scale bars: 500 nm (top right image); 1 μm (all other images). (B) Representative confocal microscopy images with NCL, HNRNPK, and DAPI and with FBL, HNRNPK and DAPI as well as representative confocal microscopy images of FBL, NCL, and DAPI staining (nuclei were stained with DAPI; biological replicates analysis, P = 0.020). HNRNPK expression was analyzed by Alexa Fluor 488 intensity (biological replicates analysis, P = 0.010); NCL expression and NCL nucleoplasm expression were accessed by dot plot analysis of Alexa Fluor 647 intensity (biological replicates analysis, P = 0.031); FBL expression was analyzed by dot plot analysis of Alexa Fluor 647 intensity (biological replicates analysis, P = 0.0002); and FBL unload was examined via Alexa Fluor 647 spot total area (biological replicates analysis, P = 0.0001); for all graphs, dot plot cell replicates, >1.000, *P < 0.05; ***P < 0.001. Scale bars: 10 μm. All graphs are shown as the mean or median.