RESUMEN
Mutations in the ER chaperone calreticulin (CALR) are common in myeloproliferative neoplasm (MPN) patients, activate the thrombopoietin receptor (MPL), and mediate constitutive JAK/STAT signaling. The mechanisms by which CALR mutations cause myeloid transformation are incompletely defined. We used mass spectrometry proteomics to identify CALR-mutant interacting proteins. Mutant CALR caused mislocalization of binding partners and increased recruitment of FLI1, ERP57, and CALR to the MPL promoter to enhance transcription. Consistent with a critical role for CALR-mediated JAK/STAT activation, we confirmed the efficacy of JAK2 inhibition on CALR-mutant cells in vitro and in vivo. Due to the altered interactome induced by CALR mutations, we hypothesized that CALR-mutant MPNs may be vulnerable to disruption of aberrant CALR protein complexes. A synthetic peptide designed to competitively inhibit the carboxy terminal of CALR specifically abrogated MPL/JAK/STAT signaling in cell lines and primary samples and improved the efficacy of JAK kinase inhibitors. These findings reveal what to our knowledge is a novel potential therapeutic approach for patients with CALR-mutant MPN.
Asunto(s)
Antineoplásicos/farmacología , Calreticulina/genética , Leucemia/genética , Trastornos Mieloproliferativos/genética , Animales , Calreticulina/antagonistas & inhibidores , Calreticulina/metabolismo , Línea Celular , Cromatina/metabolismo , Sistemas de Liberación de Medicamentos , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Quinasas Janus/antagonistas & inhibidores , Leucemia/tratamiento farmacológico , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Trastornos Mieloproliferativos/tratamiento farmacológico , Receptores de Trombopoyetina/genética , Transducción de SeñalRESUMEN
To counteract the breakdown of genome integrity, eukaryotic cells have developed a network of surveillance pathways to prevent and resolve DNA damage. Recent data has recognized the importance of RNA binding proteins (RBPs) in DNA damage repair (DDR) pathways. Here, we describe Nol12 as a multifunctional RBP with roles in RNA metabolism and genome maintenance. Nol12 is found in different subcellular compartments-nucleoli, where it associates with ribosomal RNA and is required for efficient separation of large and small subunit precursors at site 2; the nucleoplasm, where it co-localizes with the RNA/DNA helicase Dhx9 and paraspeckles; as well as GW/P-bodies in the cytoplasm. Loss of Nol12 results in the inability of cells to recover from DNA stress and a rapid p53-independent ATR-Chk1-mediated apoptotic response. Nol12 co-localizes with DNA repair proteins in vivo including Dhx9, as well as with TOPBP1 at sites of replication stalls, suggesting a role for Nol12 in the resolution of DNA stress and maintenance of genome integrity. Identification of a complex Nol12 interactome, which includes NONO, Dhx9, DNA-PK and Stau1, further supports the protein's diverse functions in RNA metabolism and DNA maintenance, establishing Nol12 as a multifunctional RBP essential for genome integrity.
Asunto(s)
ADN/metabolismo , Proteínas Nucleares/metabolismo , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/metabolismo , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular , Reparación del ADN , Humanos , Proteínas Nucleares/química , Dominios Proteicos , Proteínas de Unión al ARN/químicaRESUMEN
Ribosomal processing requires a series of endo- and exonucleolytic steps for the production of mature ribosomes, of which most have been described. To ensure ribosome synthesis, 3' end formation of rRNA uses multiple nucleases acting in parallel; however, a similar parallel mechanism had not been described for 5' end maturation. Here, we identify Rrp17p as a previously unidentified 5'-3' exonuclease essential for ribosome biogenesis, functioning with Rat1p in a parallel processing pathway analogous to that of 3' end formation. Rrp17p is required for efficient exonuclease digestion of the mature 5' ends of 5.8S(S) and 25S rRNAs, contains a catalytic domain close to its N terminus, and is highly conserved among higher eukaryotes, being a member of a family of exonucleases. We show that Rrp17p binds late pre-60S ribosomes, accompanying them from the nucleolus to the nuclear periphery, and provide evidence for physical and functional links between late 60S subunit processing and export.
Asunto(s)
Exonucleasas/fisiología , Proteínas de la Membrana/genética , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , Secuencia de Aminoácidos , Exonucleasas/genética , Exonucleasas/metabolismo , Exorribonucleasas/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de SecuenciaRESUMEN
The study of the dynamic interactome of cellular ribonucleoprotein (RNP) particles has been hampered by severe methodological limitations. In particular, the affinity purification of intact RNP complexes from cell lysates suffers from RNA degradation, loss of interacting macromolecules and poor overall yields. Here we describe a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae. Our method overcomes many of the previous limitations to produce large RNP interactomes with almost no contamination.