RESUMO
Impairment of ribosome function activates the MAPKKK ZAK, leading to activation of mitogen-activated protein (MAP) kinases p38 and JNK and inflammatory signaling. The mechanistic basis for activation of this ribotoxic stress response (RSR) remains completely obscure. We show that the long isoform of ZAK (ZAKα) directly associates with ribosomes by inserting its flexible C terminus into the ribosomal intersubunit space. Here, ZAKα binds helix 14 of 18S ribosomal RNA (rRNA). An adjacent domain in ZAKα also probes the ribosome, and together, these sensor domains are critically required for RSR activation after inhibition of both the E-site, the peptidyl transferase center (PTC), and ribotoxin action. Finally, we show that ablation of the RSR response leads to organismal phenotypes and decreased lifespan in the nematode Caenorhabditis elegans (C. elegans). Our findings yield mechanistic insight into how cells detect ribotoxic stress and provide experimental in vivo evidence for its physiological importance.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , MAP Quinase Quinase Quinases/metabolismo , Peptidil Transferases/metabolismo , RNA Ribossômico 18S/metabolismo , Ribossomos/metabolismo , Estresse Fisiológico , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Ativação Enzimática , Células HeLa , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/genética , Conformação Proteica , Domínios Proteicos , RNA Ribossômico 18S/genética , Homologia de Sequência , Transdução de SinaisRESUMO
The PR-domain (PRDM) family of genes encodes transcriptional regulators, several of which are deregulated in cancer. By using a functional screening approach, we sought to identify novel tumor suppressors among the PRDMs. Here we demonstrate oncogenic collaboration between depletion of the previously uncharacterized PR-domain family member Prdm11 and overexpression of MYC. Overexpression of PRDM11 inhibits proliferation and induces apoptosis. Prdm11 knockout mice are viable, and loss of Prdm11 accelerates MYC-driven lymphomagenesis in the Eµ-Myc mouse model. Moreover, we show that patients with PRDM11-deficient diffuse large B-cell lymphomas (DLBCLs) have poorer overall survival and belong to the nongerminal center B-cell-like subtype. Mechanistically, genome-wide mapping of PRDM11 binding sites coupled with transcriptome sequencing in human DLBCL cells evidenced that PRDM11 associates with transcriptional start sites of target genes and regulates important oncogenes such as FOS and JUN. Hence, we characterize PRDM11 as a putative novel tumor suppressor that controls the expression of key oncogenes, and we add new mechanistic insight into B-cell lymphomagenesis.
Assuntos
Proteínas de Transporte/genética , Transformação Celular Neoplásica/genética , Linfoma/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , Animais , Células Cultivadas , Embrião de Mamíferos , Deleção de Genes , Regulação Neoplásica da Expressão Gênica/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Linfoma/patologia , Linfoma Difuso de Grandes Células B/genética , Camundongos , Dados de Sequência Molecular , Fatores de Transcrição , Proteínas Supressoras de Tumor/genéticaRESUMO
PRDM family members are transcriptional regulators involved in tissue specific differentiation. PRDM5 has been reported to predominantly repress transcription, but a characterization of its molecular functions in a relevant biological context is lacking. We demonstrate here that Prdm5 is highly expressed in developing bones; and, by genome-wide mapping of Prdm5 occupancy in pre-osteoblastic cells, we uncover a novel and unique role for Prdm5 in targeting all mouse collagen genes as well as several SLRP proteoglycan genes. In particular, we show that Prdm5 controls both Collagen I transcription and fibrillogenesis by binding inside the Col1a1 gene body and maintaining RNA polymerase II occupancy. In vivo, Prdm5 loss results in delayed ossification involving a pronounced impairment in the assembly of fibrillar collagens. Collectively, our results define a novel role for Prdm5 in sustaining the transcriptional program necessary to the proper assembly of osteoblastic extracellular matrix.
Assuntos
Desenvolvimento Ósseo/genética , Colágeno Tipo I , Osteoblastos , RNA Polimerase II/genética , Transcrição Gênica , Células 3T3 , Animais , Diferenciação Celular/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Decorina/genética , Decorina/metabolismo , Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Colágenos Fibrilares , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Camundongos , Especificidade de Órgãos , Osteoblastos/citologia , Osteoblastos/metabolismo , Regiões Promotoras Genéticas , Proteoglicanas/genética , Proteoglicanas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
PRDM proteins belong to the SET domain protein family, which is involved in the regulation of gene expression. Although few PRDM members possess histone methyltransferase activity, the molecular mechanisms by which the other members exert transcriptional regulation remain to be delineated. In this study, we find that Prdm5 is highly expressed in mouse embryonic stem (mES) cells and exploit this cellular system to characterize molecular functions of Prdm5. By combining proteomics and next-generation sequencing technologies, we identify Prdm5 interaction partners and genomic occupancy. We demonstrate that although Prdm5 is dispensable for mES cell maintenance, it directly targets genomic regions involved in early embryonic development and affects the expression of a subset of developmental regulators during cell differentiation. Importantly, Prdm5 interacts with Ctcf, cohesin, and TFIIIC and cooccupies genomic loci. In summary, our data indicate how Prdm5 modulates transcription by interacting with factors involved in genome organization in mouse embryonic stem cells.