RESUMO
Protein syntheses mediated by cellular and viral internal ribosome entry sites (IRESs) are believed to have many features in common. Distinct mechanisms for ribosome recruitment and preinitiation complex assembly between the two processes have not been identified thus far. Here we show that the methylation status of rRNA differentially influenced the mechanism of 80S complex formation on IRES elements from the cellular sodium-coupled neutral amino acid transporter 2 (SNAT2) versus the hepatitis C virus mRNA. Translation initiation involves the assembly of the 48S preinitiation complex, followed by joining of the 60S ribosomal subunit and formation of the 80S complex. Abrogation of rRNA methylation did not affect the 48S complex but resulted in impairment of 80S complex assembly on the cellular, but not the viral, IRESs tested. Impairment of 80S complex assembly on the amino acid transporter SNAT2 IRES was rescued by purified 60S subunits containing fully methylated rRNA. We found that rRNA methylation did not affect the activity of any of the viral IRESs tested but affected the activity of numerous cellular IRESs. This work reveals a novel mechanism operating on a cohort of cellular IRESs that involves rRNA methylation for proper 80S complex assembly and efficient translation initiation.
Assuntos
Sistema A de Transporte de Aminoácidos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Ribossômico/metabolismo , Subunidades Ribossômicas/metabolismo , Sistema A de Transporte de Aminoácidos/genética , Transportador 1 de Aminoácidos Catiônicos/biossíntese , Células HEK293 , Células HeLa , Hepacivirus/genética , Humanos , Metilação , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/biossíntese , RNA Mensageiro/metabolismo , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Ribossômicas/biossíntese , Subunidades Ribossômicas/química , Subunidades Ribossômicas Maiores/metabolismo , Subunidades Ribossômicas Menores/metabolismo , Estresse FisiológicoRESUMO
IRAK2, a member of the interleukin-1 receptor-associated kinase (IRAK) family, has been implicated in Toll-like receptor (TLR)-mediated signaling. We generated IRAK2-deficient mice to examine its function in detail. These mice are resistant to lipopolysaccharide-induced septic shock, because of impaired TLR4-mediated induction of pro-inflammatory cytokines and chemokines. Although IRAK2 deficiency did not affect TLR4-mediated NFkappaB activation, a reduction of lipopolysaccharide (LPS)-mediated mRNA stabilization contributed to the reduced cytokine and chemokine production observed in bone marrow-derived macrophages from IRAK2-deficient mice. Furthermore, the ratios of LPS-induced cytokine and chemokine mRNAs in translation-active (polysomal) versus translation-inactive (free ribosomes) pools were reduced in IRAK2-deficient macrophages compared with wild type macrophages. Importantly, LPS-induced phosphorylation of MKK3/6, MNK1, and eIF4E was significantly reduced in IRAK2-deficient macrophages compared with wild type macrophages. Moreover, LPS stimulation induced an interaction of IRAK2 with TRAF6, MKK3/6, and MK2, implicating a critical role for mitogen-activated protein kinase signaling in LPS-induced IRAK2-mediated post-transcriptional control. These results reveal that IRAK2 is required for LPS-mediated post-transcriptional control of cytokine and chemokine expression, which plays an essential role in TLR4-induced septic shock.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Lipopolissacarídeos/farmacologia , Processamento Pós-Transcricional do RNA , Transcrição Gênica , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Quimiocinas/genética , Quimiocinas/imunologia , Citocinas/genética , Citocinas/imunologia , Quinases Associadas a Receptores de Interleucina-1/genética , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases/fisiologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
Previously, we demonstrated that treatment of monocytic cells with IFN-gamma causes release of ribosomal protein L13a from the 60S ribosome and subsequent translational silencing of Ceruloplasmin (Cp) mRNA. Here, evidence using cultured cells demonstrates that Cp mRNA silencing is dependent on L13a and that L13a-deficient ribosomes are competent for global translational activity. Human monocytic U937 cells were stably transfected with two different shRNA sequences for L13a and clonally selected for more than 98% abrogation of total L13a expression. Metabolic labeling of these cells showed rescue of Cp translation from the IFN-gamma mediated translational silencing activity. Depletion of L13a caused significant reduction of methylation of ribosomal RNA and of cap-independent translation mediated by Internal Ribosome Entry Site (IRES) elements derived from p27, p53, and SNAT2 mRNAs. However, no significant differences in the ribosomal RNA processing, polysome formation, global translational activity, translational fidelity, and cell proliferation were observed between L13a-deficient and wild-type control cells. These results support the notion that ribosome can serve as a depot for releasable translation-regulatory factors unrelated to its basal polypeptide synthetic function. Unlike mammalian cells, the L13a homolog in yeast is indispensable for growth. Thus, L13a may have evolved from an essential ribosomal protein in lower eukaryotes to having a role as a dispensable extra-ribosomal function in higher eukaryotes.