Genome-wide identification and characterization of transcripts translationally regulated by bacterial lipopolysaccharide in macrophage-like J774.1 cells

Abstract

Although Escherichia coli LPS is known to elicit various proinflammatory responses in macrophages, its effect on the translational states of transcripts has not yet been explored on a genome-wide scale. To address this, we investigated the mRNA profiles in polysomal and free messenger ribonucleoprotein particle (mRNP) fractions of mouse macrophage-like J774.1 cells, using Affymetrix Mouse Genome 430 2.0 GeneChips. Comparison of the mRNA profiles in total cellular, polysomal, and free mRNP fractions enabled us to identify transcripts that were modulated at the translational level by LPS: among 19,791 transcripts, 115 and 418 were up- and downregulated at 1, 2, or 4 h after LPS stimulation (100 ng/ml) in a translation-dependent manner. Interestingly, gene ontology-based analysis suggested that translation-dependent downregulated genes frequently include those encoding proteins in the mitochondrial respiratory chain. In fact, the mRNA levels of some transcripts for complexes I, IV, and V in the mitochondrial respiratory chain were translationally downregulated, eventually contributing to the decline of their protein levels. Moreover, the amount of metabolically labeled cytochrome oxidase subunit Va in complex IV was decreased without any change of its mRNA level in total cellular fraction after LPS stimulation. Consistently, the total amounts and activities of complexes I and IV were attenuated by LPS stimulation, and the attenuation was independent of nitric oxide. These results demonstrated that translational suppression may play a critical role in the LPS-mediated attenuation of mitochondrial oxidative phosphorylation in a nitric oxide-independent manner in J774.1 cells.

Toward a Genome-Wide Landscape of Translational Control

“…Fractionated mRNAs are then extracted and subjected to DNA microarray analysis for identification and quantification. Although a large number of fractions can be obtained to examine mRNA profiles across the entire polysome range (Arava et al 2003), most studies pool fractions because of the high cost of DNA microarrays (Johannes et al 1999;Zong et al 1999;Chen et al 2002Chen et al , 2011Jechlinger et al 2003;Preiss et al 2003;Kitamura et al 2004Kitamura et al , 2008Provenzani et al 2006;Spence et al 2006;Genolet et al 2008; Parent and Techniques to obtain genome-wide data on mRNA translation. (A) The polysome technique.…”

Transgenic mice: beyond the knockout

“…Initiation begins when the small and large ribosomal subunits (S40 and S60, respectively) bind the mRNA to form the large ribosomal complex (S80), which binds only actively translated mRNA. With use of this basic principle, actively translated mRNA can be distinguished from nontranslated mRNA by gradient centrifugation, spectroscopy, and quantitative RT-PCR or microarray analysis (28,37), which is referred to as polysomal profiling. Traditionally, polysomal profiling is performed using cell cultures, because it requires large quantities of a homogeneous population of cells, which has prevented its wide use on tissue samples or whole animals.…”

SP600125 Inhibits Cap-dependent Translation Independently of the c-Jun N-terminal Kinase Pathway

“…Polysomal and non-polysomal fractions were prepared using a 15-50% sucrose density gradient as previously described with minor modifications (Kitamura et al, 2008): we collected fractions 1-11 and 12-20 of 20 total fractions as the non-polysomal and polysomal fractions, respectively. Sucrose sedimentation analysis was performed by continuously monitoring the sucrose density gradients at 260 nm using a BioRad EM-1 monitor (Hercules, CA, USA) connected to a Piston Gradient Fractionator (BioComp Instruments, New Brunswick, Canada).…”

“…Western blot analysis was performed as previously described (Kitamura et al, 2008). Membranes were incubated with antiphospho-PDK1 (Ser241), phospho-AKT (Ser473), AKT, phosphomTOR (Ser2448), mTOR, 4EBP1, phospho-S6K (Thr389), phospho-eIF4B (Ser422), eIF4B, phospho-JNK1/2 (Thr183/ Tyr185), JNK1/2, JNK2, phospho-c-Jun (Ser63), c-Jun (Cell Signaling Technology, Beverly, MA, USA), GAPDH, S6K, and JNK1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C.…”

“…Newly synthesized proteins were metabolically labeled as previously described (Kitamura et al, 2008). To analyze GAPDH protein synthesis, 500 μg of cytoplasmic proteins, extracted with a buffer containing 10 mM Tris-HCl (pH 7.6), 50 mM NaCl, 5 mM EDTA, 1% Nonidet P40 and a complete protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), were immunoprecipitated with an anti-GAPDH antibody (Santa Cruz Biotechnology) and then subjected to Western blot analysis.…”