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1.  Nuclear translation visualized by ribosome-bound nascent chain puromycylation 
The Journal of Cell Biology  2012;197(1):45-57.
A new method for visualizing translation in cells via standard immunofluorescence microscopy provides evidence for translation in the nucleoplasm and nucleolus.
Whether protein translation occurs in the nucleus is contentious. To address this question, we developed the ribopuromycylation method (RPM), which visualizes translation in cells via standard immunofluorescence microscopy. The RPM is based on ribosome-catalyzed puromycylation of nascent chains immobilized on ribosomes by antibiotic chain elongation inhibitors followed by detection of puromycylated ribosome-bound nascent chains with a puromycin (PMY)-specific monoclonal antibody in fixed and permeabilized cells. The RPM correlates localized translation with myriad processes in cells and can be applied to any cell whose translation is sensitive to PMY. In this paper, we use the RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chemical stress.
doi:10.1083/jcb.201112145
PMCID: PMC3317795  PMID: 22472439
2.  Induction of GADD34 Is Necessary for dsRNA-Dependent Interferon-β Production and Participates in the Control of Chikungunya Virus Infection 
PLoS Pathogens  2012;8(5):e1002708.
Nucleic acid sensing by cells is a key feature of antiviral responses, which generally result in type-I Interferon production and tissue protection. However, detection of double-stranded RNAs in virus-infected cells promotes two concomitant and apparently conflicting events. The dsRNA-dependent protein kinase (PKR) phosphorylates translation initiation factor 2-alpha (eIF2α) and inhibits protein synthesis, whereas cytosolic DExD/H box RNA helicases induce expression of type I-IFN and other cytokines. We demonstrate that the phosphatase-1 cofactor, growth arrest and DNA damage-inducible protein 34 (GADD34/Ppp1r15a), an important component of the unfolded protein response (UPR), is absolutely required for type I-IFN and IL-6 production by mouse embryonic fibroblasts (MEFs) in response to dsRNA. GADD34 expression in MEFs is dependent on PKR activation, linking cytosolic microbial sensing with the ATF4 branch of the UPR. The importance of this link for anti-viral immunity is underlined by the extreme susceptibility of GADD34-deficient fibroblasts and neonate mice to Chikungunya virus infection.
Author Summary
Nucleic acids detection by multiple molecular sensors results in type-I interferon production, which protects cells and tissues from viral infections. At the intracellular level, the detection of double-stranded RNA by one of these sensors, the dsRNA-dependent protein kinase also leads to the profound inhibition of protein synthesis. We describe here that the inducible phosphatase 1 co-factor Ppp1r15a/GADD34, a well known player in the endoplasmic reticulum unfolded protein response (UPR), is activated during double-stranded RNA detection and is absolutely necessary to allow cytokine production in cells exposed to poly I:C or Chikungunya virus. Our data shows that the cellular response to nucleic acids can reveal unanticipated connections between innate immunity and fundamental stress pathways, such as the ATF4 branch of the UPR.
doi:10.1371/journal.ppat.1002708
PMCID: PMC3355096  PMID: 22615568
3.  Ribosomal protein mRNAs are translationally-regulated during human dendritic cells activation by LPS 
Immunome Research  2009;5:5.
Background
Dendritic cells (DCs) are the sentinels of the mammalian immune system, characterized by a complex maturation process driven by pathogen detection. Although multiple studies have described the analysis of activated DCs by transcriptional profiling, recent findings indicate that mRNAs are also regulated at the translational level. A systematic analysis of the mRNAs being translationally regulated at various stages of DC activation was performed using translational profiling, which combines sucrose gradient fractionation of polysomal-bound mRNAs with DNA microarray analysis.
Results
Total and polysomal-bound mRNA populations purified from immature, 4 h and 16 h LPS-stimulated human monocyte-derived DCs were analyzed on Affymetrix microarrays U133 2.0. A group of 375 transcripts was identified as translationally regulated during DC-activation. In addition to several biochemical pathways related to immunity, the most statistically relevant biological function identified among the translationally regulated mRNAs was protein biosynthesis itself. We singled-out a cluster of 11 large ribosome proteins mRNAs, which are disengaged from polysomes at late time of maturation, suggesting the existence of a negative feedback loop regulating translation in DCs and linking ribosomal proteins to immuno-modulatory function.
Conclusion
Our observations highlight the importance of translation regulation during the immune response, and may favor the identification of novel protein networks relevant for immunity. Our study also provides information on the potential absence of correlation between gene expression and protein production for specific mRNA molecules present in DCs.
doi:10.1186/1745-7580-5-5
PMCID: PMC2788525  PMID: 19943945
4.  Regulation of translation is required for dendritic cell function and survival during activation 
The Journal of Cell Biology  2007;179(7):1427-1439.
In response to inflammatory stimulation, dendritic cells (DCs) have a remarkable pattern of differentiation (maturation) that exhibits specific mechanisms to control antigen processing and presentation. Here, we show that in response to lipopolysaccharides, protein synthesis is rapidly enhanced in DCs. This enhancement occurs via a PI3K-dependent signaling pathway and is key for DC activation. In addition, we show that later on, in a manner similar to viral or apoptotic stress, DC activation leads to the phosphorylation and proteolysis of important translation initiation factors, thus inhibiting cap-dependent translation. This inhibition correlates with major changes in the origin of the peptides presented by MHC class I and the ability of mature DCs to prevent cell death. Our observations have important implications in linking translation regulation with DC function and survival during the immune response.
doi:10.1083/jcb.200707166
PMCID: PMC2373495  PMID: 18166652

Results 1-4 (4)