RNA methylation plays an important role in functional regulation of RNAs, and has thus attracted an increasing interest in biology and drug discovery. Here, we collected and collated transcriptomic, ...proteomic, structural and physical interaction data from the Harmonizome database, and applied supervised machine learning to predict novel genes associated with RNA methylation pathways in human. We selected five types of classifiers, which we trained and evaluated using cross-validation on multiple training sets. The best models reached 88% accuracy based on cross-validation, and an average 91% accuracy on the test set. Using protein-protein interaction data, we propose six molecular sub-networks linking model predictions to previously known RNA methylation genes, with roles in mRNA methylation, tRNA processing, rRNA processing, but also protein and chromatin modifications. Our study exemplifies how access to large omics datasets joined by machine learning methods can be used to predict gene function.
The generation of reactive oxygen species (ROS) by the nicotinamide adenine dinucleotide phosphate oxidase is an important mechanism by which neutrophils kill pathogens. The oxidase is composed of a ...membrane-bound cytochrome and 4 soluble proteins (p67phox, p40phox, p47phox, and GTP-Rac). These components form an active complex at the correct time and subcellular location through a series of incompletely understood mutual interactions, regulated, in part, by GTP/GDP exchange on Rac, protein phosphorylation, and binding to lipid messengers. We have used a variety of assays to follow the spatiotemporal assembly of the oxidase in genetically engineered primary mouse neutrophils, during phagocytosis of both serum- and immunoglobulin G-opsonized targets. The oxidase assembles directly on serum-Staphylococcus aureus–containing phagosomes within seconds of phagosome formation; this process is only partially dependent (∼ 30%) on PtdIns3P binding to p40phox, but totally dependent on Rac1/2 binding to p67phox. In contrast, in response to immunoglobulin G-targets, the oxidase first assembles on a tubulovesicular compartment that develops at sites of granule fusion to the base of the emerging phagosome; oxidase assembly and activation is highly dependent on both PtdIns3P-p40phox and Rac2-p67phox interactions and delivery to the phagosome is regulated by Rab27a. These results define a novel pathway for oxidase assembly downstream of FcR-activation.
Exciting research has connected specific RNA modifications to chromatin, providing evidence for co-transcriptional deposition and function in gene regulation. Here we review insights gained from ...studying the co-transcriptional roles of RNA modifications, and their influence in normal and disease contexts. We also discuss how the availability of novel technical approaches could raise the translational potential of targeting RNA-modifying enzymes for the treatment of disease.
High content cellular screening Rausch, Oliver
Current opinion in chemical biology,
08/2006, Letnik:
10, Številka:
4
Journal Article
Recenzirano
Over the past few years, high content screening has firmly established itself as a high-throughput technology for the analysis of microscopy-based cellular assays. In particular, it has opened new ...areas of cell biology for the large-scale analysis of cellular phenotypes and has enabled the application of increasingly sophisticated assays for large-scale genetic and compound screening, benefiting both the academic and pharmaceutical research environment.
N
-methyladenosine (m
A) is an abundant internal RNA modification
that is catalysed predominantly by the METTL3-METTL14 methyltransferase complex
. The m
A methyltransferase METTL3 has been linked to ...the initiation and maintenance of acute myeloid leukaemia (AML), but the potential of therapeutic applications targeting this enzyme remains unknown
. Here we present the identification and characterization of STM2457, a highly potent and selective first-in-class catalytic inhibitor of METTL3, and a crystal structure of STM2457 in complex with METTL3-METTL14. Treatment of tumours with STM2457 leads to reduced AML growth and an increase in differentiation and apoptosis. These cellular effects are accompanied by selective reduction of m
A levels on known leukaemogenic mRNAs and a decrease in their expression consistent with a translational defect. We demonstrate that pharmacological inhibition of METTL3 in vivo leads to impaired engraftment and prolonged survival in various mouse models of AML, specifically targeting key stem cell subpopulations of AML. Collectively, these results reveal the inhibition of METTL3 as a potential therapeutic strategy against AML, and provide proof of concept that the targeting of RNA-modifying enzymes represents a promising avenue for anticancer therapy.
Hemopoietic cytokines such as interleukin-3 and granulocyte colony-stimulating factor (G-CSF) are potent activators of hemopoietic
cell growth and strongly induce activation of extracellular ...signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK),
and p38 mitogen-activated protein (MAP) kinases. However, the role of these kinases is unclear. Using specific chemical inhibitors
for MEK and p38, we demonstrate here that both ERK and p38 pathways are critically involved in the transduction of a proliferative
signal and cooperate in G-CSF-induced cell proliferation. We show that, like ERK and JNK activation, activation of p38 and
its downstream substrate MAP kinase-activated protein kinase 2 by interleukin-3 or G-CSF requires Ras activation. We demonstrate
that two distinct cytoplasmic regions of the G-CSF receptor are involved in activation of the p38 pathway: a region within
the 100 membrane-proximal amino acids is sufficient to induce low levels of p38 and MAP kinase-activated protein kinase 2
activation, whereas the membrane-distal phosphorylation site Tyr 763 mediates strong activation of these kinases. The levels of p38 activation correlate closely with those of Ras activation
by G-CSF, suggesting that the degree of Ras activation is a critical determinant for the extent of p38 activation by hemopoietic
cytokines.
The neutrophil nicotinamide adenine dinucleotide phosphate-oxidase is a multisubunit enzyme (comprising gp91phox, p22phox, p67phox, p40phox, p47phox, and Rac) that plays a vital role in microbial ...killing. The recent discovery of a chronic granulomatous disease patient who expresses a mutant p40phox subunit, together with the development of mouse models of p40phox function, indicate phosphatidylinositol 3-phosphate binding to the PX domain of p40phox is an important signal for oxidase activation. However, the presence of other conserved residues and domains in p40phox suggest further regulatory roles for this protein. To test this, we introduced wild-type and mutated versions of p40phox into fully differentiated mouse neutrophils by retroviral transduction of p40phox−/− bone marrow progenitors and repopulation of the bone marrow compartment in radiation chimaeras. Phosphorylation of p40phox on threonine 154, but not serine 315, was required for full oxidase activation in response to formylated bacterial peptide fMLP, serum-opsonized S aureus, and immunoglobulin-opsonized sheep red blood cells. A functional SH3 domain was not required for oxidase activation, and deletion of the entire domain resulted in enhanced oxidase responses. Phosphorylation of threonine 154 in response to S aureus was mediated by protein kinase Cδ and was required for full translocation of p47phox to phagosomes. These results define an important new element in the physiological activation of the oxidase.
The interaction between myelin-associated glycoprotein (MAG), expressed at the periaxonal membrane of myelin, and receptors on neurons initiates a bidirectional signalling system that results in ...inhibition of neurite outgrowth and maintenance of myelin integrity. We show that this involves a lipid-raft to lipid-raft interaction on opposing cell membranes. MAG is exclusively located in low buoyancy Lubrol WX-insoluble membrane fractions isolated from whole brain, primary oligodendrocytes, or MAG-expressing CHO cells. Localisation within these domains is dependent on cellular cholesterol and occurs following terminal glycosylation in the trans-Golgi network, characteristics of association with lipid rafts. Furthermore, a recombinant form of MAG interacts specifically with lipid-raft fractions from whole brain and cultured cerebellar granule cells, containing functional MAG receptors GT1b and Nogo-66 receptor and molecules required for transduction of signal from MAG into neurons. The localisation of both MAG and MAG receptors within lipid rafts on the surface of opposing cells may create discrete areas of high avidity multivalent interaction, known to be critical for signalling into both cell types. Localisation within lipid rafts may provide a molecular environment that facilitates the interaction between MAG and multiple receptors and also between MAG ligands and molecules involved in signal transduction.