VH-DJHrecombination of the immunoglobulin heavy chain (Igh) locus is temporally and spatially controlled during early B cell development, and yet no regulatory elements other than the VHgene ...promoters have been identified throughout the entire VHgene cluster. Here, we discovered regulatory sequences that are interspersed in the distal VHgene region. These conserved repeat elements were characterized by the presence of Pax5 transcription factor-dependent active chromatin by binding of the regulators Pax5, E2A, CTCF, and Rad21, as well as by Pax5-dependent antisense transcription in pro-B cells. ThePax5-activatedintergenicrepeat (PAIR) elements were no longer bound by Pax5 in pre-B and B cells consistent with the loss of antisense transcription, whereas E2A and CTCF interacted with PAIR elements throughout early B cell development. The pro-B cell-specific and Pax5-dependent activity of the PAIR elements suggests that they are involved in the regulation of distal VH-DJHrecombination at theIghlocus.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Ceramide kinase (CerK) is a sphingolipid metabolizing enzyme very sensitive to oxidation; however, the determinants are unknown. We show here that the thiol-modifying agent
N-ethyl-maleimide ...abrogates CerK activity in vitro and in a cell based assay, implying that important cysteine residues are accessible in purified as well as endogenous CerK. We replaced every 22 residues in human CerK, by an alanine, and measured activity in the resulting mutant proteins. This led to identification of a cluster of cysteines, C
347
XXXC
351
XXC
354, essential for CerK function. These findings are discussed based on homology modeling of the catalytic domain of CerK.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
RNA-ligand binding often depends crucially on the local RNA secondary structure at the binding site. We develop here a model that quantitatively predicts the effect of RNA secondary structure on ...effective RNA-ligand binding activities based on equilibrium thermodynamics and the explicit computations of partition functions for the RNA structures. A statistical test for the impact of a particular structural feature on the binding affinities follows directly from this approach. The formalism is extended to describing the effects of hybridizing small “modifier RNAs” to a target RNA molecule outside its ligand binding site. We illustrate the applicability of our approach by quantitatively describing the interaction of the mRNA stabilizing protein
HuR with AU-rich elements Meisner, N.-C., Hackermüller, J., Uhl, V., Aszódi, A., Jaritz, M., Auer, M., 2004. mRNA openers and closers: a methodology to modulate AU-rich element controlled mRNA stability by a molecular switch in mRNA conformation. ChemBioChem 5, 1432–1447. We discuss our model and recent experimental findings demonstrating the effectivity of modifier RNAs in vitro in the context of the current research activities in the field of non-coding RNAs. We speculate that modifier RNAs might also exist in nature; if so, they present an additional regulatory layer for fine-tuning gene expression that could evolve rapidly, leaving no obvious traces in the genomic DNA sequences.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Summary The high sequence identity observed between UNC-93B of mouse and human imply common evolutionary ancestors and a conserved function. A nonconservative point mutation in the mouse Unc93b1 gene ...has been associated with defective Toll-like receptor (TLR) signaling and impaired major histocompatibility complex (MHC) I and II restricted antigen responses 1 . Like murine UNC-93B, the human homologue is predicted to form 12 transmembrane domains, and it localizes to the endoplasmic reticulum. In human beings its expression is highest in professional antigen-presenting cells such as dendritic cells and macrophages. Interestingly, UNC-93B itself is specifically induced by TLR3 signaling in monocyte-derived dendritic cells and macrophages. To study the effect of UNC-93B deficiency in TLR signaling and antigen-presentation in human beings, UNC-93B message was knocked down in monocyte-derived dendritic cells and a reduced TNFα production in response to TLR3 agonists was observed. In the same experiment, the achieved knockdown had no effect on an MHC II–dependent antigen response, suggesting that the reduced quantity of human UNC-93B was still capable of supporting class II antigen presentation or that UNC-93B is not required for class II antigen presentation in human antigen-presenting cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Ceramide kinase (CerK) produces ceramide-1-phosphate, a sphingophospholipid with recognized signaling properties. It localizes to the Golgi complex and fractionates essentially between detergent ...soluble and insoluble fractions; however the determinants are unknown. Here we made a detailed mutagenesis study of the N-terminal Pleckstrin Homology (PH) domain of CerK, based upon modeling, and identified key positively charged amino acid residues within an unusual motif in the loop interconnecting {beta}-strands 6 and 7. These residues are critical for CerK membrane association, phosphoinositide binding and activity. Their mutagenesis results in increased thermolability, sensitivity to proteolysis, reduced apparent molecular weight as well as propensity of the recombinant mutant protein to aggregate, indicating that this loop impacts the overall conformation of the CerK protein. This is in contrast to most PH domains whose function strongly relies on charges located in the {beta}1-{beta}2 loop.
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