Quantitative Proteomic Analysis of Ribosome Assembly and Turnover In Vivo

Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and ¹⁵N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with ¹⁵N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.     

Protein kinase C-epsilon is involved in the adenosine-activated signal transduction pathway conferring protection against ischemia-reperfusion injury in primary rat neuronal cultures

Adenosine activates a signal transduction pathway (STP) in the heart and the brain, conferring protection against ischemia-reperfusion insult. Activation of protein kinase C (PKC), probably mainly PKC-epsilon, has been demonstrated to be part of the heart STP, but its role in the neuronal pathway is less clear. Here, we provide proof for the participation of PKC-epsilon in the neuronal adenosine-activated STP. Primary rat neuronal cultures were exposed to chemical ischemia by iodoacetate, followed by reperfusion. The cultured neurons were protected against this insult by activation of the adenosine mechanism, by N6-(R)-phenylisopropyladenosine [R(-)-PIA], a specific A1 adenosine receptor agonist. Exposure of the cultures to bisindolylmaleimide I, a highly selective PKC inhibitor, abrogated the protection. The exposure of the cultures to R(-)-PIA was found to result in phosphorylation (activation) of PKC-epsilon. Furthermore, insertion into the cells of a specific peptide inhibitor of PKC-epsilon translocation (epsilonV1-2), also abrogated the protection conferred by R(-)-PIA. These results demonstrate that activation of PKC-epsilon is a vital step in the neuronal adenosine-activated STP.     

Cryoelectron microscopy and cryoelectron tomography of the nuclear pre-mRNA processing machine

Large nuclear ribonucleoprotein particles, which can be viewed as the naturally assembled precursor messenger RNA (pre-mRNA) processing machine, were analyzed in frozen-hydrated preparations by cryoelectron microscopy. A general and reproducible strategy for preparing ice-embedded large nuclear ribonucleoprotein (lnRNP) particles at sufficiently high concentration was developed. Taking advantage of their negatively charged components, the lnRNP particles are adsorbed and thus concentrated on a positively charged lipid monolayer while preserving their native structure. Using this approach we carried out cryoelectron tomography and three-dimensional image reconstruction of individual lnRNP particles. The study revealed a structure similar to that of negatively stained particles studied previously, yet with additional features. The small additional domain visualized in negative stain appeared to be larger in the ice preparations. In addition, using image restoration from focus series of ice-embedded lnRNP particles, new features such as holes within the subunits were visualized in two dimensions, and it was shown that the subunits are interconnected via a fiber, very likely formed by the pre-mRNA. This finding supports the model that each subunit represents a spliceosome that splices out the intron wound around it.     

Independent gene phylogenies and morphology demonstrate a malagasy origin for a wide-ranging group of swallowtail butterflies

Madagascar is home to numerous endemic species and lineages, but the processes that have contributed to its endangered diversity are still poorly understood. Evidence is accumulating to demonstrate the importance of Tertiary dispersal across varying distances of oceanic barriers, supplementing vicariance relationships dating back to the Cretaceous, but these hypotheses remain tentative in the absence of well-supported phylogenies. In the Papilio demoleus group of swallowtail butterflies, three of the five recognized species are restricted to Madagascar, whereas the remaining two species range across the Afrotropical zone and southern Asia plus Australia. We reconstructed phylogenetic relationships for all species in the P. demoleus group, as well as 11 outgroup Papilio species, using 60 morphological characters and about 4 kb of nucleotide sequences from two mitochondrial (cytochrome oxidase I and II) and two nuclear (wg and EF-1alpha) genes. Of the three endemic Malagasy species, the two that are formally listed as endangered or at risk represented the most basal divergences in the group, while the more common third endemic was clearly related to African P. demodocus. The fifth species, P. demoleus, showed little differentiation across southern Asia, but showed divergence from its subspecies sthenelus in Australia. Dispersal-vicariance analysis using cladograms derived from morphology and three independent genes indicated a Malagasy diversification of lime swallowtails in the middle Miocene. Thus, diversification processes on the island of Madagascar may have contributed to the origin of common butterflies that now occur throughout much of the Old World tropical and subtemperate regions. An alternative hypothesis, that Madagascar is a refuge for ancient lineages resulting from successive colonizations from Africa, is less parsimonious and does not explain the relatively low continental diversity of the group.     

Mutations in microcephalin cause aberrant regulation of chromosome condensation

Microcephalin (MCPH1) is a gene mutated in primary microcephaly, an autosomal recessive neurodevelopmental disorder in which there is a marked reduction in brain size. PCC syndrome is a recently described disorder of microcephaly, short stature, and misregulated chromosome condensation. Here, we report the finding that MCPH1 primary microcephaly and PCC syndrome are allelic disorders, both having mutations in the MCPH1 gene. The two conditions share a common cellular phenotype of premature chromosome condensation in the early G2 phase of the cell cycle, which, therefore, appears to be a useful diagnostic marker for individuals with MCPH1 gene mutations. We demonstrate that an siRNA-mediated depletion of MCPH1 is sufficient to reproduce this phenotype and also show that MCPH1-deficient cells exhibit delayed decondensation postmitosis. These findings implicate microcephalin as a novel regulator of chromosome condensation and link the apparently disparate fields of neurogenesis and chromosome biology. Further characterization of MCPH1 is thus likely to lead to fundamental insights into both the regulation of chromosome condensation and neurodevelopment.     

Regulation of splicing: the importance of being translatable

RNA sequences that conform to the consensus sequence of 5' splice sites but are not used for splicing occur frequently in protein coding genes. Mutational analyses have shown that suppression of splicing at such latent sites may be dictated by the necessity to maintain an open reading frame in the mRNA. Here we show that stop codon frequency in introns having latent 5' splice sites is significantly greater than that of introns lacking such sites and significantly greater than the expected occurrence by chance alone. Both observations suggest the occurrence of a general mechanism that recognizes the mRNA reading frame in the context of pre-mRNA.     

Stop codon-mediated suppression of splicing is a novel nuclear scanning mechanism not affected by elements of protein synthesis and NMD

The pre-mRNA splicing machine must frequently discriminate between normal and many potential 5'splice sites that match the consensus sequence but remain latent. Suppression of splicing (SOS) at such latent 5'splice sites is required for the maintenance of an open reading frame, and to ensure that only RNAs that encode for functional proteins will be formed. In this study we show that SOS is a novel mechanism distinct from the known RNA surveillance mechanisms. First, SOS is distinct from nonsense-mediated mRNA decay (NMD) because it is not dependent on translation and is not affected by RNAi-mediated down-regulation of hUpf1 and hUpf2--two key components of the NMD pathway. Second, SOS is distinct from nonsense-associated alternative splicing (NAS), because a mutant of hUpf1, which was shown to abrogate NAS, does not activate latent splicing. Elucidating the mechanism of SOS is pertinent to human disease in view of the large number of human genes that harbor latent splice sites.     

Percutaneous coronary intervention with adjunctive abciximab and clopidogrel in a patient with chronic idiopathic thrombocytopaenic purpura

The use of the antiplatelet agents abciximab and clopidogrel is now accepted therapy in percutaneous coronary intervention. We present a case in which these agents were used in a patient with idiopathic thrombocytopaenic purpura and a platelet count of 40x10(9)/l undergoing primary multivessel coronary stenting. This case shows that unstable coronary syndromes can occur in patients with thrombocytopaenia and that antiplatelet agents may be used safely in this context.     

CD4+ T cell and eosinophil adhesion is mediated by specific ICAM-3 ligation and results in eosinophil activation

T cells and eosinophils, which are found in close proximity in asthmatic lungs, express many surface receptors that are counterligands. These data suggest that direct interactions between these cell types could play an important role in regulating airway inflammation in asthma. We examined the effect of selective adhesion between counterligands on human eosinophils and CD4+ T cells to determine 1) the existence of specific adhesive interactions and 2) if augmented specific adhesion to CD4+ T cells also caused augmented secretion of leukotriene C4 (LTC4) from eosinophils. A new method for binding of human CD4+ T cells to microwell plates was developed, which allowed for specific quantitative assessment of eosinophil adhesion to individual CD4+ T cells in culture. Adhesion of CD4+ T cells to eosinophils was minimal in unstimulated cells but increased after activation of T cells by PMA. Augmented adhesion was regulated substantially through binding of ICAM-3 and only minimally by ICAM-1. We further evaluated whether this specific adhesion up-regulated stimulated secretion of LTC4 from eosinophils. Adhesion with CD4+ T cells augmented eosinophil secretion of LTC4 caused by FMLP plus cytochalasin. Blockade of ICAM-3, as well as ICAM-1, inhibited completely the augmented secretion of eosinophil LTC4. We demonstrate that eosinophils and CD4+ T cells are capable of ligand-specific adhesion that is mediated predominantly by ICAM-3 ligation and that this binding causes augmented eosinophil secretion.