Translation initiation factor eIF4B interacts with a picornavirus internal ribosome entry site in both 48S and 80S initiation complexes independently of initiator AUG location.
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Most eukaryotic initiation factors (eIFs) are required for internal translation initiation at the internal ribosome entry site (IRES) of picornaviruses. eIF4B is incorporated into ribosomal 48S initiation complexes with the IRES RNA of foot-and-mouth disease virus (FMDV). In contrast to the weak interaction of eIF4B with capped cellular mRNAs and its release upon entry of the ribosomal 60S subunit, eIF4B remains tightly associated with the FMDV IRES during formation of complete 80S ribosomes. Binding of eIF4B to the IRES is energy dependent, and binding of the small ribosomal subunit to the IRES requires the previous energy-dependent association of initiation factors with the IRES. The interaction of eIF4B with the IRES in 48S and 80S complexes is independent of the location of the initiator AUG and thus independent of the mechanism by which the small ribosomal subunit is placed at the actual start codon, either by direct internal ribosomal entry or by scanning. eIF4B does not greatly rearrange its binding to the IRES upon entry of the ribosomal subunits, and the interaction of eIF4B with the IRES is independent of the polypyrimidine tract-binding protein, which enhances FMDV translation. (+info)
Inhibition of muscle protein synthesis by alcohol is associated with modulation of eIF2B and eIF4E.
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The present study examined potential mechanisms for the inhibition of protein synthesis in skeletal muscle after chronic alcohol consumption. Rats were maintained on an alcohol-containing diet for 14 wk; control animals were pair fed. Alcohol-induced myopathy was confirmed by a reduction in lean body mass as well as a decrease in the weight of the gastrocnemius and psoas muscles normalized for tibial length. No alcohol-induced decrease in total RNA content (an estimate of ribosomal RNA) was detected in any muscle examined, suggesting that alcohol reduced translational efficiency but not the capacity for protein synthesis. To identify mechanisms responsible for regulating translational efficiency, we analyzed several eukaryotic initiation factors (eIF). There was no difference in the muscle content of either total eIF2alpha or the amount of eIF2alpha in the phosphorylated form between alcohol-fed and control rats. Similarly, the relative amount of eIF2Bepsilon in muscle was also not different. In contrast, alcohol decreased eIF2B activity in psoas (fast-twitch) but not in soleus or heart (slow-twitch) muscles. Alcohol feeding also dramatically influenced the distribution of eIF4E in the gastrocnemius (fast-twitch) muscle. Compared with control values, muscle from alcohol-fed rats demonstrated 1) an increased binding of the translational repressor 4E-binding protein 1 (4E-BP1) with eIF4E, 2) a decrease in the phosphorylated gamma-form of 4E-BP1, and 3) a decrease in eIF4G associated with eIF4E. In summary, these data suggest that chronic alcohol consumption impairs translation initiation in muscle by altering multiple regulatory sites, including eIF2B activity and eIF4E availability. (+info)
Alpha-momorcharin inhibits HIV-1 replication in acutely but not chronically infected T-lymphocytes.
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AIM: To identify the anti-human immunodeficiency virus type 1 (HIV-1) activities of alpha-momorcharin (alpha-MMC) from Momordica charantia in acutely and chronically infected T-lymphocytes. METHODS: The anti-HIV activities of alpha-MMC were examined by 1) the inhibition of syncytia formation induced by HIV-1 III B; 2) reduction of p24 core antigen expression level and decrease in numbers of HIV antigen positive cells in acutely and chronically infected cultures. The cytotoxic effects of alpha-MMC was tested by trypan blue dye exclusion or colorimetric MTT assay. RESULTS: alpha-MMC was found to obviously inhibit HIV-1 III B-inducing C8166 syncytia formation and markedly reduced both expression of p24 core antigen and the numbers of HIV antigen positive cells in acutely but not chronically HIV-1-infected culture. The median effective concentration (EC50) in these assays were 0.016, 0.07, and 0.32 mg.L-1, respectively. CONCLUSION: alpha-MMC is a unique component of momorcharin with anti-HIV activity, and markedly inhibited HIV-1 replication in acutely but not chronically HIV-1-infected T-lymphocytes. (+info)
Phosphorylation of yeast ribosomal proteins by CKI and CKII in the presence of heparin.
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We have found that heparin has a different effect on Trichosporon cutaneum ribosomal protein phosphorylation by CKI and by CKII. In the presence of heparin, modification of 13 kDa, 19 kDa and 38 kDa proteins catalyzed by CKII was inhibited, while in the case of CKI, in addition to protein of 15 kDa, phosphorylation of 20 kDa and 35 kDa proteins was detected. It was also found that, in the presence of heparin, phosphorylation of P proteins (13 kDa and 38 kDa) by ribosome-bound protein kinases was inhibited. Moreover at the same conditions modification of 40 kDa protein was observed in all four yeast species tested. (+info)
Direct identification of NH...N hydrogen bonds in non-canonical base pairs of RNA by NMR spectroscopy.
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It is shown that the recently developed quantitative J(NN)HNN-COSY experiment can be used for the direct identification of hydrogen bonds in non-canonical base pairs in RNA. Scalar(2h)J(NN)couplings across NH.N hydrogen bonds are observed in imino hydrogen bonded GA base pairs of the hpGA RNA molecule, which contains a tandem GA mismatch, and in the reverse Hoogsteen AU base pairs of the E-loop of Escherichia coli 5S rRNA. These scalar couplings correlate the imino donor(15)N nucleus of guanine or uridine with the acceptor N1 or N7 nucleus of adenine. The values of the corresponding(2h)J(NN)coupling constants are similar in size to those observed in Watson-Crick base pairs. The reverse Hoogsteen base pairs could be directly detected for the E-loop of E.coli 5S rRNA both in the free form and in a complex with the ribosomal protein L25. This supports the notion that the E-loop is a pre-folded RNA recognition site that is not subject to significant induced conformational changes. Since Watson-Crick GC and AU base pairs are also readily detected the HNN-COSY experiment provides a useful and sensitive tool for the rapid identification of RNA secondary structure elements. (+info)
Identification of abundantly expressed novel and conserved genes from the infective larval stage of Toxocara canis by an expressed sequence tag strategy.
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Larvae of Toxocara canis, a nematode parasite of dogs, infect humans, causing visceral and ocular larva migrans. In noncanid hosts, larvae neither grow nor differentiate but endure in a state of arrested development. Reasoning that parasite protein production is orientated to immune evasion, we undertook a random sequencing project from a larval cDNA library to characterize the most highly expressed transcripts. In all, 266 clones were sequenced, most from both 3' and 5' ends, and similarity searches against GenBank protein and dbEST nucleotide databases were conducted. Cluster analyses showed that 128 distinct gene products had been found, all but 3 of which represented newly identified genes. Ninety-five genes were represented by a single clone, but seven transcripts were present at high frequencies, each composing >2% of all clones sequenced. These high-abundance transcripts include a mucin and a C-type lectin, which are both major excretory-secretory antigens released by parasites. Four highly expressed novel gene transcripts, termed ant (abundant novel transcript) genes, were found. Together, these four genes comprised 18% of all cDNA clones isolated, but no similar sequences occur in the Caenorhabditis elegans genome. While the coding regions of the four genes are dissimilar, their 3' untranslated tracts have significant homology in nucleotide sequence. The discovery of these abundant, parasite-specific genes of newly identified lectins and mucins, as well as a range of conserved and novel proteins, provides defined candidates for future analysis of the molecular basis of immune evasion by T. canis. (+info)
Isolation of DICE1: a gene frequently affected by LOH and downregulated in lung carcinomas.
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In the development and progression of sporadic tumors multiple tumor suppressor genes are inactivated that may be distinct from predisposing cancer genes. Previously, a tumor suppressor locus on human chromosome 13q14 that is distinct from the retinoblastoma predisposing gene 1 (RB1) has been identified in lung, head and neck, breast, ovarian and prostate tumors. By an approach that combines genomic difference cloning and positional cloning we isolated the cDNA of a novel gene (DICE1) located at 13q14.12-14.2. The DICE1 gene is highly conserved in evolution and its mRNA is expressed in a wide variety of fetal and adult tissues. The DICE1 cDNA encodes a predicted protein of 887 amino acids corresponding to an 100 kD protein that shows 92.9% identity to the carboxy-terminal half of the mouse EGF repeat transmembrane protein DBI-1. The DBI-1 protein interferes with the mitogenic response to insulin-like growth factor 1 (IGF-I) and is presumably involved in anchorage-dependent growth. When compared to normal lung tissue expression of the DICE1 mRNA was reduced or undetectable in the majority of non-small cell lung carcinomas analysed. The location of the DICE1 gene in the region of allelic loss, its high evolutionary conservation and the downregulation of expression in carcinoma cells suggests that DICE1 is a candidate tumor suppressor gene in non-small cell lung carcinomas and possibly in other sporadic carcinomas. (+info)
Temperature-sensitive mutations in the Saccharomyces cerevisiae MRT4, GRC5, SLA2 and THS1 genes result in defects in mRNA turnover.
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In a screen for factors involved in mRNA turnover, four temperature-sensitive yeast strains (ts1189, ts942, ts817, and ts1100) exhibited defects in the decay of several mRNAs. Complementation of the growth and mRNA decay defects, and genetic experiments, revealed that ts1189 is mutated in the previously unknown MRT4 gene, ts942 is mutated in GRC5 (encoding the L9 ribosomal protein), ts817 contains a mutation in SLA2 (encoding a membrane protein), and ts1100 contains a mutation in THS1 (encoding the threonyl-tRNA synthetase). Three of the four mutants (mrt4, grc5, and sla2) were not defective in protein synthesis, suggesting that these strains contain mutations in factors that may play a specific role in mRNA decay. The mRNA stabilization observed in the ths1 strain, however, could be due to the significant drop in translation observed in this mutant at 37 degrees. While the three interesting mutants appear to encode novel mRNA decay factors, at least one could be linked to a previously characterized mRNA decay pathway. The growth and mRNA decay defects of ts942 (grc5) cells were suppressed by overexpression of the NMD3 gene, encoding a protein shown to participate in a two-hybrid interaction with the nonsense-mediated decay protein Upf1p. (+info)