Escherichia coli ribosomal RNA contains 10 pseudouridines, one in the 16 S RNA and nine in the 23 S RNA. Previously, the gene for the synthase responsible for the 16 S RNA pseudouridine was identified and cloned, as was a gene for a synthase that makes a single pseudouridine in 23 S RNA. The yceC open reading frame of E. coli is one of a set of genes homologous to these previously identified ribosomal RNA pseudouridine synthases. In this work, the gene was cloned, overexpressed, and shown to code for a pseudouridine synthase able to react with in vitro transcripts of 23 S ribosomal RNA. Deletion of the gene and analysis of the 23 S RNA from the deletion strain for the presence of pseudouridine at its nine known sites revealed that this synthase is solely responsible in vivo for the synthesis of three of the nine pseudouridine residues, at positions 955, 2504, and 2580. Therefore, this gene has been renamed rluC. Despite the absence of one-third of the normal complement of pseudouridines, there was no change in the exponential growth rate in either LB or M-9 medium at temperatures ranging from 24 to 42°C. From this work and our previous studies, we have now identified three synthases that account for 50% of the pseudouridines in the E. coli ribosome.Pseudouridine (⌿), 1 the 5-ribosyl isomer of uridine, occurs in rRNA (1), tRNA (2), and small nuclear and nucleolar RNA (3, 4) but not in mRNA or viral genomic RNAs. All the RNAs in which ⌿ is found share a common characteristic, namely a tertiary structure that must be maintained for proper function. ⌿ is made after the polynucleotide chain has been formed by an enzyme-catalyzed but energy-independent isomerization of uridine (reviewed in Ref. 5). A considerable amount of ⌿ is found in ribosomal RNA approaching 8% of the uridines in mammals (6). The number and distribution of ⌿ is different between the two large rRNAs. In small subunit (SSU) RNA, the number varies from 0 or 1 (yeast mitochondria) to 1 (Escherichia coli) to ϳ40 (mammals), and ⌿ are deployed throughout the molecule, whereas in the large subunit (LSU) RNA, although there is also a wide variation in the number of ⌿ from 1 (yeast mitochondria) to 4 -9 (prokaryotes) to 55-57 (mammals), the distribution is conserved in all organisms to three defined secondary structural regions at or near the peptidyl transferase center (reviewed in Ref. 5). In E. coli, the organism studied in this work, there are 10 ⌿ residues, one in the SSU RNA at position 516 (7) and nine in the LSU RNA at positions 746, 955, 1911, 1915, 1917, 2457, 2504, 2580, and 2605 (8, 9). ⌿1915 is further modified by methylation at N 3 (10).Despite the specificity implicit in the conservation of geographic localization in the LSU to the functionally important peptidyl transferase center, there is no known role for ⌿ in the ribosome. To address this issue, we have embarked on a program to identify all of the synthases responsible for formation of the 10 ⌿ in E. coli rRNA with the aim of deleting specific ⌿ residues by inactivating the genes for th...

In order to identify novel genes associated with the initiation of programmed cell death during development, we employed a differential screening protocol to isolate cDNAs that were induced when the intersegmental muscles (ISM) of the moth Manduca sexta become committed to die at the end of metamorphosis. In this report we provide the first description of Acheron (Achn), a novel protein that was isolated in this screen. Acheron contains three Lupus antigen (La) repeats, nuclear localization and export (NLS and NES) signals, and an RNA recognition motif. Achn defines a new subfamily of La proteins that appears to have branched from authentic La protein relatively late in metazoan evolution. Achn is widely expressed in various insect, mouse and human tissues. Consistent with its expression during ISM death, Achn has been shown in separate studies to control muscle differentiation and apoptosis in both mice and zebrafish. These data define Achn as a newly discovered regulatory molecule that presumably mediates a variety of developmental and homeostatic processes in animals.

During myogenesis, reductions in trophic factor availability signal most myoblasts to fuse, up-regulate the expression of muscle-specific genes, and form myotubes. Those cells failing to differentiate into myotubes initiate apoptosis and rapidly die. At present, the signal-transduction molecules that determine whether myoblasts should differentiate or die are largely unknown. In this report, we describe the cloning and characterization of DALP, a small LIM-only type zinc-finger protein that is induced when the intersegmental muscles (ISMs) of the moth Manduca sexta become committed to die at the end of metamorphosis. Forced expression of death-associated LIM-only protein (DALP) in Drosophila results in skeletal muscle atrophy. Ectopic expression of DALP, or its mammalian ortholog Hic-5, blocks differentiation and induces apoptosis in mouse C 2 C 12 myoblasts. Both of these effects can be overcome by contact with normal myoblasts or by ectopic expression of the muscle-specific transcription factor MyoD. Hic-5 expression is specifically and dramatically induced in normal myoblasts that die after removal of trophic support. Taken together, these data suggest that DALP and Hic-5 act upstream of MyoD and function as phylogenetically conserved ''switches'' to block muscle differentiation and induce death.

The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta, participate in the emergence behavior of the adult moth and then die during the subsequent 30 hours. In addition, several populations of interneurons and uniquely identified motor neurons also die after adult emergence. The trigger for all of these deaths is a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone. The ability of the muscles and neurons to die requires de novo gene expression. A differential hybridization screen of a "condemned" ISM cDNA library permitted the isolation of clones encoding four new up-regulated mRNAs. On sequencing, one of these recombinants was found to encode apolipophorin III (apoLp-III), a component of lipophorin, the major hemolymph lipoprotein of insects, previously shown to be synthesized in fat body. Although apoLp-III mRNA and protein were expressed at all stages of ISM development, levels of both molecules were dramatically elevated with the commitment of the cells to die. When ISM cell death was delayed by injection of 20-hydroxyecdysone, expression of apoLp-III at both the RNA and protein levels was markedly reduced at the normal time of cell death. Immunocytochemistry demonstrated that apoLp-III protein was abundantly expressed in the cytoplasm of dying muscles, interneurons, and identified motor neurons at the time of cell death. Apolipoproteins I and II, required components of lipophorin, were not expressed at detectable levels in the muscles or neurons. Furthermore, Western blots of native gels suggest that apoLp-III was not associated with any other proteins. These data suggest that apoLp-III has activities independent of lipid transport that may play a role in programmed cell death. ApoLp-III joins apolipoproteins E and J (clusterin, sulfated glycoprotein-2) as a group of proteins that function in both lipid transfer and cell death.

The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta participate in the emergence behavior of the adult moth at the end of metamorphosis and then die during the subsequent 30-hr period. The trigger for this death is a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone (20-HE). Previous work has demonstrated that the ability of the ISMs to die is dependent on new gene expression. Using a differential hybridization cloning strategy, a cDNA library made from the ISMs committed to die was screened, and four up-regulated clones were isolated. One clone, 18-56, was selected for this study. Northern and Western analysis demonstrated that while clone 18-56 was expressed in all tissues examined and during every stage of ISM development, there was a dramatic increase in expression at both mRNA and protein levels when the ISMs became committed to die. If ISM death was delayed by an injection of 20-HE on the day proceeding adult emergence, 18-56 expression remained at basal levels. Immunocytochemistry demonstrated that 18-56 protein was located predominantly in nuclei prior to the commitment of the ISMs to die and then accumulated to high levels in cytoplasm at the time of cell death. DNA sequence analysis revealed that 18-56 protein shares 74% identity with yeast SUG1 and 92% with human Trip1, both of which are members of the conserved CAD (Conserved ATPase-containing Domain) family of putative transcriptional regulators. To verify that these genes shared functional as well as sequence homology, Manduca clone 18-56 was transformed into a yeast mutant for SUG1 function. Manduca 18-56 was able to both complement the lethal SUG1 phenotype and to suppress the transcriptional activity of a SUG1 mutation in yeast. Taken together, these data support the hypothesis that members of the phylogenetically conserved CAD family participate in important basal and developmental processes.

RluC from E. coli is the enzyme responsible for catalyzing the isomerization of uridines 955, 2504 and 2580 in 23S rRNA to pseudouridine. Histidine-tagged RluC was cloned, overexpressed and purified by nickel-affinity chromatography. A proteolytically derived fragment of the enzyme consisting of residues 89-319 has been shown to retain catalytic activity. Crystals of this fragment, grown by precipitation with sodium acetate at pH 8.0, belong to space group P321, with unit-cell dimensions a = b = 97.1, c = 86.3 A and have two molecules in the crystallographic asymmetric unit. The flash-frozen crystals diffract X-rays to at least 2.3 A resolution and appear suitable for crystal structure determination.

The term programmed cell death (PCD) was coined in 1965 to describe the loss of the intersegmental muscles (ISMs) of moths at the end of metamorphosis. While it was subsequently demonstrated that this hormonally controlled death requires de novo gene expression, the signal transduction pathway that couples hormone action to cell death is largely unknown. Using the ISMs from the tobacco hawkmoth Manduca sexta, we have found that Acheron/LARP6 mRNA is induced ∼1,000-fold on the day the muscles become committed to die. Acheron functions as a survival protein that protects cells until cell death is initiated at eclosion (emergence), at which point it becomes phosphorylated and degraded in response to the peptide Eclosion Hormone (EH). Acheron binds to a novel BH3-only protein that we have named BBH1 (BAD/BNIP3 homology 1). BBH1 accumulates on the day the ISMs become committed to die and is presumably liberated when Acheron is degraded. This is correlated with the release and rapid degradation of cytochrome c and the subsequent demise of the cell. RNAi experiments in the fruit fly Drosophila confirmed that loss of Acheron results in precocious ecdysial muscle death while targeting BBH1 prevents death altogether. Acheron is highly expressed in neurons and muscles in humans and drives metastatic processes in some cancers, suggesting that it may represent a novel survival protein that protects terminally differentiated cells and some cancers from death.

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