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PubMed1996

The 5' part of the human H19 RNA contains cis-acting elements hampering its translatability.

H19 is an imprinted gene developmentally regulated in man and mouse and implicated in various neoplasms. No corresponding protein product has yet been detected, although several open reading frames (ORFs) could be identified along its RNA. The largest ORF found in the human gene could encode a putative 26 kDa protein. We have isolated two H19 cDNAs (AP and ES) that contain this ORF4 and correspond to incomplete copies of the unique 2.3 kb H19 RNA. In transient expression assays, AP was able to synthesize a 26 kDa protein whereas ES was not. With respect to ORF4, ES exhibits a 536 bp long GC-rich 5' untranslated region, whereas AP contains the last 22 nucleotides of this 5'UTR. Using deletions and point mutations, we have found that the length and probably the secondary structure of the 5'UTR strongly hampers the translatability of the RNA. In addition, a potential role of upstream ORFs (uORFs) was detected as stressed by the enhances translation of a construct mutated in uORF3 overlapping ORF4. Interactions between H19 and proteins are indicated by a specific binding between 5'UTR derived RNA segments and two nuclear proteins of about 27 kDa. Our results favor a potential role of these particular structures and binding properties in general trans-regulation of RNA post-transcriptional processes rather than in normal control of H19 mRNA translation.

Animals↗
PubMed2002

Evidence for translational regulation of the imprinted Snurf-Snrpn locus in mice.

In studies of genomic imprinting in the Prader-Willi/Angelman domain, an agouti coat color cassette was inserted into the downstream open reading frame (ORF) of the imprinted bicistronic Snurf-Snrpn locus in the mouse. The fusion gene was maternally silenced, as is Snurf-Snrpn, and produced a tan abdomen only when inherited paternally in otherwise-black mice. A screen for dominant epigenetic or genetic events was performed with ENU mutagenesis, using a strategy whereby variation in abdominal color was scored at weaning. One mouse with maternal origin of the fusion gene had a tan abdomen and had an imprinting defect resulting in loss of both maternal methylation and silencing of the fusion gene. One mouse with paternal origin of the fusion gene was completely yellow and was found to have an ATG-to-AAG mutation in the initiation codon of the upstream ORF encoding SNURF. Northern blotting, immunoblotting, and transfection studies indicated that the ATG-to-AAG mutation causes a 15-fold or more increase in translation of the downstream ORF in two fusion constructs, and it is likely that similar translational control affects the normal Snurf-Snrpn transcript as well.

Agouti Signaling Protein↗
PubMed1997

Disruption of ribosomal scanning on the 5'-untranslated region, and not restriction of translational initiation per se, modulates the stability of nonaberrant mRNAs in the yeast Saccharomyces cerevisiae.

Translation and mRNA decay constitute key players in the post-transcriptional control of gene expression. We examine the mechanisms by which the 5'-untranslated region (UTR) of nonaberrant mRNAs acts to modulate both these processes in Saccharomyces cerevisiae. Two classes of functional relationship between ribosome-5'-UTR interactions and mRNA decay are identifiable. In the first of these, elements in the main open reading frame (ORF) dictate how the decay process reacts to inhibitory structures in the 5'-UTR. The same types of stability modulation can be elicited by trans-regulation of translation via inducible binding of the iron-regulatory protein to an iron-responsive element located 9 nucleotides from the 5' cap. A eukaryotic translational repressor can therefore modulate mRNA decay via the 5'-UTR. In contrast, translational regulation mediated via changes in the activity of the cap-binding eukaryotic translation initiation factor eIF-4E bypasses translation-dependent pathways of mRNA degradation. Thus modulation of mRNA stability via the 5'-UTR depends on disruption of the scanning process, rather than changes in translational initiation efficiency per se. In the second class of pathway, an upstream ORF (uORF) functions as a powerful destabilizing element, inducing termination-dependent degradation that is apparently independent of any main ORF determinants but influenced by the efficiencies of ribosomal recognition of the uORF start and stop codons. This latter mechanism provides a regulatable means to modulate the stability of nonaberrant mRNAs via a UPF-dependent pathway.

Eukaryotic Initiation Factor-4E↗
PubMed2026

Ribosome stalling position, spacing, and A-site occupancy impact translation and cotranslational mRNA decay in plants.

Ribosomes can pause during mRNA translation, but what causes pausing, how pauses affect protein production, and whether they trigger cotranslational mRNA decay are poorly understood in plants. Here, we investigate the causes and consequences of ribosome pausing in Arabidopsis and maize. This is accomplished by sizing, mapping, and quantifying footprints of individual ribosomes (monosomes) and closely spaced ribosome pairs (disomes) at single-codon resolution on open reading frames (ORFs). Ribosome footprinting was combined with 5'P-degradome-seq to examine the coincidence of pausing with cotranslational decay under control conditions and brief hypoxia in Arabidopsis. The data resolve two monosome conformations and three disome configurations. These include monosomes with a vacant or occupied A-site and disomes that have collided or are separated by one or two codons. Pausing is prevalent at initiation, termination, and di-Proline codons. Di-Proline pauses do not trigger cotranslational decay but appear important in cotranslational protein processing. Brief hypoxia induces stalling of A-site vacant ribosomes at Aspartate codons, often coinciding with 5'P peaks, indicating that rate-limiting decoding can trigger cotranslational mRNA decay. Notably, actively transcribed and translated hypoxia-response mRNAs accumulate 1- to 2-codon-separated disomes and are actively degraded. Comparative analysis of footprints in the two species reveals ribosome conformations and codon-specific pausing can be conserved or lineage-specific, as exemplified by pausing at di-Prolines and on Conserved Peptide upstream ORFs. In sum, the stalling of ribosomes at specific codons, coupled with ribosome A-site occupancy and disome spacing, modulates protein production and cotranslational mRNA decay in plants.

Ribosomes↗
PubMed2006

Mutation, selection, and evolution of the Crohn disease susceptibility gene CARD15.

Three common mutations in the CARD15 (NOD2) gene are known to be associated with susceptibility to Crohn disease (CD), and genetic data suggest a gene dosage model with an increased risk of 2-4-fold in heterozygotes and 20-40-fold in homozygotes. However, the discovery of numerous rare variants of CARD15 indicates that some heterozygotes for the common mutations have a rare mutation on the other CARD15 allele, which would support a recessive model for CD. We addressed this issue by screening CARD15 for mutations in 100 CD patients who were heterozygous for one of the three common mutations. We also developed a strategy for evaluating potential disease susceptibility alleles (DSAs) that involves assessing the degree of evolutionary conservation of involved residues, predicted effects on protein structure and function, and genotyping in a large sample of cases and controls. The evolutionary analysis was aided by sequencing the entire coding region of CARD15 in three primates (chimp, gibbon, and tamarin) and aligning the human sequence with these and orthologs from other species. We found that 11 of the 100 CD patients screened had a second potential pathogenic mutation within the exonic and periexonic sequences examined. Assuming that there are no additional pathogenic mutations in noncoding regions, our study suggests that most carriers of the common DSAs are true heterozygotes, and supports previous evidence for a gene dosage model. Four novel nonsynonymous mutations were detected, one of which would produce premature termination of translation c.2686C>T (p.Arg896X). Two potential DSAs--c.2107C>T (p.Arg703Cys) and g.2238T>A (c.74-7T>A)--were significantly associated with CD in the case control sample. Analysis of the evolution of CARD15 revealed strong conservation of the encoded protein, with identity to the human sequence ranging from 99.1% in the chimp to 44.5% in fugu. Higher primates possess an open reading frame (ORF) upstream of the putative initiation site in other species that encodes a further 27 N-terminal amino acids, while four regions of high conservation are observed outside of the known domains of CARD15, indicative of additional residues of functional importance. The strategy developed here may have general application to the assessment of mutation pathogenicity and genetic models in other complex disorders.

Alleles↗
PubMed1996

The Pseudomonas aeruginosa fumc and soda genes belong to an iron-responsive operon.

Pseudomonas aeruginosa contains two superoxide dismutases (SOD), a Mn-containing SOD (Mn-SOD) and a Fe-SOD, which are encoded by sodA and sodB, respectively. We have cloned and sequenced a DNA fragment from P. aeruginosa, strain CHA, which contains the sodA gene and three other open reading frames (ORF). We report here that one of the ORFs upstream from sodA is fumC, which encodes the O2.- resistant isoform of fumarase (or fumarate hydratase). It is shown that fumC and sodA belong to the same operon. By primer extension experiments, the transcription initiation site has been located at -413 from the ATG codon of the fumC gene. The fumC-sodA operon was found to be negatively regulated in presence of iron and the E. coli FUR protein was shown to bind to the 19-bp FUR consensus sequence present at the transcription start site of the operon.

Bacterial Proteins↗
PubMed1999

Gene expression from the ORF50/K8 region of Kaposi's sarcoma-associated herpesvirus.

The ORF50 gene of Kaposi's sarcoma (KS)-associated herpesvirus, or human herpesvirus 8 (KSHV), activates viral replication and is weakly homologous to the herpesvirus family of R transactivators; therefore, the transcription and translation events from this region of KSHV are key events in viral reactivation. We demonstrate that ORF50 is expressed in a bicistronic message after induction of the viral lytic cycle. ORF50 migrated as a series of polypeptides: the major ones as 119 and 101 kDa, respectively. Using 3' rapid amplification of cDNA ends, RT-PCR, and cDNA library screening, we demonstrate that the major ORF50 transcript also encodes K8. The ORF50/K8 transcript was resistant to cyclohexamide, whereas the K8 transcript was only partially resistant to cyclohexamide at early timepoints. Both transcripts showed partial resistance after 12 h of phorbol ester induction. Using a GAL4-ORF50 fusion protein expression vector, we demonstrate that the transactivation domain of ORF50 resides within a 160-amino-acid region of the carboxyl portion of the ORF. Upstream regions of both ORF50 and K8 have basal promoter activity in KSHV-infected cells. K8, which had sequence homology to Bzip proteins, did not activate either promoter. However, both promoters were activated after cotransfection of ORF50 in BCBL-1 cells.

Alternative Splicing↗
PubMed2001

Expression of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor monocistronic and bicistronic transcripts in primary effusion lymphomas.

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) encodes a G protein-coupled receptor (vGPCR) in open reading frame (ORF) 74, which is homologous to human chemokine receptors. KSHV vGPCR is constitutively active and induces VEGF-mediated angiogenesis. Previous studies have shown that ORF 74 is transcribed as part of a bicistronic message containing ORF K14 upstream of ORF 74, with an early lytic pattern of expression. We have now extended these studies by analyzing three different KSHV-positive primary effusion lymphoma (PEL) cell lines and three PEL clinical samples. In addition, we have identified another less abundant monocistronic transcript containing only ORF 74. Both transcripts were identified at low but similar levels in two PEL clinical samples. We evaluated the degree of sequence and functional conservation of ORF74 in three additional PELs and two KS clinical specimens, demonstrating complete identity at the amino acid level among all isolates. While it is expressed as an early lytic transcript in PEL cell lines, in primary clinical PEL samples transcription of KSHV vGPCR can be readily detected.

Amino Acid Sequence↗
PubMed1992

Sex pheromone plasmid pAD1-encoded surface exclusion protein of Enterococcus faecalis.

During conjugative transfer of sex pheromone plasmids of Enterococcus faecalis a so-called surface exclusion protein reduces the frequency with which these plasmids are transferred to cells already possessing the same plasmid. We report here the DNA sequence of a 3.8 kb fragment of the sex pheromone plasmid pAD1 containing the structural gene sea1 for surface exclusion protein and a small open reading frame (ORF) upstream of sea1. Surface exclusion protein Sea1 was found to be highly homologous to the surface exclusion protein Sec10 encoded by the sex pheromone plasmid pCF10. Hybridization studies with DNA probes derived from the structural gene sea1 demonstrated that, with the exception of pAM373, all known sex pheromone plasmids carry a homologous gene. These studies also indicated that the genetic organization is similar in these plasmids, with the structural gene for surface exclusion protein being located 5' to that for aggregation substance.

Amino Acid Sequence↗
PubMed1999

A gene (tmpA) for an efflux protein of the transporter family III from Brevibacterium linens OC2, an antibacterial substance-producing strain.

A gene (tmpA) encoding a putative transmembrane protein has been cloned from B. linens OC2, an antibacterial substance-producing strain. The deduced TmpA protein sequence shares similarities to members of the transporter family III exploiting the transmembrane proton gradient to provide export of toxic compounds such as antiseptics or antibiotics. Northern blot analysis indicated that tmpA gene is expressed. Length of RNA messenger and overlapping of ORFs upstream tmpA gene suggested that it might belong to an operon. The tmpA gene is unusual among B. linens species since it was not detected among eight B. linens collection strains and 40 B. linens industrial strains.

Amino Acid Motifs↗
PubMed1996

Molecular characterization of the xerC gene of Lactobacillus leichmannii encoding a site-specific recombinase and two adjacent heat shock genes.

Sequencing of four overlapping DNA fragments comprising 3.527 kb isolated from a L. leichmannii genomic library revealed three complete open reading frames (ORFs) and one that was truncated. The deduced amino acid sequences of the complete ORFs showed considerable similarities with the already known sequences of the xerC, hslV, and hslU gene products of Escherichia coli: the site-specific XerC recombinase, a member of the lambda integrase family, and the HtpI resp. HtpO heat shock proteins. The deduced amino acid sequence of the fourth, incomplete ORF upstream the xerC gene showed strong homology with the gid A gene product of B. subtilis.

Amino Acid Sequence↗
PubMed1998

Isolation and characterisation of the acetyl-CoA carboxylase gene from Aspergillus nidulans.

The putative gene encoding acetyl-CoA carboxylase, accA, has been isolated from Aspergillus nidulans. This single-copy gene has an open reading frame (ORF) of 6864 bp and contains two small introns near the 5'-end. A short ORF upstream of the ATG start codon has been identified in this gene by RT-PCR. Based on sequence homology to acetyl-CoA carboxylases from other organisms, putative biotin-, ATP-, HCO3-- and acetyl-CoA- binding sites have been assigned. Northern data and ACC enzyme-activity measurements from A. nidulans suggested that expression of accA was higher in media containing nitrate than ammonia as a sole nitrogen source. Deletion of accA in A. nidulans was unsuccessful. The failure of A. nidulans to grow in the presence of the ACC-specific inhibitor, soraphen A, supplemented with C16-18 fatty acids suggested that ACC is an essential enzyme.

Acetyl-CoA Carboxylase↗
PubMed1999

Functional analysis of multiple AUG codons in the transcripts of the STA2 glucoamylase gene from Saccharomyces cerevisiae.

A scanning ribosome will usually initiate translation as soon as it encounters the first favourable AUG codon and only a few eukaryotic transcripts have more complex arrangements. These relatively few complex transcripts are normally characterized by structural features such as multiple AUGs and significant secondary structure. However, the functional relevance of these features has rarely been established. We present here a study of the functional significance of the multiple AUGs in the leader of STA2 transcripts of the budding yeast Saccharomyces cerevisiae, and extrapolate, where applicable, these results to a co-regulated gene, MUC1. The STA2 gene (a representative member of the polymorphic STA1-3 gene family), encodes an extracellular glucoamylase, and is evolutionarily linked to, and transcriptionally co-regulated with, the MUC1 gene, which encodes a mucin-like protein essential for pseudohyphal/invasive growth and cell-adhesion in S. cerevisiae. Each of these genes contains a putative upstream ORF, while STA2 has two additional in-frame AUG codons 5' to the major cistron. We show that utilization of the alternative translational start-sites of STA2 results in glucoamylases that differ at their N-termini, which are associated with differences in their localization patterns. Analysis of mutants revealed the presence of a putative secretion-enhancing signal that might prove to be relevant to the alternative targeting mechanism recently uncovered in S. cerevisiae. We show that a short up-stream ORF present in the leaders of STA1-3 and MUC1 is probably bypassed by a process of leaky scanning.

Amino Acid Sequence↗
PubMed1998

An onion isolate of tobacco rattle virus: reactivity with an antiserum to Hypochoeris mosaic virus, a putative furovirus, and molecular analysis of its RNA 2.

A tubular virus from onion was found to react with an antiserum to Hypochoeris mosaic virus (HyMV), a putative furovirus. Sequence analysis of its genomic RNAs and further serological tests, however, indicated it to be a tobravirus rather than a furovirus. The reactivity of the HyMV antiserum with several isolates of tobacco rattle virus (TRV) suggests that HyMV itself may be a tobravirus. The deduced amino acid sequences of the putative proteins encoded on RNA 2 of the onion virus isolate (ON) suggest close evolutionary relationships to the TRV isolate TCM from tulip. However, RNA 2 of the ON isolate contains a shorter RNA 1-like sequence on its 3'-end and an additional small ORF upstream of its RNA 1-like part. The sequence of its 315 5'-terminal nucleotides is more similar to that of RNA 2 of the PLB isolate from potato than to that of TCM RNA 2.

Amino Acid Sequence↗
PubMed1992

5' end-dependent translation initiation of hepatitis C viral RNA and the presence of putative positive and negative translational control elements within the 5' untranslated region.

Hepatitis C virus (HCV) is a distant relative of pestiviruses and flaviviruses, but it has a 5' untranslated region (UTR) with some features structurally similar to that of picornaviruses. In order to test the role of the 5' UTR in controlling the expression of the HCV polyprotein, we fused full-length or deleted versions of the 5' UTR of HCV-1 RNA to chloramphenicol acetyl transferase (CAT) mRNA to monitor CAT activity in vivo. We found: (1) the full-length 5' UTR of HCV-1 RNA is translationally inactive while 5' deletions which mimic a 5' subgenomic RNA detected in vivo are active, (2) an efficient cis-acting element which represses translation is found at the 5' terminus, (3) a putative element which enhances translation is found near the 3' terminus of the 5' UTR, (4) additional cis-acting elements including small open reading frames (ORFs) upstream from the putative enhancer element downregulate translation. We did not find evidence supporting the existence of an internal ribosome entry site in the 5' UTR of HCV-1 RNA. These data suggest that HCV may employ a distinctive translation control strategy such as the generation of subgenomic viral mRNA in infected cells. Translational control of HCV might be responsible for some of the characteristic pathobiology seen in viral infection.

Base Sequence↗
PubMed2005

Genetic and functional analysis of Haemophilus somnus high molecular weight-immunoglobulin binding proteins.

Haemophilus somnus immunoglobulin binding proteins (IgBPs) are virulence associated but only one (p76) has been genetically defined. We determined the nucleotide sequence of the 5'-flanking region of the p76 gene. This region had been identified as the coding region for a series of high molecular weight (HMW)-IgBPs. Analysis of the nucleotide sequence indicated the gene (immunoglobulin binding protein A, ibpA) encoding the HMW and p76 IgBPs comprised a single open reading frame of 12,285 base pairs (bp). The ibpA gene is flanked by an upstream ORF of 1758bp, designated ibpB. The predicted amino acid sequences of these two genes demonstrate similarity to virulence exoproteins and their transporter proteins that comprise a two-partner secretion pathway in various Gram-negative bacteria. Motifs associated with binding to mammalian cells were also identified within the sequence. Competitive inhibition studies implicated a putative heparin-binding domain in adherence to bovine endothelial cells. Expression plasmids for glutathione S-transferase (GST)-fused recombinant fragments covered amino acid residues 972-3201. IgG2 Fc binding studies identified fragment 972-1515 (GST-IbpA3) as an Fc binding peptide. This peptide and GST-IbpA5 (aa 2071-2730) reacted strongly with convalescent phase serum. In a small preliminary study, calves immunized with the purified GST-IbpA3 peptide were protected against an intrabronchial H. somnus challenge.

Amino Acid Motifs↗
PubMed2004

Immediate early genes expressed in chlorovirus infections.

Twenty-three chlorovirus genes expressed in host cells as early as 5-10 min postinfection (p.i.), or immediate early, were isolated and characterized. Some showed significant homology with those for transcriptional factors and mRNA-processing proteins including TFIIB, helicases, mRNA capping enzyme, nucleolin, and bean transcription factor. Others code for (i) factors influencing translation such as aminoacyl tRNA synthetases and ribosomal protein, and (ii) unknown proteins. Enzymes involved in polysaccharide synthesis were also found. All transcripts of these genes had a poly(A) tail, which decreased in size after 20 min p.i., possibly caused by the shortening by an exonuclease. Often, due to readthrough either from an upstream ORF or into a downstream ORF, a few extra transcripts for each gene appeared after 40 min p.i., suggesting a change in promoter selection and termination accuracy at this point. A typical TATA-box and a common element 5'-ATGACAA were in the promoter region of almost all of the immediate early genes, which may be recognized by host RNA polymerase and transcription factors.

Animals↗
PubMed1996

Identification of a gene, pilF, required for type 4 fimbrial biogenesis and twitching motility in Pseudomonas aeruginosa.

Many bacterial pathogens produce a class of surface structures called type 4 fimbriae. In Pseudomonas aeruginosa these fimbriae are responsible for adhesion and translocation across host epithelial surfaces. We have identified a novel gene involved in the complex process of type 4 fimbrial biogenesis. This gene, termed pilF, is located on SpeI fragment S at 30 min on the P. aeruginosa genomic map, which is the sixth region on the chromosome shown to contain a fimbrial-associated gene. The PilF protein has a predicted M(r) of 22402, and together with a highly homologous upstream ORF shares a chromosomal arrangement similar to that found in Haemophilus influenzae. A pilF mutant is blocked in the export/assembly of the fimbrial subunit PilA, and accumulates this protein in the membrane fraction. Complementation studies indicate that the cloned pilF gene is able to restore the expression of surface fimbriae, twitching motility and susceptibility to fimbrial-specific bacteriophage.

Amino Acid Sequence↗