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RNA editing and alternative splicing generate mRNA transcript diversity from the Drosophila 4f-rnp locus.

Extensive sequencing of genomic 4f-rnp and 15 cDNA clones isolated from libraries of 0-4-h embryos, pupae, and adult heads from Drosophila melanogaster has enabled identification of factors resulting in transcript sequence diversity. The 4f-rnp gene contains eight small introns. Among non-edited cDNAs, one transcript class potentially encodes a 943-amino-acid protein, within which several motifs are predicted, including a single C-terminal RNA-binding domain. Intron 5 is retained in all cDNAs examined except for a pupal cDNA, where it is excised. This potentially introduces an in-frame stop codon and predicts a truncated protein of 639 amino acids. One adult head transcript class is edited, some 31% of As being converted to Gs exclusive of edits within introns. An edit site in intron 4 changes a canonical 3'-terminal AG to GG, which interferes with splicing and is predicted to introduce a stop codon. A potential editing substrate recognition element in 4f-rnp contains the weak consensus sequence: 5'-G-G-G-N-A-A-G-3', which may interact with double-stranded RNA adenosine deaminase following pairing of 4f-rnp mRNA with an antisense transcript. It is possible that extensive editing in 4f-rnp destabilizes transcripts and thus provides a novel mechanism for post-transcriptional control of gene expression, although resolution of this point will require study of additional edited transcripts.

Alternative Splicing↗

Structure of the C-terminal end of the nascent peptide influences translation termination.

The efficiency of translation termination at NNN NNN UGA A stop codon contexts has been determined in Escherichia coli. No general effects are found which can be attributed directly to the mRNA sequences itself. Instead, termination is influenced primarily by the amino acids at the C-terminal end of the nascent peptide, which are specified by the two codons at the 5' side of UGA. For the penultimate amino acid (-2 location), charge and hydrophobicity are important. For the last amino acid (-1 location), alpha-helical, beta-strand and reverse turn propensities are determining factors. The van der Waals volume of the last amino acid can affect the relative efficiency of stop codon readthrough by the wild-type and suppressor forms of tRNA(Trp) (CAA). The influence of the -1 and -2 amino acids is cooperative. Accumulation of an mRNA degradation intermediate indicates mRNA protection by pausing ribosomes at contexts which give inefficient UGA termination. Highly expressed E.coli genes with the UGA A termination signal encode C-terminal amino acids which favour efficient termination. This restriction is not found for poorly expressed genes.

Amino Acid Sequence↗

Novel paraoxonase (PON1) nonsense and missense mutations predicted by functional genomic assay of PON1 status.

Paraoxonase (PON1) has been termed an environmental response enzyme for its function in the detoxification of organophosphate pesticides, nerve agents and pharmaceuticals such as glucocorticoids and statins, as well as its cardioprotective role in breaking down oxidized LDL. PON1(192) genotype can be predicted with high accuracy from an examination of the two-dimensional plot of paraoxon and diazoxon hydrolysis rates [ 1]. Individuals for whom this functional genomic assay failed to predict PON1(192) genotype, or who had a low PON activity relative to others with the same genotype, were predicted to have genetic alterations that explained the inconsistency. Sequencing of the PON1 region of 23 Caucasian individuals detected a nonsense mutation changing amino acid 194 from a Trp to a stop codon (PON1(Trp194stop)). It was predicted that subjects who genotyped as PON1(192QR) but phenotyped as PON1(192QQ) or PON1(192RR) might carry the protein truncation mutation for which the defective product failed to be detected by the phenotyping assay. Screening of the five discordant subjects resulted in the detection of a single Caucasian carrying the stop codon, and determined its phasing on the PON1(192R) allele. Sequencing confirmed the change and revealed an additional subject with a likely deletion of the 5' end of the PON1 gene. Additional sequencing of 25 subjects with low PON1 activities identified two additional previously undescribed PON1 mutations, which may affect PON1 function: PON1(Pro90Leu) associated with the PON1(192Q) allele and PON1(Asp124missplice) associated with the PON1(192R) allele.

Aryldialkylphosphatase↗

Nonsense-mediated decay of human HEXA mRNA.

Nonsense-mediated mRNA decay (NMD), the loss of mRNAs carrying premature stop codons, is a process by which cells recognize and degrade nonsense mRNAs to prevent possibly toxic effects of truncated peptides. Most mammalian nonsense mRNAs are degraded while associated with the nucleus, but a few are degraded in the cytoplasm; at either site, there is a requirement for translation and for an intron downstream of the early stop codon. We have examined the NMD of a mutant HEXA message in lymphoblasts derived from a Tay-Sachs disease patient homozygous for the common frameshift mutation 1278ins4. The mutant mRNA was nearly undetectable in these cells and increased to approximately 40% of normal in the presence of the translation inhibitor cycloheximide. The stabilized transcript was found in the cytoplasm in association with polysomes. Within 5 h of cycloheximide removal, the polysome-associated nonsense message was completely degraded, while the normal message was stable. The increased lability of the polysome-associated mutant HEXA mRNA shows that NMD of this endogenous mRNA occurred in the cytoplasm. Transfection of Chinese hamster ovary cells showed that expression of an intronless HEXA minigene harboring the frameshift mutation or a closely located nonsense codon resulted in half the normal mRNA level. Inclusion of multiple downstream introns decreased the abundance further, to about 20% of normal. Thus, in contrast to other systems, introns are not absolutely required for NMD of HEXA mRNA, although they enhance the low-HEXA-mRNA phenotype.

Cell Line↗

Conserved features of selenocysteine insertion sequence (SECIS) elements in selenoprotein W cDNAs from five species.

SECIS elements form stem-loop structures in the 3' untranslated regions (UTR) of eukaryotic mRNAs that encode selenoproteins. These elements direct incorporation of selenocysteine at UGA codons, provided the SECIS element lies a sufficient distance from the UGA. The cDNAs encoding skeletal muscle selenoprotein W from human, rhesus monkey, sheep, rat, and mouse contained highly similar SECIS elements that retained important features common to all known SECIS elements. Comparative analysis of these SECIS elements showed that in some regions both predicted secondary structure and nucleotide sequences were conserved, in other areas secondary structure was maintained using different primary sequence, and in still other portions, base pairing was not conserved. The rodent and sheep selenoprotein W mRNAs used UGA as a stop codon and as a selenocysteine codon. Thus, UGA specified both selenocysteine incorporation and termination in a single mRNA. The selenoprotein W SECIS elements contained an additional highly conserved base-paired stem that may prevent inappropriate selenocysteine incorporation at the UGA stop codons.

Amino Acid Sequence↗

Novel exon 11 skipping mutation in a patient with glycogen storage disease type IIId.

We report the molecular genetic abnormalities of a patient with GSD IIId presenting with progressive myopathy and cardiopathy leading to a fatal outcome. We identified two independent deletions including a 4 bp deletion (117-1120) and a 98 bp deletion (1135-1232) in cDNA. Sequencing of the genomic DNA of the corresponding region revealed a 4 bp deletion in exon 10; however, the other 98 bp deletion corresponding to exon 11, which was deleted in cDNA, was present in genomic DNA. We therefore concluded that skipping of exon 11 occurred in the cDNA of the patient. Intron/exon boundary analysis of the skipped exon 11 revealed no mutation in the consensus splice-site sequence. If normal splicing had occurred, a stop codon would have appeared within exon II due to frameshift mutation. The mechanism of exon skipping observed in our patient is as yet unknown, and it is still not clear whether intraexonal mutation of the preceding exon can influence splice-site selection. It is possible that a unique exon skipping occurred, preventing the appearance of a stop codon in our patient.

Adult↗

Aminoglycoside-mediated rescue of a disease-causing nonsense mutation in the V2 vasopressin receptor gene in vitro and in vivo.

Many human diseases are caused by inactivating mutations in specific G-protein-coupled receptors (GPCRs). In about 10% of these cases, a premature stop codon leads to the generation of a truncated, functionally inactive receptor protein. In this study, we tested the hypothesis that such GPCR mutations can be functionally rescued in vitro and in vivo by treatment with aminoglycoside antibiotics, which are known for their ability to suppress premature termination codons. As a model system, we studied a mutant V2 vasopressin receptor (AVPR2) containing the inactivating E242X nonsense mutation which mimics human X-linked nephrogenic diabetes insipidus (XNDI) when introduced into mice via gene targeting techniques. Studies with cultured mammalian cells expressing the E242X mutant receptor showed that G418 (geneticin) was by far the most potent aminoglycoside antibiotic capable of suppressing the E242X nonsense codon. Strikingly, G418 treatment increased AVP-mediated cAMP responses in cultured kidney collecting duct cells prepared from E242X mutant mice in vitro, and significantly improved the urine-concentrating ability of E242X mutant mice in vivo. This is the first study demonstrating that G418 (aminoglycosides) can ameliorate the clinical symptoms of a disease-causing premature stop codon in a member of the GPCR superfamily.

Aminoglycosides↗

Role of ribosome release in the basal level of expression of the Escherichia coli gene pheA.

In Escherichia coli, the expression of the phenylalanine biosynthetic operon pheA is regulated by an attenuation mechanism. In the presence of excess phenylalanine, gene expression was decreased to 10% of the fully deattenuated level. To understand the factors that determine the basal level of pheA expression, we examined the role of ribosome release from the leader peptide stop codon UGA. The transcriptional readthrough from the pheA attenuator decreased by over 2-fold in the presence of the defective release factor 2. However, a release factor 1 (UAG and UAA specific) mutation did not influence the basal level of pheA expression. These results support the proposal that the release of translating ribosomes from the leader peptide stop codon in stem 2 of the pheA attenuator plays a crucial role in determining the basal level of expression of this gene.

Alleles↗

Clinical and virological aspects of blood donors infected with hepatitis B virus genotypes B and C.

Pathogenic and therapeutic differences among hepatitis B virus (HBV) genotypes have been documented. However, the association of virological characteristics with clinical differences among HBV genotypes remains unclear. We therefore studied the clinical and virological characteristics of Taiwanese volunteer blood donors infected with HBV genotypes B and C. HBV genotypes were determined in 300 candidate blood donors positive for HBV surface antigen (HBsAg), and sequences of the precore gene of the HBV genome were determined in 50 HBV e antigen (HBeAg)-positive and 50 HBeAg-negative blood donors. Of 300 HBsAg-positive blood donors, 10% had elevated serum aminotransferase levels and 27% were positive for HBeAg. HBV genotype distribution in 264 viremic carriers was as follows: B, 221 (83.7%); C, 39 (14.8%); F, 1 (0.4%); and mixed infection, 3 (1.1%). Blood donors with genotype C infection tended to have a higher frequency of HBeAg positivity and a higher serum HBV DNA level than those with genotype B infection. The frequency of precore stop codon mutation was significantly higher in HBeAg-negative blood donors than HBeAg-positive ones, irrespective of HBV genotypes. Meanwhile, only 5% of blood donors with genotype C infection had C-1858 strains. In conclusion, mixed infection of HBV genotypes indeed occurs, and genotype C has a higher serum HBV DNA level than genotype B. Precore stop codon mutation is common in HBeAg-negative HBV carriers, irrespective of HBV genotypes. In contrast, precore C-1858 strains are rarely identified in Taiwanese HBV genotype C.

Adult↗

Bacterial and nonbacterial expression of wild-type and mutant human phospholipid hydroperoxide glutathione peroxidase and purification of the mutant enzyme in the milligram scale.

15-Lipoxygenases and phospholipid hydroperoxide glutathione peroxidases are counterparts in the metabolism of hydroperoxy lipids and a balanced regulation of both enzymes is essential for normal cell function. Glutathione peroxidases contain selenocysteine as catalytically active amino acid and this selenocysteine is encoded by a TGA stop codon. Detailed protein chemical investigations on phospholipid hydroperoxide glutathione peroxidases and crystal trials have been hampered in the past by limited protein supply. There is no efficient natural source for large-scale enzyme preparation and overexpression of the functional protein in recombinant systems has not been reported so far. To avoid problems with recognition of the selenocysteine stop codon we mutated the selenocysteine to a cysteine and expressed the Sec46Cys mutant in milligram amounts in the baculovirus/insect cell system and as His-tag fusion protein in Escherichia coli. The recombinant enzyme species were purified by conventional fast protein liquid chromatography (nonfusion protein) or by affinity chromatography on a nickel matrix (His-tag protein). Surprisingly, we found that both protein variants were functional although their specific activities were reduced when compared with the wild-type enzyme. Basic protein chemical and enzymatic properties of the purified enzyme species were determined and monoclonal antibodies which recognize the native phospholipid hydroperoxide glutathione peroxidases were raised using our enzyme preparations as antigen. The described strategies for overexpression of mutant phospholipid hydroperoxide glutathione peroxidase species and their purification from recombinant sources provide sufficient amounts of enzyme for future protein chemical investigations and detailed crystal trials.

Animals↗

Genetic structure and domains of DNA polymerase III of Bacillus subtilis.

We have determined the nucleotide sequence of the polC gene of Bacillus subtilis which codes for DNA polymerase III. Our recent analysis has revealed that the gene comprises 4311 nucleotides, from the start to the stop codon, 306 nucleotides more than we reported earlier. The plasmid reported by us and by N.C. Brown's laboratory contained a sequence at the end of the gene which is not related to the polC region of B. subtilis. We have isolated the rest of the gene, the sequence of which is presented in this paper. The new stop codon is followed by a hyphenated palindromic sequence of 13 nucleotides. The C-terminus of the coding region contains the novel mutation, dnaF, which results in a defect in the initiation of replication due to a change in the codon TCC to TTC (serine to phenylalanine). The hypermutator mutation mut-1 is due to two point mutations in the 3' to 5' exonuclease domain, the proof reading function. The codon changes are GGA to GAA (glycine to glutamic acid) and AGC to AAC (serine to asparagine). The elongation defective mutation, polC26, affecting the catalytic site that adds nucleotides to the growing chain, is due to a change in the codon GTC to GAC (valine to aspartic acid). It is separated from the mutation reported earlier, azp-12, by 306 nucleotides. Knowing the locations of the mutational sites allowed us to deduce the domains of the gene and the enzyme it encodes, and permitted us to present a precise map of the gene at the molecular level.

Amino Acid Sequence↗

Tissue-specific alternative splicing generates two synaptojanin isoforms with differential membrane binding properties.

Synaptojanin is an Src homology 3 domain-binding inositol 5-phosphatase that is thought to function in synaptic vesicle endocytosis. It is encoded by a cDNA with two open reading frames separated by an in-frame stop codon. The first open reading frame encodes a 145-kDa form of the protein, whereas a 170-kDa isoform appears to be composed of both open reading frames and contains additional Src homology 3 domain-binding consensus sequences. Here, we demonstrate that the two synaptojanin isoforms are generated by the alternative use of an exon containing the stop codon. Whereas the 145-kDa isoform is highly enriched in adult brain, the 170-kDa isoform is excluded from this tissue and has a widespread distribution in non-neuronal cells. Unlike the 145-kDa isoform, which can be removed from membranes by a low salt wash, the 170-kDa isoform remains membrane-associated, even in the presence of 1 salt. Further, the 170-kDa form, but not the 145-kDa form, can be isolated from membranes as part of a large molecular weight complex. These properties may allow the 170-kDa isoform of synaptojanin to play a unique and perhaps more general role in endocytosis as compared with the 145-kDa isoform.

Alternative Splicing↗

Clinical reactivation during lamivudine treatment correlates with mutations in the precore/core promoter and polymerase regions of hepatitis B virus in patients with anti-hepatitis B e-positive chronic hepatitis.

BACKGROUND: Drug-resistant mutants may emerge in patients with chronic hepatitis B receiving lamivudine therapy. AIM: To evaluate whether different viral mutational patterns may be associated with clinical reactivation during lamivudine treatment in patients with chronic B hepatitis. METHODS: Eight anti-hepatitis B e-positive patients with (group A) and 14 patients without clinical exacerbation (five anti-hepatitis B e-positive, group B1; nine hepatitis B e antigen-positive, group B2) during lamivudine treatment were investigated. RESULTS: 'Polymerase region': M204V/I variants were found in all group A patients, but in none of group B1 (P=0.0007) and in four of nine of group B2 (44%; P=0.02) patients. The L180M substitution was detected in four of eight (50%) of group A and in none of groups B1 and B2. 'Core promoter': the double basic core promoter (A1762T/G1764A) variant was detected in seven of eight (87%) of group A and in one of five (20%; P=0.03) of group B1 and one of nine (11%; P=0.002) of group B2 patients. 'Precore': the G1896A stop codon mutation was present in seven of eight (87%) of group A and in zero of five (P=0.004) of group B1 and one of nine (11%; P=0.002) of group B2. CONCLUSIONS: Different mutational patterns were observed in the lamivudine-treated patients with and without exacerbation. There was an association of the basic core promoter and stop codon mutations with lamivudine resistance in patients with disease exacerbation.

Adult↗

Genetic variation in the 3' non-coding region of dengue viruses.

The 3' non-coding region (3'NCR) of strains of dengue 1 (DEN 1), DEN 2, DEN 3, and DEN 4 viruses, isolated in different geographical regions, was sequenced and compared to published sequences of the four dengue viruses. A total of 50 DEN 2 strains was compared: 7 West African strains, 3 Indonesian mosquito strains, 1 Indonesian macaque isolate, and 39 human isolates from Southeast Asia, the South Pacific, and the Caribbean and Americas. Nucleotide sequence alignment revealed few deletions and no repeat sequences in the 3' NCR of DEN 2 viruses and showed that much of the 3' NCR was well conserved. The strains could be divided into two groups, sylvatic and human/mosquito/macaque, based on nucleotide sequence homology. A hypervariable region was identified immediately following the NS5 stop codon, which involved a 2-10 nucleotide deletion in human, mosquito, and macaque isolates compared with the sylvatic strains. The DEN 2 3'NCR was also compared with 3'NCR sequences from strains of DEN 1, DEN 3, and DEN 4 viruses. DEN 1 was found to have four copies of an eight nucleotide imperfect repeat following the NS5 stop codon, while DEN 4 virus had a deletion of 75 nucleotides in the 3'NCR. We propose that the variation in nucleotide sequence in the 3'NCR may have evolved as a function of DEN virus transmission and replication in different mosquito and non-human primate/human host cycles. The results from this study are consistent with the hypothesis that DEN viruses arose from sylvatic progenitors and evolved into human epidemic strains. However, the data do not support the hypothesis that variation in the 3'NCR correlates with DEN virus pathogenesis.

3' Untranslated Regions↗

Correlation of TP53 mutations and p53 expression in ovarian tumors.

To characterize the involvement of the TP53 tumor suppressor gene in ovarian cancer, mutation analysis of exons 2-11 of TP53 and immunodetection of its protein product, p53, were done in 48 ovarian tumors. Normally, p53 is not immunodetectable. Missense TP53 mutations have been reported to result in p53 accumulation and detection, but mutations generating premature stop codons have not. Mutations were identified in 19 of 41 malignant tumors but not in 5 benign tumors and 2 tumors of low malignant potential. Fifteen of the 19 tumors with mutations also stained positively by immunohistochemistry or Western blot or both. They included 11 missense mutations, 1 in-frame duplication (474ins6), and 3 frameshift mutations generating premature stop codons. The three tumors with frameshifts also had a wild-type TP53 allele and displayed normal size but not truncated p53 by Western blot. This indicates that these tumors express wild-type p53. The significance of TP53 mutations in the development of the three tumors is questionable unless there is a mechanism for inactivating wild-type p53. Nine of the 19 mutations found here, including the 3 frameshifts, were previously not reported in ovarian cancer. Thirteen of the 19 mutations were single nucleotide substitutions with 6 transitions and 7 transversions. The ratio of transversions to transitions (1.2) was different from literature reports (0.5) (P < 0.01). Thus, the spectrum of TP53 mutations in our study differed from other ovarian tumor reports. This difference may be due to population-based differences in the molecular epidemiology of TP53 mutations.

Blotting, Western↗

Bovine and ovine gonadotropin-releasing hormone (GnRH)-II ligand precursors and type II GnRH receptor genes are functionally inactivated.

The decapeptide sequence of GnRH-II is conserved in all jawed vertebrate species studied to date. New data for cattle (Bos taurus) indicates a gene encoding GnRH-II decapeptide possessing arginine (codon: CGG) rather than tryptophan (TGG) at position three in the mature peptide. This substitution is unique. We confirmed the DNA sequence after cloning part of the bovine prepro-GnRH-II gene. Bovine GnRH-II peptide was synthesized and pharmacologically characterized. It did not bind to mammalian GnRH receptors expressed in different types of cell nor did it exhibit agonist or antagonist properties on types I or II GnRH receptors expressed in COS-7 cells. Bovine primers facilitated cloning of ovine GnRH-II DNA. A premature stop codon (TGA) replaces the expected tryptophan codon at position seven of GnRH-II in sheep DNA. Thus, both species possess prepro-GnRH-II genes encoding inactive peptides, as previously described for chimpanzee GnRH-II. The updated bovine type II GnRH receptor gene sequence revealed inactivation by frame shifts, premature stop codons, and nucleotide changes specifying nonconservative replacement of amino acid residues, similar to inactivation of sheep type II GnRH receptor. Spliced RNA transcripts from the disrupted receptor gene were not detected in bovine pituitary. In contrast, bovine prepro-GnRH-I and type I GnRH receptor genes are intact, encoding well-conserved protein sequences. These findings, and previous descriptions of inactivation of the human type II GnRH receptor and deletions of prepro-GnRH-II and type II GnRH receptor in laboratory rodents, suggest the GnRH-II system has been replaced by the GnRH-I system or is redundant in certain mammals.

Amino Acid Sequence↗

Novel mutation in Hepatitis B virus preventing HBeAg production and resembling primate strains.

Chronic carriers of hepatitis B infection often harbour virus strains with mutations in the precore region. These mutations are temporally associated with the development of HBeAg loss and seroconversion to anti-HBe. The most common precore mutation is a stop codon at position 1896, but other mutations leading to abolished HBeAg secretion have been described. Here, a novel precore mutation introducing a lysine in the precore position 28, a sequence shared by non-human primates but not by other human isolates, is described. However, the insertion causes a frame-shift preventing the expression of HBeAg by introducing a stop codon 5 aa downstream of the mutation. Analysis of the predicted RNA secondary structure indicates that the insertion could occur without fatally affecting the stability of the stem-loop encapsidation signal.

Animals↗

Finding novel genes in bacterial communities isolated from the environment.

MOTIVATION: Novel sequencing techniques can give access to organisms that are difficult to cultivate using conventional methods. When applied to environmental samples, the data generated has some drawbacks, e.g. short length of assembled contigs, in-frame stop codons and frame shifts. Unfortunately, current gene finders cannot circumvent these difficulties. At the same time, the automated prediction of genes is a prerequisite for the increasing amount of genomic sequences to ensure progress in metagenomics. RESULTS: We introduce a novel gene finding algorithm that incorporates features overcoming the short length of the assembled contigs from environmental data, in-frame stop codons as well as frame shifts contained in bacterial sequences. The results show that by searching for sequence similarities in an environmental sample our algorithm is capable of detecting a high fraction of its gene content, depending on the species composition and the overall size of the sample. The method is valuable for hunting novel unknown genes that may be specific for the habitat where the sample is taken. Finally, we show that our algorithm can even exploit the limited information contained in the short reads generated by 454 technology for the prediction of protein coding genes. AVAILABILITY: The program is freely available upon request.

Algorithms↗