Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation.
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Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export. In addition, Hrplp is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues. By using functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites. These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA. To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p. Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrpl p indicating that methylation does not affect specific RNA binding. However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrpl p. Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus. (+info)
In vivo and in vitro processing of the Bacillus subtilis transcript coding for glutamyl-tRNA synthetase, serine acetyltransferase, and cysteinyl-tRNA synthetase.
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In Bacillus subtilis, the adjacent genes gltX, cysE, and cysS encoding respectively glutamyl-tRNA synthetase, serine acetyl-transferase, and cysteinyl-tRNA synthetase, are transcribed as an operon but a gltX probe reveals only the presence of a monocistronic gltX mRNA (Gagnon et al., 1994, J Biol Chem 269:7473-7482). The transcript of the gltX-cysE intergenic region contains putative alternative secondary structures forming a p-independent terminator or an antiterminator, and a conserved sequence (T-box) found in the leader of most aminoacyl-tRNA synthetase and many amino acid biosynthesis genes in B. subtilis and in other Gram-positive eubacteria. The transcription of these genes is initiated 45 nt upstream from the first codon of gltX and is under the control of a sigmaA-type promoter. Analysis of the in vivo transcript of this operon revealed a cleavage site immediately downstream from the p-independent terminator structure. In vitro transcription analysis, using RNA polymerases from Escherichia coli, B. subtilis, and that encoded by the T7 phage, in the presence of various RNase inhibitors, shows the same cleavage. This processing generates mRNAs whose 5'-end half-lives differ by a factor of 2 in rich medium, and leaves putative secondary structures at the 3' end of the gltX transcript and at the 5' end of the cysE/S mRNA, which may be involved in the stabilization of these mRNAs. By its mechanism and its position, this cleavage differs from that of the other known transcripts encoding aminoacyl-tRNA synthetases in B. subtilis. (+info)
Donor site competition is involved in the regulation of alternative splicing of the rat beta-tropomyosin pre-mRNA.
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The rat beta-tropomyosin (beta-TM) gene encodes both skeletal muscle beta-TM mRNA and nonmuscle TM-1 mRNA via alternative RNA splicing. This gene contains eleven exons: exons 1-5, 8, and 9 are common to both mRNAs; exons 6 and 11 are used in fibroblasts as well as in smooth muscle, whereas exons 7 and 10 are used in skeletal muscle. Previously we demonstrated that utilization of the 3' splice site of exon 7 is blocked in nonmuscle cells. In this study, we use both in vitro and in vivo methods to investigate the regulation of the 5' splice site of exon 7 in nonmuscle cells. The 5' splice site of exon 7 is used efficiently in the absence of flanking sequences, but its utilization is suppressed almost completely when the upstream exon 6 and intron 6 are present. The suppression of the 5' splice site of exon 7 does not result from the sequences at the 3' end of intron 6 that block the use of the 3' splice site of exon 7. However, mutating two conserved nucleotides GU at the 5' splice site of exon 6 results in the efficient use of the 5' splice site of exon 7. In addition, a mutation that changes the 5' splice site of exon 7 to the consensus U1 snRNA binding site strongly stimulates the splicing of exon 7 to the downstream common exon 8. Collectively, these studies demonstrate that 5' splice site competition is responsible, in part, for the suppression of exon 7 usage in nonmuscle cells. (+info)
A novel nucleotide incorporation activity implicated in the editing of mitochondrial transfer RNAs in Acanthamoeba castellanii.
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In Acanthamoeba castellanii, most of the mtDNA-encoded tRNAs are edited by a process that replaces one or more of the first three nucleotides at their 5' ends. As a result, base pairing potential is restored at acceptor stem positions (1:72, 2:71, and/or 3:70, in standard tRNA nomenclature) that are mismatched according to the corresponding tRNA gene sequence. Here we describe a novel nucleotide incorporation activity, partially purified from A. castellanii mitochondria, that has properties implicating it in mitochondrial tRNA editing in this organism. This activity is able to replace nucleotides at the first three positions of a tRNA (positions 1, 2, and 3), matching the newly incorporated residues through canonical base pairing to the respective partner nucleotide in the 3' half of the acceptor stem. Labeling experiments with natural (Escherichia coli tRNATyr) and synthetic (run-off transcripts corresponding to A. castellanii mitochondrial tRNALeu1) substrates suggest that the nucleotide incorporation activity consists of at least two components, a 5' exonuclease or endonuclease and a template-directed 3'-to-5' nucleotidyltransferase. The nucleotidyltransferase component displays an ATP requirement and generates 5' pppN... termini in vitro. The development of an accurate and efficient in vitro system opens the way for detailed studies of the biochemical properties of this novel activity and its relationship to mitochondrial tRNA editing in A. castellanii. In addition, the system will allow delineation of the structural features in a tRNA that identify it as a substrate for the labeling activity. (+info)
Photocrosslinking of 4-thio uracil-containing RNAs supports a side-by-side arrangement of domains 5 and 6 of a group II intron.
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Previous studies suggested that domains 5 and 6 (D5 and D6) of group II introns act together in splicing and that the two helical structures probably do not interact by helix stacking. Here, we characterized the major Mg2+ ion- and salt-dependent, long-wave UV light-induced, intramolecular crosslinks formed in 4-thiouridine-containing D56 RNA from intron 5gamma (aI5gamma) of the COXI gene of yeast mtDNA. Four major crosslinks were mapped and found to result from covalent bonds between nucleotides separating D5 from D6 [called J(56)] and residues of D6 near and including the branch nucleotide. These findings are extended by results of similar experiments using 4-thioU containing D56 RNAs from a mutant allele of aI5gamma and from the group IIA intron, aI1. Trans-splicing experiments show that the crosslinked wild-type aI5gamma D56 RNAs are active for both splicing reactions, including some first-step branching. An RNA containing the 3-nt J(56) sequence and D6 of aI5gamma yields one main crosslink that is identical to the most minor of the crosslinks obtained with D56 RNA, but in this case in a cation-independent fashion. We conclude that the interaction between J(56) and D6 is influenced by charge repulsion between the D5 and D6 helix backbones and that high concentrations of cations allow the helices to approach closely under self-splicing conditions. The interaction between J(56) and D6 appears to be a significant factor establishing a side-by-side (i.e., not stacked) orientation of the helices of the two domains. (+info)
Novel endotheliotropic herpesviruses fatal for Asian and African elephants.
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A highly fatal hemorrhagic disease has been identified in 10 young Asian and African elephants at North American zoos. In the affected animals there was ultrastructural evidence for herpesvirus-like particles in endothelial cells of the heart, liver, and tongue. Consensus primer polymerase chain reaction combined with sequencing yielded molecular evidence that confirmed the presence of two novel but related herpesviruses associated with the disease, one in Asian elephants and another in African elephants. Otherwise healthy African elephants with external herpetic lesions yielded herpesvirus sequences identical to that found in Asian elephants with endothelial disease. This finding suggests that the Asian elephant deaths were caused by cross-species infection with a herpesvirus that is naturally latent in, but normally not lethal to, African elephants. A reciprocal relationship may exist for the African elephant disease. (+info)
A concise promoter region of the heart fatty acid-binding protein gene dictates tissue-appropriate expression.
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The heart fatty acid-binding protein (HFABP) is a member of a family of binding proteins with distinct tissue distributions and diverse roles in fatty acid metabolism, trafficking, and signaling. Other members of this family have been shown to possess concise promoter regions that direct appropriate tissue-specific expression. The basis for the specific expression of the HFABP has not been previously evaluated, and the mechanisms governing expression of metabolic genes in the heart are not completely understood. We used transient and permanent transfections in ventricular myocytes, skeletal myocytes, and nonmyocytic cells to map regulatory elements in the HFABP promoter, and audited results in transgenic mice. Appropriate tissue-specific expression in cell culture and in transgenic mice was dictated by 1.2 kb of the 5'-flanking sequence of FABP3, the HFABP gene. Comparison of orthologous murine and human genomic sequences demonstrated multiple regions of near-identity within this promoter region, including a CArG-like element close to the TATA box. Binding and transactivation studies demonstrated that this element can function as an atypical myocyte enhancer-binding factor 2 site. Interactions with adjacent sites are likely to be necessary for fully appropriate, tissue-specific, developmental and metabolic regulation. (+info)
Genomic organization of the KCNQ1 K+ channel gene and identification of C-terminal mutations in the long-QT syndrome.
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The voltage-gated K+ channel KVLQT1 is essential for the repolarization phase of the cardiac action potential and for K+ homeostasis in the inner ear. Mutations in the human KCNQ1 gene encoding the alpha subunit of the KVLQT1 channel cause the long-QT syndrome (LQTS). The autosomal dominant form of this cardiac disease, the Romano-Ward syndrome, is characterized by a prolongation of the QT interval, ventricular arrhythmias, and sudden death. The autosomal recessive form, the Jervell and Lange-Nielsen syndrome, also includes bilateral deafness. In the present study, we report the entire genomic structure of KCNQ1, which consists of 19 exons spanning 400 kb on chromosome 11p15.5. We describe the sequences of exon-intron boundaries and oligonucleotide primers that allow polymerase chain reaction (PCR) amplification of exons from genomic DNA. Two new (CA)n repeat microsatellites were found in introns 10 and 14. The present study provides helpful tools for the linkage analysis and mutation screening of the complete KCNQ1 gene. By use of these tools, five novel mutations were identified in LQTS patients by PCR-single-strand conformational polymorphism (SSCP) analysis in the C-terminal part of KCNQ1: two missense mutations, a 20-bp and 1-bp deletions, and a 1-bp insertion. Such mutations in the C-terminal domain of the gene may be more frequent than previously expected, because this region has not been analyzed so far. This could explain the low percentage of mutations found in large LQTS cohorts. (+info)