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PubMed1994

Transcription of a nematode U1 small nuclear RNA in vitro. 3'-end formation requires cis-acting elements within the coding sequence.

We have used block-substitution mutagenesis and in vitro transcription assays to identify cis-acting DNA sequence elements important for initiation and 3'-end formation of a U1 small nuclear RNA (snRNA) in the parasitic nematode Ascaris lumbricoides. Efficient initiation of synthesis by RNA polymerase II requires a compact element centered approximately 50 base pairs upstream of the transcriptional start site. Surprisingly, 3'-end formation of U1 snRNAs synthesized in vitro is solely dependent upon elements within the U1 coding sequence. In all other systems studied thus far, 3'-end formation of U snRNAs requires signals present in the 3'-flanking region. We also show that sequence elements that direct 3'-end formation of the A. lumbricoides trans-spliced leader RNA function when RNA synthesis is initiated from the U1 promoter. These results indicate that 3'-end formation of U snRNAs in nematodes is mechanistically distinct from the analogous process in higher eukaryotes.

Animals↗
PubMed1994

Origin, properties, and regulated expression of multiple mRNAs encoded by the protein kinase C1 gene of Caenorhabditis elegans.

Recently, we cloned and characterized cDNA encoding a novel, protein kinase C (designated PKC1B) from Caenorhabditis elegans. PKC1B (707 amino acid residues) is a developmentally regulated, calcium-independent kinase that is expressed exclusively in sensory neurons and related interneurons. We have now discovered a mechanism by which a second, distinct mRNA (PKC1A mRNA) with increased protein coding potential is generated from the C. elegans PKC1 gene. PKC1A mRNA is produced in a process that involves the utilization of an alternative, distal promoter, the incorporation of two unique exons into the mRNA, and alternative cis/trans splicing. Diversity among PKC1 gene transcripts is increased substantially by trans-splicing. The 5' end of PKC1A mRNA contains an acceptor site that is modified by the addition of either a classical spliced leader sequence 2 or one of four novel spliced leaders. PKC1A mRNA encodes a predicted kinase that contains the entire sequence of PKC1B as well as an N-terminal extension of 56 residues. The extension contains a preponderance of basic amino acids. The levels of transcripts arising from the distal (1A) and proximal (1B) promoters for the PKC1 gene are differentially regulated during C. elegans development. The ratio of 1B mRNA:1A mRNA varies from 40:1 to unity as the nematodes progress from early larval stages to mature adults. The novel exons in the PKC1A structural gene are not contiguous with the PKC1A promoter but are instead positioned downstream from a second gene, kinase upstream gene-1, in the context of a multicystronic operon. PKC1A and kinase upstream gene-1 mRNAs are coordinately expressed in a fixed ratio throughout C. elegans post-embryonic development, suggesting that a shared upstream promoter regulates transcription of both genes. Finally, PKC1A and PKC1B mRNA levels are differentially regulated by phorbol esters in a process that may involve the participation of another PKC isoform that is analogous to mammalian PKC delta.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and expression of a Leishmania major gene encoding a single-stranded DNA-binding protein containing nine "CCHC" zinc finger motifs.

The genes encoding GP63, the major surface glycoprotein of the protozoan parasite Leishmania, are highly conserved across diverse species of Leishmania both within the protein coding region and in the immediate 5'-untranslated region. Located between the 3' trans-spliced leader acceptor site and the translational initiation codon of the GP63 gene is an area of conserved hexanucleotide direct repeats (CTCGCC) which vary in number according to species. To determine whether these repeats represent a site of protein DNA interaction, a Leishmania major lambda gt11 expression library was screened with a radiolabeled synthetic oligodeoxynucleotide probe containing multiple CTCGCC repeats to detect clones expressing functional DNA-binding proteins. Using this approach a gene was isolated which encodes a sequence-specific DNA-binding protein referred to as HEXBP (hexamer-binding protein). Sequence analysis of the HEXBP gene showed that HEXBP contains nine cysteine-rich motifs which are identical to a consensus sequence known as the "CCHC type" zinc finger. This motif is present in a number of nucleic acid-binding proteins including the nucleocapsid protein of retroviruses. In accordance with the activity exhibited by other proteins containing the "CCHC" motif, HEXBP binds to single-stranded nucleic acids as demonstrated by gel mobility shift assays and Southwestern blot assays.

Amino Acid Sequence↗
PubMed1993

Expression of a globin gene in Caenorhabditis elegans.

Oligonucleotides related to parts of a globin-like sequence in the genome of Caenorhabditis elegans were used to probe a cDNA library from the same species. A complete globin-like sequence was found in the cDNA, showing that a globin gene appears to be expressed. The hypothetical protein was compatible with the conventional globin fold but may be truncated in the B helix, as in Chironomus globin III. An intron in the codon for residue E3 in the E helix was removed in expression. An initiation codon preceded the globin but the sequence upstream (extending for 30 nucleotides to the vector ligation site) had characteristics both of the code for a protein hydrophobic leader and of a trans-spliced RNA leader. The evidence indicates that C. elegans globin has a single domain, unlike some nematodes that express two tandem globin domains in a continuous translation product, and from its sequence may be predicted to have a high affinity for oxygen.

Amino Acid Sequence↗
PubMed1993

Splicing and 3'-processing of the tyrosine tRNA of Trypanosoma brucei.

Trypanosoma brucei belongs to a family of protozoa that is characterized as having diverged early in the evolution of eukaryotes. Many unusual forms of RNA processing have been discovered in these cells, one of which is trans-splicing, and, although a great number of genes have been sequenced, no evidence for cis-splicing has yet been found. This study shows that tRNA(Tyr) of T. brucei contains an 11-nucleotide intron. tRNA of the size predicted for unspliced precursor was detected by Northern analysis using an oligonucleotide probe complementary to the putative intervening region. Direct sequence analysis of mature tRNA(Tyr) showed that the predicted intron region is absent from this form of the molecule. Mutated versions of the gene encoding tRNA(Tyr) were introduced into trypanosomes by DNA transformation and shown to be expressed in vivo. Introduction into the genome of a tRNA(Tyr) gene containing an amber suppressor mutation led to a significant accumulation of unspliced precursor molecules. Analysis of the tRNA(Tyr) species from both the wild type and transformed cells revealed unspliced and spliced processing intermediates. The relative abundance of each of these intermediates suggests that 3'-processing and splicing occur independently and that, in the wild type, splicing tends to precede 3'-processing and CCA addition.

Animals↗
PubMed1993

Two wnt genes in Caenorhabditis elegans.

wnt genes encode secretory glycoproteins that have been implicated in growth control and development in mice, frogs and insects. In this report we examine properties of two wnt genes recently identified in the nematode Caenorhabditis elegans. The first gene, Ce-wnt-1, was previously identified by a polymerase chain reaction-based screen of genomic DNA, and the second, Ce-wnt-2, was fortuitously encountered in a survey of clones in a cDNA library by the Caenorhabditis Genome Project. Full-length or nearly full-length cDNAs representing both mRNAs encode proteins that are similar in length, sequence and functional domains to other Wnt proteins. Primary products of 372 and 362 amino acids begin with a hydrophobic signal peptide, include two potential N-linked glycosylation sites and contain the 22 cysteine residues conserved throughout the wnt family. In contrast to mammalian and insect wnt genes with four or five exons and conserved intron-exon boundaries, Ce-wnt-1 has nine coding exons; only one of the eight identified introns interrupts the coding sequence at a position homologous to an intron position in other wnt genes. The major transcript derived from Ce-wnt-1 is 1.4 kb in length, and the 22 nucleotides at its 5' end are added by a trans-splicing mechanism. Ce-wnt-2 is also expressed via a single major transcript, 1.5 kb in length. Both RNAs are detectable in all larval forms and adults, but they are most abundant at the embryonic stage. Ce-wnt-1 is localized to the left arm of chromosome II and Ce-wnt-2 maps to a cluster of genes on chromosome IV.

Amino Acid Sequence↗
PubMed1993

Assembly of the U2 small nuclear ribonucleoprotein from Trypanosoma brucei. A mutational analysis.

trans-Splicing in trypanosomes requires the functions of U2 and U4/U6 small nuclear (sn) RNPs. We have analyzed protein binding and assembly of the Trypanosoma brucei U2 snRNP, using specific antibodies against U2 snRNP proteins and in vitro reconstitution assays of U2 deletion derivatives and human-trypanosome hybrid RNAs. Stable binding of both the U2-specific 40-kDa and the common proteins requires only the 3'-terminal domain (stem-loop IIb, single-stranded region, and stem-loop IV), with loop IV providing the critical sequence determinant; stem-loop IV suffices for binding of the 40 kDa-protein, but not of the common proteins; surprisingly, the sequence of the "Sm-analogous" single-stranded region between stem-loops IIb and IV is not essential for protein binding. Our mutational analysis further indicates that interactions between common and specific proteins play an important role in the assembly of a stable core complex. Finally, a partially assembled U2 RNP complex could be identified as a kinetic intermediate of U2 snRNP assembly. We propose a model of the domain structure and assembly of the trans-spliceosomal U2 snRNP, which deviates in several aspects from that of the cis-spliceosomal U2 snRNP; these differences may be related to the trans-splicing-specific functions of the trypanosomal U2 snRNP.

Animals↗
PubMed1996

The canonical GU dinucleotide at the 5' splice site is recognized by p220 of the U5 snRNP within the spliceosome.

Specific recognition of the 5' splice site (5'SS) by the spliceosome components was studied using a simple in vitro system in which a short 5'SS RNA oligonucleotide specifically induces the assembly of snRNP particles into spliceosome-like complexes and actively participates in a trans-splicing reaction. Short-range cross-liking demonstrates that a U5 snRNP protein component, p220 (the human analogue of the yeast Prp8) specifically interacts with the invariant GU dinucleotide at the 5' end of the intron. The GU:p220 interaction can be detected in the functional splicing complex B. Although p220 has been known to contact several nucleotides around the 5' splice junction, the p220:GU dinucleotide interaction described here is remarkably specific. Consistent with the high conservation of the GU, even minor modifications of this element affect recognition of the 5'SS RNA by p220. Substitution of uridine at the GU with base analogues containing a large methyl or iodo group, but not a smaller flouro group at base position 5, interferes with association of 5'SS RNA with snRNP complexes and their functional participation in splicing.

Base Sequence↗
PubMed1996

Evidence for the presence of a small U5-like RNA in active trans-spliceosomes of Trypanosoma brucei.

The existence of the Trypanosoma brucei 5' splice site on a small RNA of uniform sequence (the spliced leader or SL RNA) has allowed us to characterize the RNAs with which it interacts in vivo by psoralen crosslinking treatment. Analysis of the most abundant crosslinks formed by the SL RNA allowed us previously to identify the spliced leader-associated (SLA) RNA. The role of this RNA in trans-splicing, as well as the possible existence of an analogous RNA interaction in cis-splicing, is unknown. We show here that the 5' splice site region of the SL RNA is also crosslinked in vivo to a second small RNA. Although it is very small and lacks a 5' trimethylguanosine (TMG) cap, the SLA2RNA possesses counterparts of the conserved U5 snRNA stem-loop 1 and internal loop 1 sequence elements, as well as a potential trypanosome snRNA core protein binding site; these combined features meet the phylogenetic definition of U5 snRNA. Like U5, the SLA2 RNA forms an RNP complex with the U4 and U6 RNAs, and interacts with the 5' splice site region via its putative loop 1 sequence. In a final analogy with U5, the SLA2 RNA is found crosslinked to a molecule identical to the free 5' exon splicing intermediate. These data present a compelling case for the SLA2 RNA not only as an active trans-spliceosomal component, but also for its identification as the trypanosome U5 structural homolog. The presence of a U5-like RNA in this ancient eukaryote establishes the universality of the spliceosomal RNA core components.

Animals↗
PubMed1995

The chloroplast genome.

The chloroplast genome consists of homogeneous circular DNA molecules. To date, the entire nucleotide sequences (120-190 kbp) of chloroplast genomes have been determined from eight plant species. The chloroplast genomes of land plants and green algae contain about 110 different genes, which can be classified into two main groups: genes involved in gene expression and those related to photosynthesis. The red alga Porphyra chloroplast genome has 70 additional genes, one-third of which are related to biosynthesis of amino acids and other low molecular mass compounds. Chloroplast genes contain at least three structurally distinct promoters and transcribe two or more classes of RNA polymerase. Two chloroplast genes, rps12 of land plants and psaA of Chlamydomonas, are divided into two to three pieces and scattered over the genome. Each portion is transcribed separately, and two to three separate transcripts are joined together to yield a functional mRNA by trans-splicing. RNA editing (C to U base changes) occurs in some of the chloroplast transcripts. Most edited codons are functionally significant, creating start and stop codons and changing codons to retain conserved amino acids.

Chloroplasts↗
PubMed1996

[Molecular mechanisms for immunoglobulin class switching and IgE production].

Immunoglobulin (Ig) E, which causes the immediate allergic reaction, is produced by B lymphocytes through the class switching. Constant (C) region gene for epsilon heavy (H) chain is lying between C gamma and C alpha genes. Upon stimulation germline forms of the target CH genes of the class switch recombination are first specifically transcribed and spliced to form the germline transcripts in which upstream and downstream of the recombination target region (S region) are joined. These transcription and splicing are essential for class switching. Then, the second isotype Ig is simultaneously expressed with IgM most likely by trans-splicing. This process might be a regulatable intermediate. Finally, DNA recombination between S regions leads the CH genes lying in-between to looping-out deletion.

Animals↗
PubMed1996

Mutations of the two-nucleotide bulge of D5 of a group II intron block splicing in vitro and in vivo: phenotypes and suppressor mutations.

Domain 5 (D5) is a highly conserved substructure of group II introns that is essential for catalysis of both steps of the splicing pathway. Here we studied the effects of mutations of the conserved 2-nt bulge in the binding and catalytic functions of D5 of intron aI5gamma of yeast mitochondrial DNA. Sequence variants of the 2-nt bulge reduced the rate of self-splicing somewhat. Deletion of one or both bulge nucleotides inhibited splicing more than 10(4)-fold, whereas mutants with a 3-nt bulge were much less debilitated. A novel allele with a symmetrical internal loop in place of the bulge self-splices nearly as actively as the control. Trans-splicing assays of D5 function showed some mutations primarily affect the catalytic function of D5, whereas others chiefly affect its binding function. Representative alleles were transformed into mtDNA and each inhibited splicing and respiratory growth. Pseudo-revertants included suppressor mutations nearby in D5 and one mutant yielded a dominant nuclear suppressor. These experiments provide new evidence that the D5 bulge is crucial for D5 binding and catalysis. It is possible that the bulge bends the D5 helix and that the extent of bending is especially important for D5 binding. The presence and exact nature of the bulge is also likely to have local structural consequences (besides bending) that influence the participation of specific backbone groups in binding and catalysis.

Base Sequence↗
PubMed1999

Genomic organization of human myristoyl-CoA: protein N-myristoyltransferase-1.

Myristoylation is a biochemical modification of proteins in which the lipid myristate becomes covalently bound to various cellular, viral, and oncoproteins catalyzed by a monomeric enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT). This modification is important for the biological activity of several proteins, especially the regulation of several oncoproteins involved in various types of cancers. Complementary DNA encoding human NMT-1 (hNMT-1) has been previously reported; however, the genomic organization of hNMT-1 has not been available. Attempts to amplify genomic fragments corresponding to hNMT-1 cDNA sequence yielded only one fragment. We have searched databases using both the cDNA and sequence of one of the intron sequence and this identified a human BAC clone sequence from chromosome 17. Alignment of hNMT-1 cDNA coding information on human chromosome 17 resulted in the complete structural identity of 23,960 bp of the hNMT-1 gene. The hNMT-1 gene is composed of 11 exons and 10 introns with consensus GT/AG boundaries. Finally, we show that 140 bp from the 5' end of recently reported full-length cDNA of hNMT-1 was not part of this genomic region raising the possibility for posttranscriptional modification in generating larger transcripts likely by trans splicing. Further, the availability of this genomic sequence will assist in unraveling the molecular basis for several observed NMT isoforms.

Acyl Coenzyme A↗
PubMed1999

Structure and promoter activity of the 5' flanking region of ace-1, the gene encoding acetylcholinesterase of class A in Caenorhabditis elegans.

We report the structure and the functional activity of the promoter region of ace-1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans. We found that ace-1 was trans -spliced to the SL1 spliced leader and that transcription was initiated at a cluster of multiple starts. There was neither a TATA nor a CAAT box at consensus distances from these starts. Interspecies sequence comparison of the 5' regions of ace-1 in C. elegans and in the related nematode Caenorhabditis briggsae identified four blocks of conserved sequences located within a sequence of 2.4 kilobases upstream from the initiator ATG. In vitro expression of CAT reporter genes in mammalian cells allowed the determination of a minimal promoter in the first 288 nucleotides. In phenotype rescue experiments in vivo, the ace-1 gene containing 2.4 kilobases of 5' flanking region of either C. elegans or C. briggsae was found to restore a coordinated mobility to the uncoordinated double mutants ace-1(-);ace-2(-)of C. elegans. This showed that the ace-1 promoter was contained in 2.4 kilobases of the 5' region, and indicated that cis -regulatory elements as well as coding sequences of ace-1 were functionally conserved between the two nematode species. The pattern of ace-1 expression was established through microinjection of Green Fluorescent Protein reporter gene constructs and showed a major mesodermal expression. Deletion analysis showed that two of the four blocks of conserved sequences act as tissue-specific activators. The distal block is a mesodermal enhancer responsible for the expression in body wall muscle cells, anal sphincter and vulval muscle cells. Another block of conserved sequence directs expression in pharyngeal muscle cells pm5 and three pairs of cephalic sensory neurons.

5' Untranslated Regions↗
PubMed1999

Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel.

The novel locus Prnd is 16 kb downstream of the mouse prion protein (PrP) gene Prnp and encodes a 179 residue PrP-like protein designated doppel (Dpl). Prnd generates major transcripts of 1.7 and 2.7 kb as well as some unusual chimeric transcripts generated by intergenic splicing with Prnp. Like PrP, Dpl mRNA is expressed during embryogenesis but, in contrast to PrP, it is expressed minimally in the CNS. Unexpectedly, Dpl is upregulated in the CNS of two PrP-deficient (Prnp(0/0)) lines of mice, both of which develop late-onset ataxia, suggesting that Dpl may provoke neurodegeneration. Dpl is the first PrP-like protein to be described in mammals, and since Dpl seems to cause neurodegeneration similar to PrP, the linked expression of the Prnp and Prnd genes may play a previously unrecognized role in the pathogenesis of prion diseases or other illnesses.

Amino Acid Motifs↗
PubMed1999

Molecular characterization of McArdle's disease in two large Finnish families.

We have studied two large unrelated Finnish families with myophosphorylase deficiency (McArdle's disease). In one, we identified a new nonsense mutation at codon 540 in exon 14 of the myophosphorylase gene, changing an encoded glutamic acid to a stop codon (E540X). The second family carried a splice-junction mutation at the 5' splice site of intron 14 (1844+G-->A), previously reported in one Caucasian patient and in a consanguineous Druze family. These data further enlarge the list of mutations associated with McArdle's disease and establish that McArdle's disease is genetically heterogeneous also within the Finnish population.

Adult↗
PubMed1999

Caenorhabditis elegans mRNAs that encode a protein similar to ADARs derive from an operon containing six genes.

The Caenorhabditis elegans T20H4.4 open reading frame (GenBank accession no. U00037) predicted by Genefinder encodes a 367 amino acid protein that is 32-35% identical to the C-terminal domain of adenosine deaminases that act on RNA. We show that T20H4.4 cDNAs (GenBank accession no. AF051275) encode a larger 495 amino acid protein that is extended at its N-terminus to include a single double-stranded RNA-binding motif, and that T20H4.4 occupies the second position in a six-gene operon (5'-T20H4.5, T20H4.4, R151.8A, R151.8B, R151.7, R151.6-3'). Ten different spliced-leader (SL) sequences were found attached to T20H4.4 mRNAs, including SL1, SL2 and eight SL2-like leaders that include two new variants. Characterization of cDNAs derived from all six genes confirmed the essential features of C.elegans operons: intercistronic distances in the range of 104-257 nt between the upstream polyadenylation sites and the downstream trans -splice sites; SL2, or SL2-like leaders, attached to the downstream mRNAs. Polycistronic mRNA fragments revealed a 5'-untranslated region (5'-UTR) >705 nt. The 5'-UTR is removed in mature mRNAs from the first gene (T20H4.5) and replaced primarily by SL1, and to a lesser extent by SL2. Our study provides new information regarding operons and how they are processed.

5' Untranslated Regions↗
PubMed1999

Delta ribozyme has the ability to cleave in transan mRNA.

We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy.

Base Sequence↗