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At least 253 records · Page 14Linked to original sources
PubMed1990

Mechanism of 3' splice site selection by the catalytic core of the sunY intron of bacteriophage T4: the role of a novel base-pairing interaction in group I introns.

The catalytic core of the sunY intron of bacteriophage T4 is separated from its 3' exon by 837 nucleotides, most of which are part of an open reading frame (ORF). Here, we report that transcripts truncated within the sunY ORF self-splice in vitro to a variety of sites in the segment immediately 3' of the core. Recognition of these proximal splice sites is shown to depend on (1) the presence on the intron side of a terminal G, which must not be part of a secondary structure; and (2) the ability of the penultimate intron nucleotide to base-pair with a 3' splice site-binding sequence (3'SSBS) located within the core. The counterpart of the 3'SSBS can be identified in most group I introns. The possible significance of such alternative splicing events for in vivo expression of intron-encoded proteins is discussed.

Base Composition↗
PubMed1991

The phage T4 nrdB intron: a deletion mutant of a version found in the wild.

Bacteriophage T4 possesses three self-splicing group I introns. Two of the three introns are mobile elements; the third, in the gene encoding a subunit of the phage nucleotide reductase (nrdB), is not mobile. Because intron mobility offers a reasonable explanation for the paradoxical occurrence of large intervening sequences in a space-efficient eubacterial phage, it is puzzling that the nrdB intron is not mobile like its compatriots. We have discovered a larger nrdB intron in a closely related phage, and we infer from comparative sequence data that the T4 intron is a deletion mutant derived from this larger intron. This larger nrdB intron encodes an open reading frame of 269 codons, which we have cloned and overexpressed. The overexpressed protein shows a dsDNA endonuclease activity specific for the intronless nrdB gene, typical of mobile introns. Thus, we believe that all three introns of T4 are or were mobile "infectious introns" and that they have entered into and been maintained in the phage population by virtue of this efficient mobility.

Amino Acid Sequence↗
PubMed1992

Activation of the catalytic core of a group I intron by a remote 3' splice junction.

Over 1000 nucleotides may separate the ribozyme core of some group I introns from their 3' splice junctions. Using the sunY intron of bacteriophage T4 as a model system, we have investigated the mechanisms by which proximal splicing events are suppressed in vitro, as well as in vivo. Exon ligation as well as cleavage at the 5' splice site are shown to require long-range pairing between one of the peripheral components of the ribozyme core and some of the nucleotides preceding the authentic 3' splice junction. Consistent with our three-dimensional modeling of the entire sunY ribozyme, we propose that this novel interaction is necessary to drive 5' exon-core transcripts into an active conformation. A requirement for additional stabilizing interactions, either RNA-based or mediated by proteins, appears to be a general feature of group I self-splicing. A role for these interactions in mediating putative alternative splicing events is discussed.

Base Sequence↗
PubMed1992

Selection of a ribozyme that functions as a superior template in a self-copying reaction.

The sunY ribozyme is derived from a self-splicing RNA group I intron. This ribozyme was chosen as a starting point for the design of a self-replicating RNA because of its small size. As a means of facilitating the self-replication process, the size of this ribozyme was decreased by the deletion of nonconserved structural domains; however, when such deletions were made, there were severe losses of enzymatic activity. In vitro genetic selection was used to identify mutations that reactivate a virtually inactive sunY deletion mutant. A selected mutant with five substitution mutations scattered throughout the primary sequence showed greater catalytic activity than the original ribozyme under the selection conditions. The sunY ribozyme and its small selected variant can both catalyze template-directed oligonucleotide assembly. The small size and reduced secondary structure of the selected variant results in an enhancement, relative to that of the original ribozyme, of its rate of self-copying. This engineered ribozyme is able to function effectively both as a catalyst and as a template in self-copying reactions.

Bacteriophage T4↗
PubMed1992

Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites.

A simple and efficient method for synthesizing long, site-specifically modified RNA molecules was developed whereby segments of RNA were joined with the use of bacteriophage T4 DNA ligase. A single hydrogen or O-methyl group was substituted for the 2'-hydroxyl group at either splice site of a nuclear pre-messenger RNA substrate. Splicing of the modified pre-messenger RNA's in vitro revealed that, although a 2'-hydroxyl is not absolutely required at either splice site, the 2'-hydroxyl at the 3' splice site is important for the second step of splicing. These results are compared to previous studies of analogous 2'-hydroxyl groups in the self-splicing Tetrahymena group I intron.

Animals↗
PubMed1991

A multisubunit ribozyme that is a catalyst of and template for complementary strand RNA synthesis.

Derivatives of the sunY self-splicing intron efficiently catalyzed the synthesis of complementary strand RNA by template-directed assembly of oligonucleotides. These ribozymes were separated into three short RNA fragments that formed active catalytic complexes. One of the multisubunit sunY derivatives catalyzed the synthesis of a strand of RNA complementary to one of its own subunits. These results suggest that prebiotically synthesized oligonucleotides might have been able to assemble into a complex capable of self-replication.

Animals↗
PubMed1991

Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein.

The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.

Adenosine Triphosphate↗
PubMed1991

Visualizing the higher order folding of a catalytic RNA molecule.

The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed. Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition. The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active. Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.

Animals↗
PubMed1985

Coupling of Tetrahymena ribosomal RNA splicing to beta-galactosidase expression in Escherichia coli.

Splicing of the Tetrahymena ribosomal RNA precursor is mediated by the folded structure of the RNA molecule and therefore occurs in the absence of any protein in vitro. The Tetrahymena intervening sequence (IVS) has been inserted into the gene for the alpha-donor fragment of beta-galactosidase in a recombinant plasmid. Production of functional beta-galactosidase is dependent on RNA splicing in vivo in Escherichia coli. Thus RNA self-splicing can occur at a rate sufficient to support gene expression in a prokaryote, despite the likely presence of ribosomes on the nascent RNA. The beta-galactosidase messenger RNA splicing system provides a useful method for screening for splicing-defective mutations, several of which have been characterized.

Base Sequence↗
PubMed1988

A specific amino acid binding site composed of RNA.

A specific, reversible binding site for a free amino acid is detectable on the intron of the Tetrahymena self-splicing ribosomal precursor RNA. The site selects arginine among the natural amino acids, and prefers the L- to the D-amino acid. The dissociation constant is in the millimolar range, and amino acid binding is at or in the catalytic rG splicing substrate site. Occupation of the G site by L-arginine therefore inhibits splicing by inhibiting the binding of rG, without inhibition of later reactions in the splicing reaction sequence. Arginine binding specificity seems to be directed at the side chain and the guanidino radical, and the alpha-amino and carboxyl groups are dispensable for binding. The arginine site can be placed within the G site by structural homology, with consequent implications for RNA-amino acid interaction, for the origin of the genetic code, for control of RNA activities, and for further catalytic capabilities for RNA.

Animals↗
PubMed1987

Splicing of messenger RNA precursors.

A general mechanism for the splicing of nuclear messenger RNA precursors in eukaryotic cells has been widely accepted. This mechanism, which generates lariat RNAs possessing a branch site, seems related to the RNA-catalyzed reactions of self-splicing introns. The splicing of nuclear messenger RNA precursors involves the formation of a multicomponent complex, the spliceosome. This splicing body contains at least three different small nuclear ribonucleoprotein particles (snRNPs), U2, U5, and U4 + U6. A complex containing precursor RNA and the U2 snRNP particle is a likely intermediate in the formation of the spliceosome.

Animals↗
PubMed1984

A labile phosphodiester bond at the ligation junction in a circular intervening sequence RNA.

The excised intervening sequence of the Tetrahymena ribosomal RNA precursor mediates its own covalent cyclization in the absence of any protein. The circular molecule undergoes slow reopening at a single phosphodiester bond, the one that was formed during cyclization. The resulting linear molecule has 5'-phosphate and 3'-hydroxyl termini; these are unusual products for RNA hydrolysis but are typical of the other reactions mediated by this molecule. The reopened circle retains cleavage-ligation activity, as evidenced by its ability to undergo another round of cyclization and reopening. The finding that an RNA molecule can be folded so that a specific phosphate can be strained or activated helps to explain how the activation energy is lowered for RNA self-splicing. The proposed mechanisms may be relevant to several other RNA cleavage reactions that are RNA-mediated.

Animals↗
PubMed1993

Movement of the guide sequence during RNA catalysis by a group I ribozyme.

Ribozymes derived from the self-splicing pre-ribosomal RNA of Tetrahymena act as sequence-specific endonucleases. The reaction involves binding an RNA or DNA substrate by base pairing to the internal guide sequence (IGS) to form helix P1. Site-specific photo-crosslinking localized the 5' end of the IGS in helix P1 to the vicinity of conserved bases between helices P4 and P5, supporting a major feature of the Michel-Westhof three-dimensional structure model. The crosslinked ribozyme retained catalytic activity. When not base-paired, the IGS was still specifically crosslinked, but the major site was 37 A distant from the reactive site in the experimentally supported three-dimensional model. The data indicate that a substantial induced-fit conformational change accompanies P1 formation, and they provide a physical basis for understanding the transport of oligonucleotides to the catalytic core of the ribozyme. The ability of RNA to orchestrate large-scale conformational changes may help explain why the ribosome and the spliceosome are RNA-based machines.

Animals↗
PubMed1991

The product of the split sunY gene of bacteriophage T4 is a processed protein.

The sunY gene of bacteriophage T4 contains a self-splicing group I intron. The ligated exons encode an open reading frame of 605 amino acids, whose inferred molecular mass is 68 kDa. However, none of the proteins made following T4 infection have been assigned to the sunY gene, and no mutations have been mapped to this locus. We show here that the primary product of the sunY gene is a protein with an apparent molecular mass of 64 kDa, which is processed to a protein approximately 4 kDa smaller. Unlike most other processed T4 proteins, cleavage occurs independently of both the T4 processing protease, the product of gene 21, and late phage protein synthesis. Insertional mutagenesis demonstrated that the sunY protein is not necessary for normal T4 growth under the conditions tested.

Genes, Viral↗
PubMed1990

Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.

A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains.

Amino Acid Sequence↗
PubMed1987

Pre-mRNA splicing and the nuclear matrix.

We examined the relationship between pre-mRNA splicing and the nuclear matrix by using an in vivo system that we have developed. Plasmids containing the inducible herpesvirus tk gene promoter linked to an intron-containing segment of the rabbit beta-globin gene were transfected into HeLa cells, and then the promoter was transactivated by infection with a TK- virus. Northern analysis revealed that the globin pre-mRNA and all its splicing intermediates and products are associated with the nuclear matrix prepared from such transfected cells. When the nuclear matrix was incubated with a HeLa cell in vitro splicing extract in the presence of ATP, the amount of matrix-associated precursor progressively decreased without a temporal lag in the reaction, with a corresponding increase in free intron lariat. Thus, most of the events of the splicing process (endonucleolytic cuts and branching) occur in this in vitro complementation reaction. However, ligation of exons cannot be monitored in this system because of the abundance of preexisting mature mRNA. Since the matrix is not a self-splicing entity, whereas the in vitro splicing system cannot process efficiently deproteinized matrix RNA, we conclude from our in vitro complementation results (which can be reproduced by using micrococcal nuclease-treated splicing extract) that the nuclear matrix preparation retains parts of preassembled ribonucleoprotein complexes that have the potential to function when supplemented with soluble factors (presumably other than most of the small nuclear ribonucleoproteins known to participate in splicing) present in the HeLa cell extract.

Animals↗
PubMed1987

In vitro splicing of the terminal intervening sequence of Saccharomyces cerevisiae cytochrome b pre-mRNA.

A region of the Saccharomyces cerevisiae mitochondrial cytochrome b gene encompassing the entire terminal intron plus flanking exonic sequences has been cloned in an SP6 vector. A runoff transcript prepared from this construct as well as the native cytochrome b pre-mRNA containing the terminal intervening sequence were found to act as substrates for the autocatalytic excision of the intervening sequence in vitro. This reaction proceeds under conditions previously shown by Cech and co-workers to promote protein-independent excision of the Tetrahymena rRNA intervening sequence. The 5' and 3' termini of the excised intervening sequence, determined by S1 nuclease mapping and sequence analysis, are consistent with the known sequence of the cytochrome b mRNA. The same region of the cytochrome b gene from a yeast mutant, defective in splicing due to a mutation in a critical sequence inside the terminal intron, has also been cloned in an SP6 vector. The mutant transcript fails to self-splice in the in vitro assay. These observations provide strong presumptive evidence that in vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA occurs by an autocatalytic mechanism analogous to that shown for other group I introns. In vivo processing of the terminal intervening sequence of the cytochrome b pre-mRNA, however, exhibits complete dependence on a protein factor previously shown to be encoded by the nuclear gene CBP2.

Base Sequence↗
PubMed1988

Group II intron domain 5 facilitates a trans-splicing reaction.

A self-splicing group II intron of yeast mitochondrial DNA (aI5g) was divided within intron domain 4 to yield two RNAs that trans-spliced in vitro with associated trans-branching of excised intron fragments. Reformation of the domain 4 secondary structure was not necessary for the trans reaction, since domain 4 sequences were shown to be dispensable. Instead, the trans reaction depended on a previously unpredicted interaction between intron domain 5, the most highly conserved region of group II introns, and another region of the RNA. Domain 5 was shown to be essential for cleavage at the 5' splice site. It stimulated that cleavage when supplied as a trans-acting RNA containing only 42 nucleotides of intron sequence. The relevance of our findings to in vivo trans-splicing mechanisms is discussed.

Electrophoresis, Polyacrylamide Gel↗