Intron Domain Research Articles

Sequence specificity of a group II intron ribozyme: multiple mechanisms for promoting unusually high discrimination against mismatched targets.

Group II intron ai5 gamma was reconstructed into a multiple-turnover ribozyme that efficiently cleaves small oligonucleotide substrates in-trans. This construct makes it possible to investigate sequence specificity, since second-order rate constants (kcat/K(m), or the specificity constant) can be obtained and compared with values for mutant substrates and with other ribozymes. The ribozyme used in this study consists of intron domains 1 and 3 connected in-cis, together with domain 5 as a separate catalytic cofactor. This ribozyme has mechanistic features similar to the first step of reverse-splicing, in which a lariat intron attacks exogenous RNA and DNA substrates, and it therefore serves as a model for the sequence specificity of group II intron mobility. To quantitatively evaluate the sequence specificity of this ribozyme, the WT kcat/Km value was compared to individual kcat/Km values for a series of mutant substrates and ribozymes containing single base changes, which were designed to create mismatches at varying positions along the two ribozyme-substrate recognition helices. These mismatches had remarkably large effects on the discrimination index (1/relative kcat/K(m)), resulting in values > 10,000 in several cases. The delta delta G++ for mismatches ranged from 2 to 6 kcal/mol depending on the mismatch and its position. The high specificity of the ribozyme is attributable to effects on duplex stabilization (1-3 kcal/mol) and unexpectedly large effects on the chemical step of reaction (0.5-2.5 kcal/mol). In addition, substrate association is accompanied by an energetic penalty that lowers the overall binding energy between ribozyme and substrate, thereby causing the off-rate to be faster than the rate of catalysis and resulting in high specificity for the cleavage of long target sequences (> or = 13 nucleotides).

Ribozyme Catalysis from the Major Groove of Group II Intron Domain 5

The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examined. We observed that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the critical G (specifically N7 or O6) is essential for chemistry. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the critical G could engage in critical tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.

Splice-junction elements and intronic sequences regulate alternative splicing of the Drosophila myosin heavy chain gene transcript.

The Drosophila muscle myosin heavy chain (Mhc) gene primary transcript contains five alternatively spliced exon groups (exon 3, 7, 9, 11 and 15), each of which contains two to five mutually exclusive members. Individual muscles typically select a specific alternative exon from each group for incorporation into the processed message. We report here on the cis-regulatory mechanisms that direct the processing of alternative exons in Mhc exon 11 in individual muscles using transgenic reporter constructs, RT-PCR and directed mutagenesis. The 6.0-kilobase exon 11 domain is sufficient to direct the correct processing of exon 11 alternatives, demonstrating that the alternative splicing cis-regulatory elements are local to Mhc exon 11. Mutational analysis of Mhc exon 11 reveals that the alternative exon nonconsensus 5'-splice donors are essential for alternative splicing regulation in general, but do not specify alternative exons for inclusion in individual muscles. Rather, we show, through exon substitutions and deletion analyses, that a 360-nucleotide intronic domain precisely directs the normal processing of one exon, Mhc exon 11e, in the indirect flight muscle. These and other data indicate that alternative exons are regulated in appropriate muscles through interactions between intronic alternative splice-specificity elements, nonconsensus exon 11 splice donors and, likely, novel exon-specific alternative splicing factors.

RNA structure: crystal clear?

Structured RNAs play an essential role in chromosome maintenance, RNA processing, protein biosynthesis, and protein transport. To understand RNA function in these diverse biological systems, the rules for RNA folding and recognition must be learned. Recent crystal structures of hammerhead ribozymes, a group I intron domain, and RNA duplexes provide new insights into the principles of RNA folding and function.

Intron-dependent enzymatic formation of modified nucleosides in eukaryotic tRNAs: A review

In eukaryotic cells, especially in yeast, several genes encoding tRNAs contain introns. These are removed from pre-tRNAs during the maturation process by a tRNA-specific splicing machinery that is located within the nucleus at the nuclear envelope. Before and after the intron removal, several nucleoside modifications are added in a stepwise manner, but most of them are introduced prior to intron removal. Some of these early nucleoside modifications are catalyzed by intron-dependent enzymes while most of the others are catalyzed in an intron-independent manner. In the present paper, we review all known cases where the nucleoside modifications were shown to depend strictly on the presence of an intron. These are pseudouridines at anticodon positions 34, 35 and 36 and 5-methylcytosine at position 34 of several eukaryotic tRNAs. One common property of the corresponding intron-dependent modifying enzymes is that their activities are essentially dependent on the local specific architecture of the pre-tRNA molecule that comprises the anticodon stem and loop prolonged by the intron domain. Thus introns clearly serve as internal ( cis-type) RNAs that guide nucleoside modifications by providing transient target sites in tRNA for selected nuclear modifying enzymes. This situation may be similar to the recently discovered ( trans-type) snoRNA-guided process of ribose methylations of ribosomal RNAs within the nucleolus of eukaryotic cells.

Stopped-flow fluorescence spectroscopy of a group II intron ribozyme reveals that domain 1 is an independent folding unit with a requirement for specific Mg2+ ions in the tertiary structure.

A stopped-flow fluorescence spectroscopic assay for RNA folding was used to monitor the association of a multicomponent ribozyme derived from group II intron ai5gamma. In the presence of Mg2+, association of a short fluorescein-labeled oligonucleotide substrate with intron domain 1 (D1) resulted in a unique fluorescein emission enhancement, which reflected ribozyme folding and substrate binding. It was found that substrate binding follows a simple bimolecular encounter model, with k(on) approaching the rate of simple duplex formation. The Kd between substrate and D1 was determined to be 11 nM, which is in close agreement with the Kd obtained through oligonucleotide cleavage assays requiring catalytic domain 5 (D5). Ribozyme variants D13 and D135, which contain D3 and/or D5 attached to D1 in-cis, bound substrate with very similar Kd values, suggesting that D1 can fold independently and contains all residues important for ground-state binding to substrate. Both stopped-flow fluorescence assays and chemical modification footprinting data showed that, in all three ribozymes, Mg2+ was required and sufficient for folding. The rates of substrate association and the fraction of active ribozyme showed similar [Mg2+] dependencies, indicating that folding and substrate binding in these three ribozymes are the result of similar processes involving specific, weakly bound Mg2+ ions. The apparent binding constants for the Mg2+ ions were found to be approximately 70 mM in each case. Together, these data show that D1 is an independent folding unit with respect to substrate binding and that specific Mg2+ ions are required for the formation of a distinct tertiary structure in group II introns.

Evolution of trans-splicing plant mitochondrial introns in pre-Permian times.

Trans-splicing in angiosperm plant mitochondria connects exons from independent RNA molecules by means of group II intron fragments. Homologues of trans-splicing introns in the angiosperm mitochondrial nad2 and nad5 genes are now identified as uninterrupted group II introns in the ferns Asplenium nidus and Marsilea drummondii. These fern introns are correctly spliced from the pre-mRNA at the sites predicted from their well-conserved secondary structures. The flanking exon sequences of the nad2 and nad5 genes in the ferns require RNA editing, including the removal of in-frame stop codons by U-to-C changes for correct expression of the genetic information. We conclude that cis-splicing introns like the ones now identified in ferns are the ancestors of trans-splicing introns in angiosperm mitochondria. Intron disruption is apparently due to a size increase of the structurally variable group II intron domain IV followed by DNA recombination in the plant mitochondrial genome.

Molecular characterization of a brown midrib3 deletion mutation in maize

The caffeic acid O-methyltransferase (COMT) gene plays an important role in the synthesis of lignin. We have used the polymerase chain reaction in conjuction with genomic analysis to characterize deletion mutations of this gene in maize. In addition, we have analyzed and compared regions of the COMT gene from three distinct heterotic groups. Both PCR and Southern analysis indicate that the active wild-type COMT gene can be polymorphic. We suggest that the intron domain of at least one heterotic inbred can contribute to the alteration of the wild-type gene. In addition, multiple deletion mutations have occurred at this locus. We have found a previously uncharacterized deletion mutation in which segments of both the intron and exon have been deleted and replaced by other sequences. Precise knowledge of its sequence has allowed us to develop an assay by which we can follow this mutation in a breeding program.

Alternatively spliced mRNAs predicted to yield frame-shift proteins and stable intron 1 RNAs of the herpes simplex virus 1 regulatory gene alpha 0 accumulate in the cytoplasm of infected cells.

The infected cell protein no. 0 (ICP0), the product of the alpha 0 gene, and an important herpes simplex virus 1 regulatory protein is encoded by three exons. We report that intron 1 forms a family of four stable nonpolyadenylylated cytoplasmic RNAs sharing a common 5' end but differing in 3' ends. The 5' and 3' ends correspond to the accepted splice donor and four splice acceptor sites within the mapped intron domain. The most distant splice acceptor site yields the mRNA encoding the 775-aa protein known as ICP0. The mRNAs resulting from the use of alternative splice acceptor sites were also present in the cytoplasm of infected cells and would be predicted to encode proteins of 152 (ICP0-B), 87 (ICP0-C), and 90 (ICP0-D) amino acids, respectively. Both the stability of the alpha 0 mRNA and the utilization of at least one splice acceptor site was regulated by ICP22 and or US1.5 protein inasmuch as cells infected with a mutant from which these genes had been deleted accumulated smaller amounts of alpha 0 mRNA than would be predicted from the amounts of accumulated intron RNAs. In addition, one splice acceptor site was at best underutilized. These results indicate that both the splicing pattern and longevity of alpha 0 mRNA are regulated. These and other recent examples indicate that herpes simplex virus 1 regulates its own gene expression and that of the infected cells through control of mRNA splicing and longevity.

Conversion of a Group II Intron into a New Multiple-Turnover Ribozyme that Selectively Cleaves Oligonucleotides: Elucidation of Reaction Mechanism and Structure/Function Relationships

The self-splicing ai5g group II intron was transformed into a three-part ribozyme that site-specifically cleaves small oligonucleotide substrates with multiple turnover. The ribozyme is composed of intron domain 1 (D1, 425 nucleotides), with catalytically essential domain 5 (D5, 58 nucleotides) provided separately as a reaction cofactor. Together, the D1/D5 complex cleaves small substrates analogous in sequence to the 5'-splice site of the intron. Activity of the ribozyme was studied using a combination of single- and multiple-turnover experiments in which the concentrations of the RNA components were varied in order to probe their individual role in the overall mechanism. Values for kcat, Km, and kcat/Km were the same within experimental error for the two enzymological approaches. These kinetic analyses reveal that the ribozyme utilizes a classic Michaelis-Menten reaction mechanism in which the chemical step of catalysis (kcat = kchem = approximately 0.03 min-1 at full saturation) is rate limiting for the overall reaction. The D1/D5 complex binds tightly to the substrate (Km = 6.3 nM) and specifically recognizes sequences both 5' and 3' to the ribozyme cleavage site. These studies represent the first quantitative analysis of group II recognition and affinity for the 5'-splice site. As observed in previous studies on the role of D5 RNA, D5 binds tightly to the ternary complex (Km = 870 nM). The second-order rate constant for RNA cleavage (kcat/Km = 3.3 x 10(6)) is an order of magnitude slower than that observed for other ribozymes in this mechanistic class, all of which are rate-limited by steps other than chemistry. That kcat equals kchem in this ribozyme is supported by the overall kinetic analysis and by the observation that an Rp phosphorothioate is cleaved approximately 3-fold more slowly than a phosphate at the cleavage site. These studies represent a preliminary examination of stereochemical preference by a group II intron active site in the transition state. The substrate specificity, reaction conditions, and mutational sensitivity of this ribozyme are consistent with a reaction analogous to the first step of group II intron self-splicing, although its stereochemical preference is analogous to a second-step reversal.

GAAA Tetraloop and Conserved Bulge Stabilize Tertiary Structure of a Group I Intron Domain

The 413 nucleotide self-splicing group I intron from Tetrahymena thermophila pre-rRNA contains a 160 nucleotide independently folding domain of RNA tertiary structure, the P4-P6 domain. This domain consists of sequence elements highly conserved among group I introns (P4 and P6) and peripheral extensions conserved in certain subgroups of these introns (P5abc and P6ab). The effect of mutation of selected bases on the formation of domain structure was analyzed using two probes: solvent-based Fe(II)-EDTA, which monitors backbone accessibility and dimethyl sulfate, which monitors availability of N (1) of adenine and N (3) of cytosine. A GAAA tetraloop and an adenosine-rich bulge were found to stabilize domain tertiary structure in a sequence-specific manner. A single base change in the GAAA tetraloop disrupted Fe(II)-EDTA protection both locally and in P6a, and a specific base-pair substitution in P6a similarly disrupted protection locally and in the tetraloop; thus remote elements of the secondary structure are linked in tertiary structure. Our model of the domain's tertiary structure is refined to include this long-range tertiary interaction. The interaction requires severe bending of the domain RNA such that sequences separated by approximately 50 bases of largely double-stranded RNA are in proximity in the tertiary structure. The bending causes or allows for contact between sequences of the conserved core and sequences of the P5 extension. Thus the P5 extension may serve to stabilize the structure of the intron core in vivo.

Differential RNA editing in closely related introns in Oenothera mitochondria

Introns a/b of the nad2 gene and b/c of the nad1 gene in Oenothera mitochondria were found to be closely related. Within a scaffold of conserved sequence regions, a 48 bp sequence element covering intron domain V and flanking nucleotides is identical in both group II introns. The third nucleotide of this element is edited in the nad2, but not in the nad1 intervening sequence. The C to U editing event compensates an nad2-specific nucleotide mismatch in the stem domain IV and thus improves secondary structure stability. This differential editing event indicates that the identical upstream 2 and downstream 45 nucleotides are not sufficient to specify this editing site. Comparison of adjacent exon editing patterns in spliced and unspliced transcripts shows a higher degree of editing in processed sequences, confirming that RNA editing is a posttranscriptional process in plant mitochondria.

Kinetic analysis of the 5' splice junction hydrolysis of a group II intron promoted by domain 5.

The 5' splice junction (5'SJ) of Group II intron transcripts is subject to a specific hydrolysis reaction (SJH). This reaction occurs either within a single transcript containing intron sequences through domain 5 (D5) or by cooperation of two separate transcripts, one bearing the 5'SJ and another contributing D5 (1). In this report we describe the latter reaction in terms of its kinetic parameters. A minimal D5 RNA of 36 nts (GGD5) was sufficient to promote SJH of a second transcript containing the 5' exon plus intron domains 1, 2, and 3 (E1:123). Equimolar production of two RNAs, the 5' exon (E1) and an intron fragment containing domains 1, 2, and 3 (123) was observed. The kinetic coefficients were evaluated by an excess GGD5 approach. The apparent Km was complex, varying with GGD5 concentration. This behavior indicates heterogeneity in E1:123 with respect to GGD5 binding. The binding heterogeneity may result from formation of E1:123 dimers or from nicks in some molecules of each E1:123 preparation. The heterogeneity was always evident, but to a variable degree, regardless of the procedure by which E1:123 was isolated. The system may be described in terms of parameters analogous to kcat and Km. At infinite dilution of GGD5, the characterizing values were: k2 degrees (the analog of kcat) = 0.0055 min-1 and Km degrees = 0.22 microM. In the limit of GGD5 saturation, the values were: k2 infinity = 0.012 min-1 and Km infinity = 4.5 microM. A natural variant D5, representing the sequence from intron 1 of the yeast cytochrome-b gene, was also functional in SJH. This GGD5b1 was governed by similar Km degrees and Km infinity values, but was only one-third as active over the entire D5 concentration range. A different D5 isomer was entirely ineffective for SJH.

Group II introns deleted for multiple substructures retain self-splicing activity.

Group II introns can be folded into highly conserved secondary structures with six major substructures or domains. Domains 1 and 5 are known to play key roles in self-splicing, while the roles of domains 2, 3, 4, and 6 are less clear. A trans assay for domain 5 function has been developed which indicates that domain 5 has a binding site on the precursor RNA that is not predicted from any secondary structure element. In this study, the self-splicing group II intron 5 gamma of the coxI gene of yeast mitochondrial DNA was deleted for various intron domains, singly and in combinations. Those mutant introns were characterized for self-splicing reactions in vitro as a means of locating the domain 5 binding site. A single deletion of domain 2, 3, 4, or 6 does not block in vitro reactions at either splice junction, though the deletion of domain 6 reduces the fidelity of 3' splice site selection somewhat. Even the triple deletion lacking domains 2, 4, and 6 retains some self-splicing activity. The deletion of domains 2, 3, 4, and 6 blocks the reaction at the 3' splice junction but not at the 5' junction. From these results, we conclude that the binding site for domain 5 is within domain 1 and that the complex of 5' exon, domain 1, and domain 5 (plus short connecting sequences) constitutes the essential catalytic core of this intron.

Group II introns deleted for multiple substructures retain self-splicing activity.

Group II introns can be folded into highly conserved secondary structures with six major substructures or domains. Domains 1 and 5 are known to play key roles in self-splicing, while the roles of domains 2, 3, 4, and 6 are less clear. A trans assay for domain 5 function has been developed which indicates that domain 5 has a binding site on the precursor RNA that is not predicted from any secondary structure element. In this study, the self-splicing group II intron 5 gamma of the coxI gene of yeast mitochondrial DNA was deleted for various intron domains, singly and in combinations. Those mutant introns were characterized for self-splicing reactions in vitro as a means of locating the domain 5 binding site. A single deletion of domain 2, 3, 4, or 6 does not block in vitro reactions at either splice junction, though the deletion of domain 6 reduces the fidelity of 3' splice site selection somewhat. Even the triple deletion lacking domains 2, 4, and 6 retains some self-splicing activity. The deletion of domains 2, 3, 4, and 6 blocks the reaction at the 3' splice junction but not at the 5' junction. From these results, we conclude that the binding site for domain 5 is within domain 1 and that the complex of 5' exon, domain 1, and domain 5 (plus short connecting sequences) constitutes the essential catalytic core of this intron.

Evidence for a reverse transcription intermediate for a marked line transposon in tumoral rat cells

We have marked a “reconstituted” LINE element from rat with an intron-containing indicator gene, to test for its RNA-mediated transposition in tumoral rat cells in culture. Three cloned LINE promoter-containing fragments were tested by a transient transfection assay using a LacZ reporter gene, and the promoter with maximum expression was substituted -in an homologous manner- to the 5′ domain of a close to full-length genomic LINE. The resulting marked LINE was stably introduced by transfection into tumoral rat cells. PCR amplification of the DNA from several transfected clones, using primers bracketting the intronic domain of the indicator gene, yielded fragments with a reduced size: their DNA sequencing, in four cases out of four, demonstrated splicing out of the intron as expected for the passage of the marked LINE through an RNA intermediate and its reverse transcription. Fractionation of cellular DNA by the Hirt procedure indicated that reverse transcribed copies are present in the “extrachromosomal” fraction. Their abundance is close to 1 copy per 10 4 cells. These results strongly suggest that rat LINEs transpose through an RNA intermediate and its reverse transcription, as previously demonstrated for the Drosophila LINE I element and, further, that reverse transcription might take place prior to integration, resulting in extrachromosomal DNA molecules as preintegrative transposition intermediates.

Duplicated Sequence Elements and Their Function in Plant Mitochondria

Abstract A considerable portion of the plant mitochondrial DNA is derived from genome internal duplications. Many of these amplified sequences determine functions of transcription and processing. Among these are promoter regions, sequences defining the 3′ ends of stable mRNAs, potential RNA processing sites and intron domains. Simultaneously, some of these repeated sequences can be active sites of recombination in plant mitochondria. Such duplicat­ed control regions may simplify coordinate expression of different genes.

A Muscle-Specific Intron Enhancer Required for Rescue of Indirect Flight Muscle and Jump Muscle Function Regulates Drosophila Tropomyosin I Gene Expression

The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless and jumpless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of our analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70-Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes. One of the intron domains is required for expression in the indirect flight muscle of the adult. The function of the second domain is unknown, but it could regulate the level of expression or be required for expression in other muscle.

A muscle-specific intron enhancer required for rescue of indirect flight muscle and jump muscle function regulates Drosophila tropomyosin I gene expression.

The control of expression of the Drosophila melanogaster tropomyosin I (TmI) gene has been investigated by P-element transformation and rescue of the flightless and jumpless TmI mutant strain, Ifm(3)3. To localize cis-acting DNA sequences that control TmI gene expression, Ifm(3)3 flies were transformed with P-element plasmids containing various deletions and rearrangements of the TmI gene. The effects of these mutations on TmI gene expression were studied by analyzing both the extent of rescue of the Ifm(3)3 mutant phenotypes and determining TmI RNA levels in the transformed flies by primer extension analysis. The results of our analysis indicate that a region located within intron 1 of the gene is necessary and sufficient for directing muscle-specific TmI expression in the adult fly. This intron region has characteristics of a muscle regulatory enhancer element that can function in conjunction with the heterologous nonmuscle hsp70 promoter to promote rescue of the mutant phenotypes and to direct expression of an hsp70-Escherichia coli lacZ reporter gene in adult muscle. The enhancer can be subdivided further into two domains of activity based on primer extension analysis of TmI mRNA levels and on the rescue of mutant phenotypes. One of the intron domains is required for expression in the indirect flight muscle of the adult. The function of the second domain is unknown, but it could regulate the level of expression or be required for expression in other muscle.

Regulation of the human beta-actin promoter by upstream and intron domains.

We have identified three regulatory domains of the complex human beta-actin gene promoter. They span a region of about 3000 bases, from not more than -2011 bases upstream of the mRNA cap site to within the 5' intron (832 bases long). A distal upstream domain contains at least one enhancer-like element. A proximal upstream domain, with a CArG [for CC(A + T rich)6GG] motif found in all known mammalian actin genes, seems to confer serum, but not growth factor, inducibility. The third domain is within the evolutionarily conserved 3' region of the first intron and contains a 13 base-pair sequence, identical to the upstream sequence with the CArG motif. This domain also contains sequences that are both serum and fibroblast growth factor inducible.