Autocatalytic activities of intron 5 of the cob gene of yeast mitochondria.

The terminal intron of the mitochondrial cob gene of Saccharomyces cerevisiae can undergo autocatalytic splicing in vitro. Efficient splicing of this intron required a high concentration of monovalent ion (1 M). We found that at a high salt concentration this intron was very active and performed many of the reactions described for other group I introns. The rate of the splicing reaction was dependent on the choice of the monovalent ion; the reaction intermediate, the intron-3' exon molecule, accumulated in NH4Cl but not in KCl. In addition, the intron was more reactive in KCl, accumulating in two different circular forms: one cyclized at the 5' intron boundary and the other at 236 nucleotides from the 5' end. These circular forms were able to undergo the opening and recyclization reactions previously described for the Tetrahymena rRNA intron. Cleavage of the 5' exon-intron boundary by the addition of GTP did not require the 3' terminus of the intron and the downstream exon. An anomalous guanosine addition at the 3' exon and at the middle of the intron was also detected. Hence, this intron, which requires a functional protein to splice in vivo, demonstrated a full spectrum of characteristic reactions in the absence of proteins.     

A self-splicing group I intron in the nuclear pre-rRNA of the green alga, Ankistrodesmus stipitatus.

The nuclear small subunit ribosomal RNA gene of the unicellular green alga Ankistrodesmus stipitatus contains a group I intron, the first of its kind to be found in the nucleus of a member of the plant kingdom. The intron RNA closely resembles the group I intron found in the large subunit rRNA precursor of Tetrahymena thermophila, differing by only eight nucleotides of 48 in the catalytic core and having the same peripheral secondary structure elements. The Ankistrodesmus RNA self-splices in vitro, yielding the typical group I intron splicing intermediates and products. Unlike the Tetrahymena intron, however, splicing is accelerated by high concentrations of monovalent cations and is rate-limited by the exon ligation step. This system provides an opportunity to understand how limited changes in intron sequence and structure alter the properties of an RNA catalytic center.     

Group II intron RNA-catalyzed recombination of RNA in vitro.

We report the first evidence for a novel reaction mediated by the self-splicing yeast mitochondrial group II intron bl1; the site-specific recombination of RNA molecules in vitro. Upon incubation of the intron lariat with two different RNAs, each harbouring a short sequence complementary to exon binding site 1 (EBS1) of the intron, novel recombined RNAs are formed. As a result of this intron-mediated shuffling of gene segments, the 5' part of RNA1 is ligated to the 3' part of RNA2 and, reciprocally, the 5' part of RNA2 to the 3' part of RNA1. Sequence analysis of the recombinant junction shows that the site of recombination is precisely located 3' to intron binding site 1 (IBS1). The hypothesized mechanism of recombination involves exchange of RNA 5' parts after the first step of a reverse splicing reaction. The possible role of this mechanism in vivo and during prebiotic evolution is discussed.     

Self-splicing of a group II intron in yeast mitochondria: dependence on 5'' exon sequences.

It has been previously suggested that self-splicing of group II introns starts with a nucleophilic attack of the 2' OH group from the branchpoint adenosine on the 5' splice junction. To investigate the sequences governing the specificity of this attack, a series of Bal31 nuclease deletion mutants was constructed in which progressively larger amounts of 5' exon have been removed starting from its 5' end. The ability of mutant RNAs to carry out self-splicing in vitro was studied. Involvement of 5' exon sequences in self-splicing activity is indicated by the fact that a mutant in which as many as 18 nucleotides of 5' exon remain is seriously disturbed in splicing, while larger deletions eliminate splicing entirely. Mutants containing a truncated 5' exon form aberrant RNAs. One of these is a 425-nucleotide RNA containing the 5' exon as well as sequences of the 5' part of the intron. Its 3' end maps at position 374 of the 887-nucleotide intron. The other is a less abundant lariat RNA probably originating from the remainder of the intron linked to the 3' exon. We interpret this large dependence of reactivity of the intron on 5' exon and adjoining intron sequences as evidence for base-pairing interactions between the exon and parts of the intron, leading to an RNA folding necessary for splicing. Possible folding models are discussed.     

Splice site selection by intron aI3 of the COX1 gene from Saccharomyces cerevisiae.

Interactions of the 5' and 3' splice sites with intron internal sequences of the yeast mitochondrial group I intron aI3 were studied using mutation analysis. The results can be fully explained by the splice guide model in which the splice sites are defined by the Internal Guide Sequence. No evidence was found for an alternative interaction between intron nucleotides preceding the 3' splice site and nucleotides in the vicinity of the core region as was found for the Tetrahymena intron. Our results also suggest that binding of the 5' and 3' splice site nucleotides to the IGS can not take place simultaneously. The intron must therefore undergo conformational changes as the reaction proceeds from the first step of self splicing, GTP attack at the 5' splice site, to exon ligation, the second step.     

Determination of an RNA structure involved in splicing inhibition of a muscle-specific exon

We have investigated the RNA structure of the region surrounding the muscle-specific exon 6B of the chicken beta-tropomyosin gene. We have used a variety of chemical and enzymatic probes: dimethylsulfate, N-cyclohexyl-N'-(2-(N-methylmorpholino)-ethyl)-carbodiimide-p-tolu enesulfonate) , RNase T1 and RNase V1. Lead acetate was also used to obtain some information on the tertiary structure of this region. Probing the wild-type sequence suggests a model involving one-stem and three-stem-loop structures in and around this exon. Two of these, hairpin I and stem III, have previously been implicated in repression of splicing of the intron following exon 6B in a HeLa nuclear extract. Stem I includes sequences at the beginning of exon 6B and stem III results from interaction of the intron upstream from exon 6B with sequences in the middle of the intron downstream from this exon (the intron whose splicing is repressed). Neither stem I nor stem III directly involves the consensus sequences (5' splice site, branch-point, 3' splice site) of the repressed intron. Probing RNAs that are derepressed for splicing of this intron show that there are structural changes around the 5' splice site and branch-point sequence that correlate with the derepression. This is true, despite the fact that the derepressed RNAs are altered in a region far from these consensus sequences. The most striking structural correlation with splicing capacity of the intron downstream from exon 6B is seen by probing with lead acetate. Lead ions cut RNA at specific residues; these sites are very sensitive to RNA tertiary structure. Repressed and derepressed RNAs show entirely different cleavage patterns after incubation with lead acetate. Remarkably, hybridizing a derepressed RNA with an RNA comprising the ascending arm of stem III not only re-establishes repression, but also converts the pattern of susceptibility to attack by lead ions over the whole molecule. We suggest that RNA conformation plays a role in keeping exon 6B from being spliced into non-muscle cell mRNA.     

Integration of group II intron bI1 into a foreign RNA by reversal of the self-splicing reaction in vitro

Group II intron bI1, the first intron of the COB gene in the mitochondria of S. cerevisiae, is able to self-splice in vitro with the basic pathway similar to nuclear pre-mRNA splicing. We show that incubation of the intron lariat with ligated exons bE1 and bE2 leads to a complete reversal of the splicing reaction. The integration of the intron into the ligated exons is correct; the reconstituted preRNA of the reverse reaction can undergo a self-splicing reaction anew. When incubated with a foreign RNA species bearing a sequence motif that is complementary to exon binding site 1, the lariat can integrate into this RNA with the position of insertion immediately downstream of this sequence. This result implies that transposition of group II introns on the RNA level by reversal of the splicing reaction is, in principle, conceivable.     

Fate of the junction phosphate in alternating forward and reverse self-splicing reactions of group II intron RNA.

The RNA-catalysed self-splicing reaction of group II intron RNA is assumed to proceed by two consecutive transesterification steps, accompanied by lariat formation. This is effectively analogous to the small nuclear ribonucleoprotein (snRNP)-mediated nuclear pre-mRNA splicing process. Upon excision from pre-RNA, a group II lariat intervening sequence (IVS) has the capacity to re-integrate into its cognate exons, reconstituting the original pre-RNA. The process of reverse self-splicing is presumed to be a true reversion of both transesterification steps used in forward splicing. To investigate the fate of the esterified phosphate groups in splicing we assayed various exon substrates (5'E-*p3'E) containing a unique 32P-labelled phosphodiester at the ligation junction. In combined studies of alternating reverse and forward splicing we have demonstrated that the labelled phosphorus atom is displaced in conjunction with the 3' exon from the ligation junction to the 3' splice site and vice versa. Neither the nature of the 3' exon sequence nor its sequence composition acts as a prominent determinant for both substrate specificity and site-specific transesterification reactions catalysed by bI1 IVS. A cytosine ribonucleotide (pCp; pCOH) or even deoxyoligonucleotides could function as an efficient substitute for the authentic 3' exon in reverse and in forward splicing. Furthermore, the 3' exon can be single monophosphate group. Upon incubation of 3' phosphorylated 5' exon substrate (5'E-*p) with lariat IVS the 3'-terminal phosphate group is transferred in reverse and forward splicing like an authentic 3' exon, but with lower efficiency. In the absence of 3' exon nucleotides, it appears that substrate specificity is provided predominantly by the base-pairing interactions of the intronic exon binding site (EBS) sequences with the intron binding site (IBS) sequences in the 5' exon. These studies substantiate the predicted transesterification pathway in forward and reverse splicing and extend the catalytic repertoire of group II IVS in that they can act as a potential and sequence-specific transferase in vitro.     

5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon

The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro. The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing. Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon. RNA was transcribed from these plasmids in vitro and tested for self-splicing activity. The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences. Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain. When only two nucleotides of the natural exon remain, no ligated exons are observed. As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts. The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA. Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site. The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA. The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site. This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site. The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon. A small circular RNA was isolated and partially sequenced and found to support the model. So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site. Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.     

Trans splicing of mRNA precursors in vitro

Two exon segments from two separate RNA molecules can be joined in a trans splicing process. In trans splicing reactions, an RNA molecule containing an exon, a 5' splice site, and adjacent intron sequences was mixed with an RNA molecule containing an exon, a 3' splice site, and adjacent intron sequences. The efficiency of trans splicing of these two RNAs increased if the two termini of the intervening sequences were paired in a short RNA duplex. However, trans splicing of two RNA molecules with no significant complementarity was also observed. These results strongly suggest that significant secondary structures within intervening sequences could affect the splicing of flanking exons. Similarly, RNAs that are complementary to segments within the intervening sequences could potentially regulate the selection of splice sites. Finally, some organisms might use trans splicing to distribute a single exon to many different mRNAs.     

Restoration of the self-splicing activity of a defective group II intron by a small trans-acting RNA

The yeast mitochondrial group II intron bI1 is self-splicing in vitro. We have introduced a deletion of hairpin C1 within the structural domain 1 that abolishes catalytic activity of the intron in the normal splicing reaction in cis, but does less severely affect a reaction in trans, the reopening of ligated exons. Since exon reopening is supposed to correspond to a reverse 3' cleavage this suggests that the deletion specifically blocks the first reaction step. The intron regains its activity to self-splice in cis by intermolecular complementation with a small RNA harbouring sequences lacking in the mutant intron. These results demonstrate the feasibility to reconstitute a functionally active structure of the truncated intron by intermolecular complementation in vitro. Furthermore, the data support the hypothesis that group II introns are predecessors of nuclear pre-mRNA introns and that the small nuclear RNAs of the spliceosome arose by segregation from the original intron.     

Alternative secondary structures in the 5' exon affect both forward and reverse self-splicing of the Tetrahymena intervening sequence RNA

The natural splice junction of the Tetrahymena large ribosomal RNA is flanked by hairpins that are phylogenetically conserved. The stem immediately preceding the splice junction involves nucleotides that also base pair with the internal guide sequence of the intervening sequence during splicing. Thus, precursors which contain wild-type exons can form two alternative helices. We have constructed a series of RNAs where the stem-loop in the 5' exon is more or less stable than in the wild-type precursor, and tested them in both forward and reverse self-splicing reactions. The presence of a stable hairpin in ligated exon substrates interferes with the ability of the intervening sequence to integrate at the splice junction. Similarly, the presence of the wild-type hairpin in the 5' exon reduces the rate of splicing 20-fold in short precursors. The data are consistent with a competition between unproductive formation of a hairpin in the 5' exon and productive pairing of the 5' exon with the internal guide sequence. The reduction of splicing by a hairpin that is a normal feature of rRNA structure is surprising; we propose that this attenuation is relieved in the natural splicing environment.     

Control of hnRNP A1 alternative splicing: an intron element represses use of the common 3' splice site

Alternative splicing of exon 7B in the hnRNP A1 pre-mRNA produces mRNAs encoding two proteins: hnRNP A1 and the less abundant A1B. We have reported the identification of several intron elements that contribute to exon 7B skipping. In this study, we report the activity of a novel element, conserved element 9 (CE9), located in the intron downstream of exon 7B. We show that multiple copies of CE9 inhibit exon 7B-exon 8 splicing in vitro. When CE9 is inserted between two competing 3' splice sites, a single copy of CE9 decreases splicing to the distal 3' splice site. Our in vivo results also support the conclusion that CE9 is a splicing modulator. First, inserting multiple copies of CE9 into an A1 minigene compromises the production of fully spliced products. Second, one copy of CE9 stimulates the inclusion of a short internal exon in a derivative of the human beta-globin gene. In this case, in vitro splicing assays suggest that CE9 decreases splicing of intron 1, an event that improves splicing of intron 2 and decreases skipping of the short internal exon. The ability of CE9 to act on heterologous substrates, combined with the results of a competition assay, suggest that the activity of CE9 is mediated by a trans-acting factor. Our results indicate that CE9 represses the use of the common 3' splice site in the hnRNP A1 alternative splicing unit.     

Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo.

The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA-protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5' splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.     

Identification of phosphate groups important to self-splicing of the Tetrahymena rRNA intron as determined by phosphorothioate substitution.

The group I intron from the rRNA precursor of Tetrahymena undergoes self-splicing. The intron RNA catalyst contains about 400 phosphate groups. Their role in catalysis has been investigated using phosphorothioate substituted RNA. In such RNA one of the peripheral oxygens of the phosphodiesters is replaced with sulfur. Incorporation of adenosine 5' phosphorothioate in either the 5' or 3' half of the ribozyme blocked splicing whereas incorporation of uridine 5' phosphorothioate only blocked splicing if the substitution was in the 3' half of the molecule. Modification-interference assays located two major and three minor inhibitory phosphorothioate substitutions suggesting that the corresponding phosphates play a significant role in self-splicing. These are all located in the most highly conserved region of the intron.