The interaction between the first and last intron nucleotides in the second step of pre-mRNA splicing is independent of other conserved intron nucleotides.

Virtually all pre-mRNA introns begin with the sequence /GU and end with AG/ (where / indicates a border between an exon and an intron). We have previously shown that the G residues at the first and last positions of the yeast actin intron interact during the second step of splicing. In this work, we ask if other highly conserved intron nucleotides also take part in this /G-G/ interaction. Of special interest is the penultimate intron nucleotide (AG/), which is important for the second step of splicing and is in proximity to other conserved intron nucleotides. Therefore, we tested interactions of the penultimate intron nucleotide with the second intron nucleotide (/GU) and with the branch site nucleotide. We also tested two models that predict interactions between sets of three conserved intron nucleotides. In addition, we used random mutagenesis and genetic selection to search for interactions between nucleotides in the pre-mRNA. We find no evidence for other interactions between intron nucleotides besides the interaction between the first and last intron nucleotides.     

The linear form of a group II intron catalyzes efficient autocatalytic reverse splicing, establishing a potential for mobility

Self-splicing group II introns catalyze their own excision from pre-RNAs, thereby joining the flanking exons. The introns can be released in a lariat or linear form. Lariat introns have been shown to reverse the splicing reaction; in contrast, linear introns are generally believed to perform no or only poor reverse splicing. Here, we show that a linear group II intron derived from ai5γ can reverse the second step of splicing with unexpectedly high efficiency and precision. Moreover, the linear intron generates dramatically more reverse-splicing product than its lariat equivalent. The finding that linear group II introns can readily undergo the critical first step of mobility by catalyzing efficient reverse splicing into complementary target molecules demonstrates their innate potential for mobility and transposition and raises the possibility that reverse splicing by linear group II introns may have played a significant role in certain forms of intron mobility and lateral gene transfer during evolution.     

Self-splicing of group II introns in vitro: mapping of the branch point and mutational inhibition of lariat formation

Group II intron bl1 from yeast mitochondria can undergo self-splicing in vitro. Exons become correctly ligated, and the excised intron has a lariat structure similar to that of introns from nuclear mRNA. The branch point of the bl1 lariat is located eight or nine nucleotides upstream of the 3' end of the intron and is part of a hairpin structure that is well conserved among group II introns. Several mutations next to the branch point and in other parts of the core structure of group II introns are shown to affect lariat formation. One of them, carried by strain M4873, abolishes splicing in vivo and in vitro, apparently by changing the architecture of the hairpin structure containing the branch point. Similarities between group II introns and nuclear pre-mRNA introns are discussed in terms of evolutionary relatedness.     

Unexpected metal ion requirements specific for catalysis of the branching reaction in a group II intron

The splicing process catalyzed by group II intron ribozymes follows the same two-step pathway as nuclear pre-mRNA splicing. In vivo, the first splicing step of wild-type introns is a transesterification reaction giving rise to a branched lariat intron-3'-exon intermediate characteristic of this splicing mode. In the wild-type introns, the ribozyme core and the substrate intron-exon junctions are carried by the same precursor molecule, making it difficult to distinguish between RNA folding and catalysis under normal splicing reactions. To characterize the catalytic step of the first transesterification reaction, we studied the reversal of this reaction, reverse branching. In this reverse reaction, the excised lariat intron and the substrate 5'-exon can be preincubated and folded separately, allowing the measure of the catalytic rate of the reaction. To measure the catalytic rate of the second splicing step, purified lariat intron-3'-exon intermediate molecules were preincubated and folded prior to the addition of 5'-exon. Conditions could be found where chemistry appeared rate limiting for both catalytic steps. Study of the metal ion requirements under these conditions resulted in the unexpected finding that, for the intron studied, substitution of magnesium ions by manganese ions enhanced the rate of the first transesterification reaction by two orders of magnitude but had virtually no effect on the second transesterification reaction or the 5' splice site cleavage by hydrolysis. Finally, the catalytic rates measured under optimal conditions for both splicing steps were faster by three orders of magnitude in the branching pathway than in the hydrolytic pathway.     

Excision of the Sinorhizobium meliloti group II intron RmInt1 as circles in vivo

Excision of group II introns as circles has been described only for a few eukaryotic introns and little is known about the mechanisms involved, the relevance or consequences of the process. We report that splicing of the bacterial group II intron RmInt1 in vivo leads to the formation of both intron lariat and intron RNA circles. We determined that besides being required for the intron splicing reaction, the maturase domain of the intron-encoded protein also controls the balance between lariat and RNA intron circle production. Furthermore, comparison with in vitro self-splicing products indicates that in vivo, the intron-encoded protein appears to promote the use of a correct EBS1/IBS1 intron-exon interaction as well as cleavage at, or next to, the expected 3' splice site. These findings provide new insights on the mechanism of excision of group II introns as circles.     

Mutations at the lariat acceptor site allow self-splicing of a group II intron without lariat formation.

The fifth intron in the gene for cytochrome c oxidase subunit I in yeast mitochondrial DNA is of the group II type and is capable of self-splicing in vitro. The reaction results in lariat formation, concomitant with exon-exon ligation and does not require a guanosine nucleotide for its initiation. It is generally assumed, but not formally proven, that the first step in splicing is a nucleophilic attack of the 2'-hydroxyl of the branchpoint nucleotide (A) on the 5'-exon-intron junction. To investigate the role of intron sequences in recognition of the 5'-splice junction and the ensuing event of cleavage and lariat formation, mutations have been introduced at and around the branchsite. Results obtained show that although branchpoint attack and subsequent lariat formation are strongly preferred events under conditions normally used for self-splicing, addition of a single T residue at intron position 856, a mutation which brings the branchpoint adenosine into a basepair, leads to a conditionally active intron, which at high ionic strength catalyses exon-exon ligation in the absence of lariat formation. Comparable behaviour is also observed with the branchpoint A deletion mutant. The implications of these findings for the mechanism of self-splicing of group II introns are discussed.     

Mutations in a yeast intron demonstrate the importance of specific conserved nucleotides for the two stages of nuclear mRNA splicing

Mutations were introduced at all positions of the internal conserved sequence (ICS) and at three positions in the 5' junction sequence of a Saccharomyces cerevisiae actin intron contained within an actin-thymidine kinase fusion gene. Stage I of splicing is reduced by changes at all these positions. C or A replacement at the fifth nucleotide of the 5' sequence reduces the fidelity of RNA cleavage at the 5' exon-intron junction and results in an accumulation of aberrant lariat intermediate. Stage II of splicing is affected by changes in the first and second residues of the 5' sequence and in the penultimate position of the ICS. An A to G transition at the branch point of the ICS causes a major accumulation of lariat intermediate.     

Interactions between the terminal bases of mammalian introns are retained in inosine-containing pre-mRNAs.

Nuclear pre-mRNA splicing has a fundamentally similar two-step mechanism to that employed by group II self-splicing introns. It is believed that nuclear pre-mRNA splicing involves a network of RNA-RNA interactions which form the catalytic core of the active spliceosome. We show here a non-Watson-Crick interaction between the first and last guanosine residues of a mammalian intron. As in Saccharomyces cerevisiae, substitution of the conserved guanosines at the 5' and 3' splice sites by A and C respectively, specifically suppresses step 2 splicing defects resulting from the individual mutations. No other combination of terminal nucleotides was able to restore splicing. We additionally provide independent evidence for an indirect interaction between other nucleotides of the consensus splice sites during step 2 of splicing. Substitution of the nucleotide in the +3 position of the 5' splice site affects competition between closely spaced AG dinucleotides at the 3' splice site, although the interaction is not via direct differential base pairing. Finally, we show that complete substitution of guanosine residues by inosine in a pre-mRNA has only a modest effect upon step 2 of splicing, although earlier spliceosome assembly steps are impaired. Predictions can thus be made about the precise configuration of the non-Watson-Crick interaction between the terminal residues.     

Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency

Intron lariat formation between the 5' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3' splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.     

Metal ion catalysis during the exon-ligation step of nuclear pre-mRNA splicing: extending the parallels between the spliceosome and group II introns

Mechanistic analyses of nuclear pre-mRNA splicing by the spliceosome and group II intron self-splicing provide insight into both the catalytic strategies of splicing and the evolutionary relationships between the different splicing systems. We previously showed that 3'-sulfur substitution at the 3' splice site of a nuclear pre-mRNA has no effect on splicing. We now report that 3'-sulfur substitution at the 3' splice site of a nuclear pre-mRNA causes a switch in metal specificity when the second step of splicing is monitored using a bimolecular exon-ligation assay. This suggests that the spliceosome uses a catalytic metal ion to stabilize the 3'-oxyanion leaving group during the second step of splicing, as shown previously for the first step. The lack of a metal-specificity switch under cis splicing conditions indicates that a rate-limiting conformational change between the two steps of splicing may mask the subsequent chemical step and the metal-specificity switch. As the group II intron, a true ribozyme, uses identical catalytic strategies for splicing, our results strengthen the argument that the spliceosome is an RNA catalyst that shares a common molecular ancestor with group II introns.     

The different intron 2 species excised in vivo from the E2A premRNA of adenovirus-2: an approach to analyse alternative splicing.

In the early period of cellular infection by adenovirus 2, the E2A region gives rise to 2 major mRNA species of 2.0 and 2.3 kilobases, formed by alternative excisions of intron 2 (Gattoni et al., 1986, J. Mol. Biol. 187, 379-307). We have analysed the excision pathways of this intron. Two major intron species of 626 and 337 nucleotides, generated by the use of 2 consensus 3' splicing sites and a minor intron species of 520 nucleotides, generated by the use of another weaker 3' splicing site, are identified, the 3 species sharing a common 5' splicing site. They are detected predominantly in the lariat form. For the 2 major species we analyzed, the branched nucleotides are localized at consensus branching sequences, 26 or 25 nucleotides upstream from the 3' terminal AG. Our results confirm that the first reactions of cleavage at the 5' end of introns and branching occur in vivo as described in in vitro systems. The second predominant form of intron 2 is the linear segment, whereas the nicked lariat form which is very minor, might not be a genuine product of in vivo splicing. All intron 2 molecules show practically intact 5' and 3' terminal sequences, indicating that they are well protected against nuclease attack throughout their life. Therefore, these results indicate that the primary reaction following the excision of the lariat intron is debranching. In addition, the existence of a potential 5' splicing site contiguous to the major internal 3' splicing site raised the possibility of an elimination of the major 626 nucleotide intron in 2 cycles of excision. However, we demonstrate that intron 2 is systematically excised by a one cycle process, which is likely to represent the general rule for the production of correctly spliced mRNA.     

Multiple physical forms of excised group II intron RNAs in wheat mitochondria

Plant mitochondrial group II introns do not all possess hallmark ribozymic features such as the bulged adenosine involved in lariat formation. To gain insight into their splicing pathways, we have examined the physical form of excised introns in germinating wheat embryos. Using RT–PCR and cRT–PCR, we observed conventional lariats consistent with a two-step transesterification pathway for introns such as nad2 intron 4, but this was not the case for the cox2 intron or nad1 intron 2. For cox2, we detected full-length linear introns, which possess non-encoded 3′terminaladenosines, as well as heterogeneous circular introns, which lack 3′ nucleotide stretches. These observations are consistent with hydrolytic splicing followed by polyadenylation as well as an in vivo circularization pathway, respectively. The presence of both linear and circular species in vivo is supported by RNase H analysis. Furthermore, the nad1 intron 2, which lacks a bulged nucleotide at the branchpoint position, comprised a mixed population of precisely full-length molecules and circular ones which also include a short, discrete block of non-encoded nucleotides. The presence of these various linear and circular forms of excised intron molecules in plant mitochondria points to multiple novel group II splicing mechanisms in vivo.     

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.     

A maturase-encoding group IIA intron of yeast mitochondria self-splices in vitro.

Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or KCl or 2M (NH4)2SO4) for ready detection of splicing activity. In KCl-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4Cl or KCl, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AI1 RNA is most reactive at 37-42 degrees C, as compared with 45 degrees C for the group IIB introns; and it lacks the KCl- or NH4Cl-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron aI5 gamma, the domain 4 of aI1 can be largely deleted in cis, without blocking splicing; also, trans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.     

U5 snRNA interacts with exon sequences at 5' and 3' splice sites.

U5 snRNA is an essential pre-mRNA splicing factor whose function remains enigmatic. Specific mutations in a conserved single-stranded loop sequence in yeast U5 snRNA can activate cleavage of G1----A mutant pre-mRNAs at aberrant 5' splice sites and facilitate processing of dead-end lariat intermediates to mRNA. Activation of aberrant 5' cleavage sites involves base pairing between U5 snRNA and nucleotides upstream of the cleavage site. Processing of dead-end lariat intermediates to mRNA correlates with base pairing between U5 and the first two bases in exon 2. The loop sequence in U5 snRNA may therefore by intimately involved in the transesterification reactions at 5' and 3' splice sites. This pattern of interactions is strikingly reminiscent of exon recognition events in group II self-splicing introns and is consistent with the notion that U5 snRNA may be related to a specific functional domain from a group II-like self-splicing ancestral intron.     

Group II introns in wheat mitochondria have degenerate structural features and varied splicing pathways

Mitochondrial introns in flowering plant genes are virtually all classified as members of the group II ribozyme family although certain structural features have degenerated to varying degrees over evolutionary time. We are interested in the impact that unconventional intron architecture might have on splicing biochemistry in vivo and we have focused in particular on intronic domains V and VI, which for self-splicing introns provide a key component of the catalytic core and the bulged branchpoint adenosine, respectively. Notably, the two transesterification steps in classical group II splicing are the same as for nuclear spliceosomal introns and release the intron as a lariat. Using RT-PCR and circularized RT-PCR, we had previously demonstrated that several wheat mitochondrial introns which lack a branchpoint adenosine have atypical splicing pathways, and we have now extended this analysis to the full set of wheat introns, namely six trans-splicing and sixteen cis-splicing ones. A number of introns are excised using non-lariat pathways and interestingly, we find that several introns which do have a conventional domain VI also use pathways that appear to exploit other internal or external nucleophiles, with the lariat form being relatively minor. Somewhat surprisingly, several introns with weakly-structured domain V/VI helices still exhibit classical lariat splicing, suggesting that accessory factors aid in restoring a splicing-competent conformation. Our observations illustrate that the loss of conventional group II features during evolution is correlated with altered splicing biochemistry in an intron-distinctive manner.     

A group III twintron encoding a maturase-like gene excises through lariat intermediates.

The 1605 bp intron 4 of the Euglena gracilis chloroplast psbC gene was characterized as a group III twintron composed of an internal 1503 nt group III intron with an open reading frame of 1374 nt (ycf13, 458 amino acids), and an external group III intron of 102 nt. Twintron excision proceeds by a sequential splicing pathway. The splicing of the internal and external group III introns occurs via lariat intermediates. Branch sites were mapped by primer extension RNA sequencing. The unpaired adenosines in domains VI of the internal and external introns are covalently linked to the 5' nucleotide of the intron via 2'-5' phosphodiester bonds. This bond is susceptible to hydrolysis by the debranching activity of the HeLa nuclear S100 fraction. The internal intron and presumptive ycf13 mRNA accumulates primarily as a linear RNA, although a lariat precursor can also be detected. The ycf13 gene encodes a maturase-like protein that may be involved in group III intron metabolism.     

Branch-point attack in group II introns is a highly reversible transesterification, providing a potential proofreading mechanism for 5'-splice site selection.

By examining the first step of group II intron splicing in the absence of the second step, we have found that there is an interplay of three distinct reactions at the 5'-splice site: branching, reverse branching, and hydrolytic cleavage. This approach has yielded the first kinetic parameters describing eukaryotic branching and establishes that group II intron catalysis can proceed on a rapid timescale. The efficient reversibility of the first step is due to increased conformational organization in the branched intermediate and it has several important mechanistic implications. Reversibility in the first step requires that the second step of splicing serve as a kinetic trap, thus driving splicing to completion and coordinating the first and second step of splicing. Facile reverse branching also provides the intron with a proofreading mechanism to control the fidelity of 5'-splice site selection and it provides a kinetic basis for the apparent mobility of group II introns.