Control of branch-site choice by a group II intron.

The branch site of group II introns is typically a bulged adenosine near the 3'-end of intron domain 6. The branch site is chosen with extraordinarily high fidelity, even when the adenosine is mutated to other bases or if the typically bulged adenosine is paired. Given these facts, it has been difficult to discern the mechanism by which the proper branch site is chosen. In order to dissect the determinants for branch-point recognition, new mutations were introduced in the vicinity of the branch site and surrounding domains. Single mutations did not alter the high fidelity for proper branch-site selection. However, several combinations of mutations moved the branch site systematically to new positions along the domain 6 stem. Analysis of those mutants, together with a new alignment of domain 5 and domain 6 sequences, reveals a set of structural determinants that appear to govern branch-site selection by group II introns.     

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.     

The receptor for branch-site docking within a group II intron active site

The distinguishing feature of group II introns, and the property that links them with spliceosomal catalysis, is their ability to undergo splicing through branching. In this reaction, the 2'-hydroxyl group of a specific adenosine within intron domain 6 serves as the nucleophile for attack on the 5' splice site. We know less about branching than any other feature of group II intron catalysis, largely because the receptor structure for activating the branch site is unknown. Here, we identify the intronic region that binds the branch site of a group IIB intron. Located in domain 1, close to receptors for intron domain 5 and both splice sites, we demonstrate that the branch-site receptor is a functional element required for transesterification. Furthermore, we show that crosslinked branch sites can carry out both steps of splicing, suggesting that the conformational state of the intron core is set early and that it persists throughout the entire splicing process.     

A catalytically active group II intron domain 5 can function in the U12-dependent spliceosome

Both spliceosomal and self-splicing group II introns require the function of similar small, metal binding RNA stem-loop elements located in U6 or U6atac snRNAs of the spliceosome or domain 5 (D5) of group II introns. Here we report that two different D5 elements can functionally replace the U6atac snRNA stem-loop in an in vivo splicing assay. For efficient function in vivo, a single base pair from the upper helical section of the D5 sequence had to be removed. Introducing the equivalent base pair deletion into the D5 element of a group II intron reduced but did not eliminate self-splicing activity. Our results strengthen the case that these RNA elements play similar roles in the catalytic centers of both the spliceosome and a self-splicing ribozyme.     

Lariat formation and a hydrolytic pathway in plant chloroplast group II intron splicing

Lariat formation has been studied intensively only with a few self-splicing group II introns, and little is known about how the numerous diverse introns in plant organelles are excised. Several of these introns have branch-points that are not a single bulge but are adjoined by A:A, A:C, A:G and G:G pairs. Using a highly sensitive in vivo approach, we demonstrate that all but one of the barley chloroplast introns splice via the common pathway that produces a branched product. RNA editing does not improve domain 5 and 6 structures of these introns. The conserved branch-point in tobacco rpl16 is chosen even if an adjacent unpaired adenosine is available, suggesting that spatial arrangements in domain 6 determine correct branch-point selection. Lariats were not detected for the chloroplast trnV intron, which lacks an unpaired adenosine in domain 6. Instead, this intron is released as linear molecules that undergo further polyadenylation. trnV, which is conserved throughout plant evolution, constitutes the first example of naturally occurring hydrolytic group II intron splicing in vivo.     

Predicted group II intron lineages E and F comprise catalytically active ribozymes

Group II introns are self-splicing, retrotransposable ribozymes that contribute to gene expression and evolution in most organisms. The ongoing identification of new group II introns and recent bioinformatic analyses have suggested that there are novel lineages, which include the group IIE and IIF introns. Because the function and biochemical activity of group IIE and IIF introns have never been experimentally tested and because these introns appear to have features that distinguish them from other introns, we set out to determine if they were indeed self-splicing, catalytically active RNA molecules. To this end, we transcribed and studied a set of diverse group IIE and IIF introns, quantitatively characterizing their in vitro self-splicing reactivity, ionic requirements, and reaction products. In addition, we used mutational analysis to determine the relative role of the EBS-IBS 1 and 2 recognition elements during splicing by these introns. We show that group IIE and IIF introns are indeed distinct active intron families, with different reactivities and structures. We show that the group IIE introns self-splice exclusively through the hydrolytic pathway, while group IIF introns can also catalyze transesterifications. Intriguingly, we observe one group IIF intron that forms circular intron. Finally, despite an apparent EBS2-IBS2 duplex in the sequences of these introns, we find that this interaction plays no role during self-splicing in vitro. It is now clear that the group IIE and IIF introns are functional ribozymes, with distinctive properties that may be useful for biotechnological applications, and which may contribute to the biology of host organisms.     

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.     

Functional group substitutions of the branchpoint adenosine in a nuclear pre-mRNA and a group II intron.

Splicing of nuclear mRNA precursors (pre-mRNAs) takes place in the spliceosome, a large and complex ribonucleoprotein. Nuclear pre-mRNA splicing and group II intron self-splicing occur by a chemically identical pathway involving recognition of a specific branchpoint adenosine and nucleophilic activation of its 2'-hydroxyl group. The chemical similarity between these two splicing reactions, as well as other considerations, have suggested that the catalytic core of the spliceosome and group II introns may be related. Here we test this hypothesis by analyzing splicing and RNA branch formation of a pre-mRNA and a group II intron in which the branchpoint adenosine was substituted with purine base analogues. We find that replacement of the branchpoint adenosine with either of two modified adenosine analogues or guanosine leads to remarkably similar patterns of splicing and RNA branch formation in the two systems.     

Adenosine is inherently favored as the branch-site RNA nucleotide in a structural context that resembles natural RNA splicing

We previously used in vitro selection to identify the 7S11 deoxyribozyme, which catalyzes formation of 2',5'-branched RNA using a branch-site adenosine nucleophile and a 5'-triphosphate electrophile. An unanswered question is whether the use of branch-site adenosine is inherently preferred or a chance event during the particular selection experiment. Here we have found that deoxyribozymes newly selected to use uridine as the branch-site RNA nucleotide in a structural context that resembles natural RNA splicing instead prefer a branch-site adenosine, although adenosine was never available during the selection itself. Our results support a chemical basis for nature's choice of the branch-site nucleotide, which is almost always adenosine in group II introns and the spliceosome.     

Interaction of intronic boundaries is required for the second splicing step efficiency of a group II intron.

Group II and nuclear pre-mRNAs introns share a common splicing pathway involving a lariat intermediate, as well as some primary sequence similarities at the splice junctions. In this work, we analyze the role of the conserved nucleotides at the first and penultimate positions (G1 and A886) of a group II self-splicing intron. We show that the G1 nucleotide is essential for the efficiency of both the first and the second splicing steps, while substitutions at the penultimate nucleotide affect mostly the efficiency of the second step. A reciprocal suppression of the second splicing step defect can be observed in some double mutants. This result is best explained by a non-Watson-Crick interaction between the first and the penultimate nucleotides of the intron, which occurs after lariat formation. The finding that an interaction between intron boundaries is required for the second splicing step in both group II and nuclear pre-mRNA introns strengthens the idea that both systems employ similar mechanisms, albeit with differences in the details of the nucleotide interactions.     

Relevance of the branch point adenosine, coordination loop, and 3' exon binding site for in vivo excision of the Sinorhizobium meliloti group II intron RmInt1

Excision of the bacterial group II intron RmInt1 has been demonstrated in vivo, resulting in the formation of both intron lariat and putative intron RNA circles. We show here that the bulged adenosine in domain VI of RmInt1 is required for splicing via the branching pathway, but branch site mutants produce small numbers of RNA molecules in which the first G residue of the intron is linked to the last C residue. Mutations in the coordination loop in domain I reduced splicing efficiency, but branched templates clearly predominated among splicing products. We also found that a single substitution at the EBS3 position (G329C), preventing EBS3-IBS3 pairing, resulted in the production of 50 to 100 times more RNA molecules in which the 5' and 3' extremities were joined. We provide evidence that these intron molecules may correspond to both, intron circles linked by a 2'-5' phosphodiester bond, and tandem, head-to-tail intron copies.     

Length changes in the joining segment between domains 5 and 6 of a group II intron inhibit self-splicing and alter 3' splice site selection.

Domain 5 (D5) and domain 6 (D6) are adjacent folded hairpin substructures of self-splicing group II introns that appear to interact within the active ribozyme. Here we describe the effects of changing the length of the 3-nucleotide segment joining D5 to D6 [called J(56)3] on the splicing reactions of intron 5 gamma of the COXI gene of yeast mitochondrial DNA. Shortened variants J(56)0 and J(56)1 were defective in vitro for branching, and the second splicing step was performed inefficiently and inaccurately. The lengthened variant J(56)5 had a milder defect-splicing occurred at a reduced rate but with correct branching and a mostly accurate 3' splice junction choice. Yeast mitochondria were transformed with the J(56)5 allele, and the resulting yeast strain was respiration deficient because of ineffective aI5 gamma splicing. Respiration-competent revertants were recovered, and in one type a single joiner nucleotide was deleted while in the other type a nucleotide of D6 was deleted. Although these revertants still showed partial splicing blocks in vivo and in vitro, including a substantial defect in the second step of splicing, both spliced accurately in vivo. These results establish that a 3-nucleotide J(56) is optimal for this intron, especially for the accuracy of 3' splice junction selection, and indicate that D5 and D6 are probably not coaxially stacked.     

Self-splicing group II and nuclear pre-mRNA introns: how similar are they?

The splicing pathway of pre-mRNA introns bears similarities to that of the group II introns, some members of which undergo self-splicing. The snRNAs may provide the pre-mRNA introns with RNA structures in trans comparable to those available in cis in group II introns. This article examines the available evidence for the hypothesis that the catalysis of these two splicing pathways is fundamentally equivalent.     

Group II intron splicing in Escherichia coli: phenotypes of cis-acting mutations resemble splicing defects observed in organelle RNA processing.

The mitochondrial group IIB intron rI1, from the green algae Scenedesmus obliquus ' LSUrRNA gene, has been introduced into the lacZ gene encoding beta-galacto-sidase. After DNA-mediated transformation of the recombinant lacZ gene into Escherichia coli, we observed correct splicing of the chimeric precursor RNA in vivo. In contrast to autocatalytic in vitro self-splicing, intron processing in vivo is independent of the growth temperature, suggesting that in E.coli, trans -acting factors are involved in group II intron splicing. Such a system would seem suitable as a model for analyzing intron processing in a prokaryotic host. In order to study further the effect of cis -mutations on intron splicing, different rI1 mutants were analyzed (with respect to their splicing activity) in E.coli. Although the phenotypes of these E. coli intron splicing mutants were identical to those which can be observed during organellar splicing of rI1, they are different to those observed in in vitro self-splicing experiments. Therefore, in both organelles and prokaryotes, it is likely that either similar splicing factors or trans -acting factors exhibiting similar functions are involved in splicing. We speculate that ubiquitous trans -acting factors, via recent horizontal transfer, have contributed to the spread of group II introns.     

Mimicking the first step of RNA splicing: an artificial DNA enzyme can synthesize branched RNA using an oligonucleotide leaving group as a 5'-exon analogue

The 7S11 deoxyribozyme synthesizes 2',5'-branched RNA by mediating the nucleophilic attack of an internal 2'-hydroxyl group of one RNA substrate into the 5'-triphosphate of a second RNA substrate, with pyrophosphate as the leaving group. Here we comprehensively examined the role of the leaving group in the 7S11-catalyzed reaction by altering the 5'-phosphorylation state and the length of the second RNA substrate. When the leaving group is the less stabilized phosphate or hydroxide anion as provided by a 5'-diphosphate or 5'-monophosphate, the same 2',5'-branched product is formed as when pyrophosphate is the leaving group, but with an approximately 50- or approximately 1000-fold lower rate (Br?nsted beta(LG) = -0.40). When the 5'-end of the RNA substrate that bears the leaving group is longer by one or more nucleotides, either the new 5'-terminal alpha-phosphate or the original alpha-phosphate can be attacked by the branch-site 2'-hydroxyl group; in the latter case, the leaving group is an oligonucleotide. The choice between these alpha-phosphate reaction sites is determined by the subtle balance between the length of the single-stranded 5'-extension and the stability of the leaving group. Because the branch-site adenosine is a bulged nucleotide flanked by Watson-Crick duplex regions, we earlier concluded that 7S11 structurally mimics the first step of natural RNA splicing. The observation of 7S11-catalyzed branch formation with an oligonucleotide leaving group strengthens this resemblance to natural RNA splicing, with the oligonucleotide playing the role of the 5'-exon in the first step. These findings reinforce the notion that splicing-related catalysis can be achieved by artificial nucleic acid enzymes that are much smaller than the spliceosome and group II introns.     

Toward predicting self-splicing and protein-facilitated splicing of group I introns

In the current era of massive discoveries of noncoding RNAs within genomes, being able to infer a function from a nucleotide sequence is of paramount interest. Although studies of individual group I introns have identified self-splicing and nonself-splicing examples, there is no overall understanding of the prevalence of self-splicing or the factors that determine it among the >2300 group I introns sequenced to date. Here, the self-splicing activities of 12 group I introns from various organisms were assayed under six reaction conditions that had been shown previously to promote RNA catalysis for different RNAs. Besides revealing that assessing self-splicing under only one condition can be misleading, this survey emphasizes that in vitro self-splicing efficiency is correlated with the GC content of the intron (>35% GC was generally conductive to self-splicing), and with the ability of the introns to form particular tertiary interactions. Addition of the Neurospora crassa CYT-18 protein activated splicing of two nonself-splicing introns, but inhibited the second step of self-splicing for two others. Together, correlations between sequence, predicted structure and splicing begin to establish rules that should facilitate our ability to predict the self-splicing activity of any group I intron from its sequence.     

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.     

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.     

The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo.

RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.