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.     

Exon sequence requirements for excision in vivo of the bacterial group II intron RmInt1

Background

Group II intron splicing proceeds through two sequential transesterification reactions in which the 5' and 3'-exons are joined together and the lariat intron is released. The intron-encoded protein (IEP) assists the splicing of the intron in vivo and remains bound to the excised intron lariat RNA in a ribonucleoprotein particle (RNP) that promotes intron mobility. Exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain dI known as EBS1 and EBS2 and two stretches of sequence known as IBS1 and IBS2 on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from EBS1 [(δ-δ' interaction (subgroup IIA)] or with a nucleotide [(EBS3-IBS3 interaction (subgroup IIB and IIC))] located in the coordination-loop of dI. The δ nucleotide is involved in base pairing with another intron residue (δ') in subgroup IIB introns and this interaction facilitates base pairing between the 5' exon and the intron.

Results

In this study, we investigated nucleotide requirements in the distal 5'- and 3' exon regions, EBS-IBS interactions and δ-δ' pairing for excision of the group IIB intron RmInt1 in vivo. We found that the EBS1-IBS1 interaction was required and sufficient for RmInt1 excision. In addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo.

Conclusions

The excision in vivo of the RmInt1 intron is a favored process, with very few constraints for sequence recognition in both the 5' and 3'-exons. Our results contribute to understand how group II introns spread in nature, and might facilitate the use of RmInt1 in gene targeting.     

An alternative intron-exon pairing scheme implied by unexpected in vitro activities of group II intron RmInt1 from Sinorhizobium meliloti

RmInt1 is a mobile group II intron which interrupts ISRm2011-2, another mobile element from the bacterium Sinorhizobium meliloti. Ribozyme constructs derived from intron RmInt1 self-splice in vitro when incubated under permissive conditions, but the excised intron and ligated exons are largely replaced by unconventional products. These include a slightly shorter, 5'-end truncated 3' exon, truncated variants of the linear and lariat forms of the intron-3' exon reaction intermediate, as well as presumably circular molecules derived from the latter. Two factors explain the abundance of these products: (i) nucleotides 5-11 of the 3' exon (IBS1*) provide a better match to the EBS1 5'-exon-binding site than the authentic IBS1 sequence in the 5' exon; (ii) exon ligation is unusually inefficient, and especially so when the 5' exon is truncated close to the second (IBS2) intron-binding site. We propose that reactions at the IBS1* site play a part in the regulation of the intron ISRm2011-2 host in vivo.     

In vitro characterization of the splicing efficiency and fidelity of the RmInt1 group II intron as a means of controlling the dispersion of its host mobile element

Group II introns are catalytic RNAs that are excised from their precursors in a protein-dependent manner in vivo. Certain group II introns can also react in a protein-independent manner under nonphysiological conditions in vitro. The efficiency and fidelity of the splicing reaction is crucial, to guarantee the correct formation and expression of the protein-coding mRNA. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. The RmInt1 intron self-splices in vitro, but this reaction generates side products due to a predicted cryptic IBS1* sequence within the 3′ exon. We engineered an RmInt1 intron lacking the cryptic IBS1* sequence, which improved the fidelity of the splicing reaction. However, atypical circular forms of similar electrophoretic mobility to the lariat intron were nevertheless observed. We analyzed a run of four cytidine residues at the 3′ splice site potentially responsible for a lack of fidelity at this site leading to the formation of circular intron forms. We showed that mutations of residues base-pairing in the tertiary EBS3–IBS3 interaction increased the efficiency and fidelity of the splicing reaction. Our results indicate that RmInt1 has developed strategies for decreasing its splicing efficiency and fidelity. RmInt1 makes use of unproductive splicing reactions to limit the transposition of the insertion sequence into which it inserts itself in its natural context, thereby preventing potentially harmful dispersion of ISRm2011-2 throughout the genome of its host.     

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.     

Potential for alternative intron-exon pairings in group II intron RmInt1 from Sinorhizobium meliloti and its relatives

Ribozyme constructs derived from group II intron RmInt1 of Sinorhizobium meliloti self-splice in vitro when incubated under permissive conditions, but exon ligation is unusually inefficient when the 5' exon is truncated close to the IBS2 intron-binding site. One plausible explanation for this observation is the presence of an alternative intron-exon pairing between an intron segment that overlaps with the EBS2 exon-binding site and a 5' exon site located just distal of IBS2 relative to the splice junction. Strikingly, the existence of this pairing is supported by comparative sequence analysis of introns related to RmInt1.     

Circle reopening in the Tetrahymena ribozyme resembles site-specific hydrolysis at the 3' splice site.

The Tetrahymena intron, after splicing from its flanking exons, can mediate its own circularization. This is followed by site-specific hydrolysis of the phosphodiester bond formed during the circularization reaction. The structural components involved in recognition of this bond for hydrolysis have not been established. We have made base substitutions to the P9.0 pairing and at the 3'-terminal guanosine residue (G414) of the intron to investigate their effects on circle formation and reopening. We have found that disruption of either P9.0 pairing or binding of the terminal nucleotide result in the formation of a large circle, C-413:5E23 from precursor RNA molecules that have undergone hydrolysis at the 3' splice site. This circle is formed at the phosphodiester bond of the 5'-terminal guanosine residue of the upstream exon via nucleophilic attack by the 3'-terminal nucleotide of the intron. The large circle is novel since it can reopen eight bases downstream from the original circularization junction at a site resembling the normal 3' splice site, restoring a guanosine to the 3' terminus and re-establishing P9.0 pairing. The new 3' terminus of the intron is capable of recircularization at any of the three normal wild-type sites. We conclude that both P9.0 and the 3'-terminal guanosine residue are required for the selection of the phosphodiester bond hydrolysed during circle reopening.     

Two domains for splicing in the intron of the phage T4 thymidylate synthase (td) gene established by nondirected mutagenesis

Of 97 nondirected T4 thymidylate synthase-defective (td) mutations, 27 were mapped to the intron of the split td gene. Clustering of these intron mutations defined two domains that are functional in splicing, each within approximately 220 residues of the respective splice sites. Two selected mutations, tdN57 and tdN47, fell within phylogenetically conserved pairings, with tdN57 disrupting the exon I-internal guide pairing (P1) in the 5' domain and tdN47 destabilizing the P9 helix in the 3' domain. A splicing assay with synthetic oligonucleotides complementary to RNA junction sequences revealed processing defects for T4tdN57 and T4tdN47, both of which are impaired in cleavage at the 5' and 3' splice sites. Thus prokaryotic genetics facilitates association of specific residue changes with their consequences to splicing.     

Interlocked circle formation by group I introns: structural requirements and mechanism.

Precursor RNA transcribed from the yeast mitochondrial gene coding for the large ribosomal RNA contains a group I intron that can excise itself in vitro. Apart from group I specific sequence elements the intron also contains a gene encoding a DNA endonuclease involved in intron dispersal. A precursor RNA derivative from which this gene has been removed self-splices efficiently, but due to activation of cryptic opening sites located in the 5' exon, the 3' part of this exon is sometimes co-excised with the intron. Upon further reaction, this enlarged intron molecules give rise to interlocked circles, comprising small circles derived from 5' exon parts and large circles of the intron. Sequence comparison between cryptic opening sites and authentic splice sites reveals in most cases homology with the 3' exon part that is capable of interacting with the Internal Guide Sequence. The role of the IGS was further substantiated by replacing the cryptic opening sites with well defined sequences of authentic splice sites: one corresponding to the 3' splice site and its mutant derivatives, the other to a fragment containing the natural 5'-3' exon junction. Precursor RNAs derived from these constructs give rise to interlocked circles, and mutation studies confirm that the 3' exon nucleotides flanking a 3' splice site are essential for their formation. The results underline the crucial role of the IGS in interlocked circle formation which behaves similarly as in the normal self-splicing reactions. It has been proposed that the two short helices formed by basepairing of the IGS with the 5' and 3' exon can co-axially stack on top of each other forming a quasi continuous RNA double helix or pseudoknot. We present a model explaining how transesterification reactions of a mutant precursor RNA in such a pseudoknot can lead to interlocked circles. The experiments support the notion that a similar structure is also operative in splicing of wild type precursor RNA.     

A mutational analysis of U12-dependent splice site dinucleotides

Introns spliced by the U12-dependent minor spliceosome are divided into two classes based on their splice site dinucleotides. The /AU-AC/ class accounts for about one-third of U12-dependent introns in humans, while the /GU-AG/ class accounts for the other two-thirds. We have investigated the in vivo and in vitro splicing phenotypes of mutations in these dinucleotide sequences. A 5' A residue can splice to any 3' residue, although C is preferred. A 5' G residue can splice to 3' G or U residues with a preference for G. Little or no splicing was observed to 3' A or C residues. A 5' U or C residue is highly deleterious for U12-dependent splicing, although some combinations, notably 5' U to 3' U produced detectable spliced products. The dependence of 3' splice site activity on the identity of the 5' residue provides evidence for communication between the first and last nucleotides of the intron. Most mutants in the second position of the 5' splice site and the next to last position of the 3' splice site were defective for splicing. Double mutants of these residues showed no evidence of communication between these nucleotides. Varying the distance between the branch site and the 3' splice site dinucleotide in the /GU-AG/ class showed that a somewhat larger range of distances was functional than for the /AU-AC/ class. The optimum branch site to 3' splice site distance of 11-12 nucleotides appears to be the same for both classes.     

Use of RmInt1, a group IIB intron lacking the intron-encoded protein endonuclease domain, in gene targeting

The group IIA intron Ll.LtrB from Lactococcus lactis and the group IIB intron EcI5 from Escherichia coli have intron-encoded proteins (IEP) with a DNA-binding domain (D) and an endonuclease domain (En). Both have been successfully retargeted to invade target DNAs other than their wild-type target sites. RmInt1, a subclass IIB3/D intron with an IEP lacking D and En domains, is highly active in retrohoming in its host, Sinorhizobium meliloti. We found that RmInt1 was also mobile in E. coli and that retrohoming in this heterologous host depended on temperature, being more efficient at 28°C than at 37°C. Furthermore, we programmed RmInt1 to recognize target sites other than its wild-type site. These retargeted introns efficiently and specifically retrohome into a recipient plasmid target site or a target site present as a single copy in the chromosome, generating a mutation in the targeted gene. Our results extend the range of group II introns available for gene targeting.     

Formation of lariats and circles in self-splicing of the precursor to the large ribosomal RNA of yeast mitochondria

Self-splicing of the precursor to large ribosomal RNA of yeast mitochondria leads not only to circles but also to lariats, structures that have not been observed before as products of self-splicing. Lariats were studied by electron microscopy after hybridization with an RNA complementary to the 3' half of the precursor. This leads to differentiation in at least two classes of lariats that vary in the position of the branch point. In all lariats the tail carries the 3' end, which suggests that a 5' end is used for branch formation with an internal nucleotide. The circles are formed from excised introns. They lack only three nucleotides encoded by mitochondrial DNA along with the 5'-terminal G added in the course of self-splicing. The diverse number of self-splicing products arising in vitro testifies to the considerable reactivity of this intron. The formation of lariats in an RNA catalyzed reaction may have implications for views on the mechanism of splicing of nuclear pre-mRNAs.