BACKGROUND
group ii introns are ribozymes and mobile retroelements consisting of a highly structured rna organized into six distinct domains .
they have a multifunctional intron-encoded protein  orf in div, encoding a reverse transcriptase-maturase that is required in vivo for the folding of the intron rna into a catalytically active structure  <cit> .
group ii introns splice via a lariat intermediate, in a mechanism similar to that of spliceosomal introns, via two sequential transesterification reactions  <cit> .
in the first step, the 2'-oh group of a branch-point nucleotide residue, usually a bulged adenosine in dvi, attacks the 5' splice junction, resulting in cleavage of the 5' exon and the formation of an intron-3' exon branched lariat intermediate.
the released 5' exon remains associated with the intron via base pairing of the intron binding sites  to the exon binding sites  located in ribozyme domain di  <cit> .
in the second step, the free 3'-oh of the 5' exon attacks the 3' splice junction, leading to the release of the intron lariat and the ligation of the 5' and 3' exons.
recognition of the 3'exon involves two additional base-pair interactions: the first involves the formation of a single tertiary base-pair between the last position of the intron  and an intron nucleotide  between di and dii, whereas the second involves the first positions of the exon and specific intron nucleotides.
the identity of the intron-binding site in the 3'exon differs between the major classes of group ii introns.
for iia introns, the sequence immediately upstream from ebs <dig>  base pairs with one to three nucleotide residues  of the 3' exon for intron splicing .
by contrast, most iib and iic introns display no statistical evidence of pairing of this δ position with the 3' exon.
these group ii ribozymes recognize the first nucleotide in the 3' exon, referred to as ibs <dig>  rather than δ', by canonical base-pairing with an intron nucleotide, denoted ebs <dig> rather than δ, located in the "coordination loop" of di  <cit> .
the δ position is involved in a new tertiary interaction with another residue  also located in the coordination loop  <cit> .
these loop nucleotides are involved in aligning the two exons for the second step of splicing .
the rmint <dig> intron of sinorhizobium meliloti, a group iib intron, is a mobile intron that self-splices in vitro in the absence of the iep  <cit> .
recently, we investigated rmint <dig> splicing activity in vivo and observed a low efficiency; only  <dig> % of exons were joined  <cit> .
this result suggests that bacterial group ii introns function more like retroelements than spliceosomal introns.
like other mobile group ii introns, rmint <dig> recognizes dna target sequences through both the iep and base pairing of the intron rna.
group ii ieps appear to recognize sequences in both the distal 5' and 3' exons, but appear to have no critical nucleotide residues in common.
rmint <dig> has less stringent requirements for recognition of the distal 5' and 3' exon regions, with t- <dig> and g+ <dig> the only critical nucleotide residues  <cit> , but is sufficiently long to confer high specificity.
the rmint <dig> iep may also interact with nucleotide residues - <dig> to - <dig> in the distal 5'-exon, because the elimination of these residues decreases retrohoming efficiency.
despite our knowledge of rmint <dig> dna target-site recognition requirements for reverse splicing and intron mobility, little is known about the exon sequences required for intron excision due to forward splicing.
in this study, we analyzed the nucleotide requirements in the distal 5' and 3' exon regions, ebs-ibs interactions and δ-δ' pairing for excision of the iib intron rmint <dig> in vivo.
we found that exon recognition by the intron is subject to few constraints, the most critical of which is the ebs1-ibs <dig> interaction and the δ-δ' pairing.
our findings might have practical implications for the development of rmint <dig> as a highly efficient gene targeting vector and support the extraordinary ability of rmint <dig> for its dispersal in nature.
RESULTS
5' exon nucleotide sequence requirements for rmint <dig> excision in vivo
initial studies with the yeast ai5γ group ii intron showed that in vitro trans-splicing depends on exon-intron binding sequences, the ebs1-ibs <dig> and ebs2-ibs <dig> pairings, whereas normal cis-splicing requires only the ebs1-ibs <dig> pairings  <cit> .
on the basis of these results, we analyzed the 5' exon nucleotide sequence requirements for rmint <dig> excision in vivo, by introducing single and multiple nucleotide substitutions into the ibs <dig> and ibs <dig> elements  that in the dna target site blocks intron homing  <cit> .
the linker nucleotide separating the ibs <dig> and ibs <dig> elements and the critical position - <dig> in the distal 5' exon for homing,  <cit> , which has previously been suggested to be involved in a secondary ibs2* element  <cit> , were also analyzed.
the excision reaction was characterized by primer extension; with a primer p complementary to a sequence located 80- <dig> nt from the 5' end of the intron.
in these assays, the excised intron rna was detected as a  <dig> nt extension product .
in addition to the excised intron lariat, the former product could potentially be generated from either linear intron or lariat-3' exon intermediates.
however, we have no evidence that rmint <dig> generates excised linear intron molecules in vivo: no linear intron rna was detected by 5'rlm-race , primer extension or crt-pcr .
moreover, we showed previously that the lariat form accumulates in the splicing process and that this product is free of lariat-3' exon reaction intermediates likewise, the rmint <dig> splicing process was inefficient, shown by a low detection of ligated exons, more likely to be susceptible to cell degradation  <cit> .
the ibs <dig> stretch encompasses  <dig> nt  in the 5' exon of rmint <dig> that form watson-crick pairs with the ebs <dig> intron rna sequence .
as shown in figure  <dig>  the individual replacement, within ibs <dig>  of the u- <dig> nucleotide by a g residue , the combined replacements of the u- <dig> and u- <dig> residues with ac , the replacement of the c- <dig>  a- <dig> and u- <dig> nucleotides with auc  and complete replacement of the ibs <dig> sequence uucau with acauc  in a construct  in which the intron is flanked by long stretches of exon sequences , moderately decreased excised intron levels .
a similar result was obtained when the ibs <dig> sequence was replaced in a construct in which rmint <dig> was flanked by short stretches of its natural 5'  and 3' exon  sequences .
over the distal 5' region, the replacement of u- <dig> with a g residue resulted in the retention of ~67% of wild-type intron rna excision activity .
the extension of the linker nucleotide between ibs <dig> and ibs <dig> by the addition of one nucleotide  led to a moderate decrease in excision efficiency .
thus, in contrast to the critical positions of the ebs-ibs interactions in the dna target  <cit> , neither the distal 5'exon region, ibs2-ebs <dig> nor putative ibs2*-ebs <dig>  <cit>  pairings are required for intron excision.
by contrast, more heterogeneous results were obtained for nucleotide substitutions within the ibs <dig> element.
the ibs <dig> stretch encompasses  <dig> nt, positions - <dig> to - <dig> with respect for the intron insertion site, that base-pair with the ebs <dig> sequence in the intron rna .
single substitutions at the 5' end, such as the replacement of g- <dig> with a c residue and of a- <dig> with a u residue, resulted in excision efficiencies of  <dig> and 86% those of the wild-type, respectively .
similar results were obtained for individual nucleotide substitutions at the 3' end of the ibs <dig> sequence: g- <dig> to c  and a- <dig> to c .
by contrast, the replacement of the u- <dig> nucleotide with a g residue strongly reduced excision to 12% wild-type levels , suggesting that this is a critical nucleotide position for cleavage, presumably at the 5' splice site.
simultaneous changes were also made within the ibs <dig> element.
we focused on simultaneous substitutions in positions - <dig>  - <dig> and - <dig>  that rendered excision undetectable, and simultaneous changes of the - <dig>  - <dig> and - <dig> nucleotides , which strongly reduced intron excision .
these results identified the ibs <dig> element in the 5' exon as the most critical for the intron excision reaction.
a combined mutant  lacking the canonical ibs <dig> sequence plus the substitution of the u- <dig> nucleotides by a g residue in ibs <dig> rendered intron excision barely detectable , which reveals the additional contribution of the ibs2-ebs <dig> pairing to the efficiency of the intron excision reaction.
3' exon nucleotide sequence requirements for rmint <dig> excision in vivo
in class iib introns, position + <dig> at the 3' exon, known as ibs <dig> , base-pairs with the ebs <dig> element in the intron rna , which is located in the "coordination loop" at di and displaced only one nucleotide from the 3' branch of helix id <cit> .
the ibs3-ebs <dig> interaction has been shown to be important for the second step of splicing  <cit>  and for rmint <dig> target-site recognition for intron mobility  <cit> .
interestingly, the rmint <dig> donor construct in which ibs <dig> c+ <dig> was replaced by an a residue displayed no significant impairment of excision activity .
in addition, replacement of the g+ <dig> residue in the 3' exon with an a residue, which has a 40% of reduction on homing efficiency when the target is mutated  <cit> , resulted in an excision efficiency 87% that of the wild type .
one possible interpretation of our data is that the substitution of ibs <dig> c+ <dig> by an a residue blocks second step of splicing and the  <dig> nt extension product is generated from lariat-3'exon intermediates.
to solve this question, we performed qrt-pcr and measured exon ligation by the a+ <dig> mutant.
as shown in table  <dig> the splicing activity of the former mutant was equivalent to that by the wild-type intron.
we therefore conclude that neither ibs3-ebs <dig> pairing nor 3' exon sequences are required for rmint <dig> excision in vivo.
a rmint <dig> splicing is calculated by 2-ΔΔct method  using qrt-pcr primers and conditions specified in methods section.
the excision process is a limiting step for rmint <dig> intron mobility
the above results suggest that the rmint <dig> excision in vivo is a highly favored process, with ibs1-ebs <dig> pairing the most critical step.
as the intron lariat released from the 3'exon remains associated with the iep, forming a ribonucleoprotein particle complex  that mediates intron mobility, we investigated whether the observed decrease in excision was correlated with reduced intron mobility.
we carried out a homing assay with intron donor and recipient plasmids, in which intron mobility was assessed by dna hybridization.
once rmint <dig> was excised in vivo from the exon-mutant donor constructs, the rnps produced were wild-type.
following introduction of either the wild-type intron  or mutant constructs into a s. meliloti rmo <dig> strain harboring the target-recipient plasmid pjb <dig> as, a broad correspondence was observed between the excision level detected by primer extension and homing efficiency .
requirement for δ-δ' interactions in the excision in vivo of the rmint <dig> intron
the δ-δ' interaction is thought to be required for the splicing reaction and seems to facilitate base pairing between the 5'exon and the intron; disruption of this interaction interferes with exon binding  <cit> .
in the case of rmint <dig>  this interaction involves residues 266g  and 227c  .
we found that the disruption of this pairing in the rmint <dig> intron , through the replacement of the δ residue g <dig> nucleotide with a c residue  strongly decreased excision , whereas the introduction of a compensatory δ'c to g mutation  restored excision to the level of the wild-type intron .
consistent with the lower excision efficiency observed following disruption of the δ-δ' interaction , mutant mobility was undetectable at both the wild-type dna target site and a mutant target site at which the ibs <dig> nucleotide +1c was replaced with a +1g nucleotide .
as expected, the introduction into the δc mutant of the compensatory δ'c to g mutation to restore δ-δ' pairing,  re-established mobility at the wild-type target , but not at the mutant target carrying a g nucleotide at position + <dig> .
these results provide evidence for the occurrence of intradomain di canonical δ-δ' pairing in vivo and its importance for rmint <dig> excision.
CONCLUSIONS
we show here that the intron-exon interaction ibs1-ebs <dig> and δ-δ' interaction in the domain di of the rybozime, play an important role in the rmint <dig> excision in vivo.
by contrast, however, ibs <dig>  residue u- <dig> in the distal 5' exon and ibs <dig> or g+ <dig> in the 3' exon are not required for the excision reaction, despite the requirement of these elements for the homing process.
nucleotide replacements causing a moderate or severe decrease in the excision efficiency of the intron displayed a similar decrease in homing efficiencies, probably due to a decrease in the production of rnps.
thus, rmint <dig> has different exon nucleotide sequence requirements in either both forward  and reverse  splicing reactions.
our results contribute to understand how group ii introns spread in nature, and might facilitate the use of rmint <dig> in gene targeting.
group ii introns can move site-specifically to homologous intronless genes, in a process known as retrohoming or, at much lower frequencies, to novel ectopic sites, in a process known as retrotransposition.
it is believed that the latter process has led to the wide dispersal of mobile group ii introns among bacterial species and also may have been used to invade eukaryotic genomes, where mobile group ii intron are thought to have evolved into spliceosomal introns and non-long-terminal-repeat retrotransposons  <cit> .
intron insertion-sites with good ebs/ibs pairings suggest insertion via retrohoming, whereas poorly matched ibs sequences suggest insertion via retrotransposition.
it is generally accepted that only those introns inserted by retrohoming at sites with complementary ibs sequences are expected to splice efficiently.
however, we show here that rmint <dig> excision in vivo is subject to few constraints, the most critical of which is the ebs1-ibs <dig> interaction.
thus, the spread of group ii introns into bacterial genomes from ectopic sites to homing sites may occur efficiently, increasing their ability to spread in nature.
gene targeting technology based on group ii introns requires the modification of ibs <dig>  ibs <dig> and ibs <dig>  to render these sites complementary to the retargeted ebs <dig>  ebs <dig> and ebs <dig> sequences for efficient rna splicing  <cit> .
we have recently demonstrated that the rmint <dig> intron could be used for gene targeting, following the design of introns for insertion into different target sites  <cit> .
as the predicted base-pairing interactions with the rmint <dig> intron rna -- ebs2-ibs <dig> and ebs3-ibs <dig> -- have only a small effect on overall intron-splicing efficiency, we hypothesize that the provision of ibs <dig> and ibs <dig> nucleotide combinations complementary to modified intron ebs <dig> and ebs <dig> sequences in intron plasmid constructs may not be necessary, potentially facilitating the use of rmint <dig> in gene targeting.

 group ii introns are ribozymes and mobile retroelements consisting of a highly structured rna organized into six distinct domains. they have a multifunctional intron-encoded protein orf in div, encoding a reverse transcriptase-maturase that is required in vivo for the folding of the intron rna into a catalytically active structure. group ii introns splice via a lariat intermediate, in a mechanism similar to that of spliceosomal introns, via two sequential transesterification reactions. in the first step, the 2'-oh group of a branch-point nucleotide residue, usually a bulged adenosine in dvi, attacks the 5' splice junction, resulting in cleavage of the 5' exon and the formation of an intron-3' exon branched lariat intermediate. the released 5' exon remains associated with the intron via base pairing of the intron binding sites to the exon binding sites located in ribozyme domain di. in the second step, the free 3'-oh of the 5' exon attacks the 3' splice junction, leading to the release of the intron lariat and the ligation of the 5' and 3' exons. recognition of the 3'exon involves two additional base-pair interactions: the first involves the formation of a single tertiary base-pair between the last position of the intron and an intron nucleotide between di and dii, whereas the second involves the first positions of the exon and specific intron nucleotides. the identity of the intron-binding site in the 3'exon differs between the major classes of group ii introns. for iia introns, the sequence immediately upstream from ebs base pairs with one to three nucleotide residues of the 3' exon for intron splicing. by contrast, most iib and iic introns display no statistical evidence of pairing of this δ position with the 3' exon. these group ii ribozymes recognize the first nucleotide in the 3' exon, referred to as ibs rather than δ', by canonical base-pairing with an intron nucleotide, denoted ebs rather than δ, located in the "coordination loop" of di. the δ position is involved in a new tertiary interaction with another residue also located in the coordination loop. these loop nucleotides are involved in aligning the two exons for the second step of splicing. the rmint intron of sinorhizobium meliloti, a group iib intron, is a mobile intron that self-splices in vitro in the absence of the iep. recently, we investigated rmint splicing activity in vivo and observed a low efficiency; only % of exons were joined. this result suggests that bacterial group ii introns function more like retroelements than spliceosomal introns. like other mobile group ii introns, rmint recognizes dna target sequences through both the iep and base pairing of the intron rna. group ii ieps appear to recognize sequences in both the distal 5' and 3' exons, but appear to have no critical nucleotide residues in common. rmint has less stringent requirements for recognition of the distal 5' and 3' exon regions, with t- and g+ the only critical nucleotide residues, but is sufficiently long to confer high specificity. the rmint iep may also interact with nucleotide residues - to - in the distal 5'-exon, because the elimination of these residues decreases retrohoming efficiency. despite our knowledge of rmint dna target-site recognition requirements for reverse splicing and intron mobility, little is known about the exon sequences required for intron excision due to forward splicing. in this study, we analyzed the nucleotide requirements in the distal 5' and 3' exon regions, ebs-ibs interactions and δ-δ' pairing for excision of the iib intron rmint in vivo. we found that exon recognition by the intron is subject to few constraints, the most critical of which is the ebs1-ibs interaction and the δ-δ' pairing. our findings might have practical implications for the development of rmint as a highly efficient gene targeting vector and support the extraordinary ability of rmint for its dispersal in nature.  5' exon nucleotide sequence requirements for rmint excision in vivo initial studies with the yeast ai5γ group ii intron showed that in vitro trans-splicing depends on exon-intron binding sequences, the ebs1-ibs and ebs2-ibs pairings, whereas normal cis-splicing requires only the ebs1-ibs pairings. on the basis of these results, we analyzed the 5' exon nucleotide sequence requirements for rmint excision in vivo, by introducing single and multiple nucleotide substitutions into the ibs and ibs elements that in the dna target site blocks intron homing. the linker nucleotide separating the ibs and ibs elements and the critical position - in the distal 5' exon for homing,, which has previously been suggested to be involved in a secondary ibs2* element, were also analyzed. the excision reaction was characterized by primer extension; with a primer p complementary to a sequence located 80- nt from the 5' end of the intron. in these assays, the excised intron rna was detected as a nt extension product. in addition to the excised intron lariat, the former product could potentially be generated from either linear intron or lariat-3' exon intermediates. however, we have no evidence that rmint generates excised linear intron molecules in vivo: no linear intron rna was detected by 5'rlm-race, primer extension or crt-pcr. moreover, we showed previously that the lariat form accumulates in the splicing process and that this product is free of lariat-3' exon reaction intermediates likewise, the rmint splicing process was inefficient, shown by a low detection of ligated exons, more likely to be susceptible to cell degradation. the ibs stretch encompasses nt in the 5' exon of rmint that form watson-crick pairs with the ebs intron rna sequence. as shown in figure the individual replacement, within ibs of the u- nucleotide by a g residue, the combined replacements of the u- and u- residues with ac, the replacement of the c- a- and u- nucleotides with auc and complete replacement of the ibs sequence uucau with acauc in a construct in which the intron is flanked by long stretches of exon sequences, moderately decreased excised intron levels. a similar result was obtained when the ibs sequence was replaced in a construct in which rmint was flanked by short stretches of its natural 5' and 3' exon sequences. over the distal 5' region, the replacement of u- with a g residue resulted in the retention of ~67% of wild-type intron rna excision activity. the extension of the linker nucleotide between ibs and ibs by the addition of one nucleotide led to a moderate decrease in excision efficiency. thus, in contrast to the critical positions of the ebs-ibs interactions in the dna target, neither the distal 5'exon region, ibs2-ebs nor putative ibs2*-ebs pairings are required for intron excision. by contrast, more heterogeneous results were obtained for nucleotide substitutions within the ibs element. the ibs stretch encompasses nt, positions - to - with respect for the intron insertion site, that base-pair with the ebs sequence in the intron rna. single substitutions at the 5' end, such as the replacement of g- with a c residue and of a- with a u residue, resulted in excision efficiencies of and 86% those of the wild-type, respectively. similar results were obtained for individual nucleotide substitutions at the 3' end of the ibs sequence: g- to c and a- to c. by contrast, the replacement of the u- nucleotide with a g residue strongly reduced excision to 12% wild-type levels, suggesting that this is a critical nucleotide position for cleavage, presumably at the 5' splice site. simultaneous changes were also made within the ibs element. we focused on simultaneous substitutions in positions - - and - that rendered excision undetectable, and simultaneous changes of the - - and - nucleotides, which strongly reduced intron excision. these results identified the ibs element in the 5' exon as the most critical for the intron excision reaction. a combined mutant lacking the canonical ibs sequence plus the substitution of the u- nucleotides by a g residue in ibs rendered intron excision barely detectable, which reveals the additional contribution of the ibs2-ebs pairing to the efficiency of the intron excision reaction. 3' exon nucleotide sequence requirements for rmint excision in vivo in class iib introns, position + at the 3' exon, known as ibs, base-pairs with the ebs element in the intron rna, which is located in the "coordination loop" at di and displaced only one nucleotide from the 3' branch of helix id. the ibs3-ebs interaction has been shown to be important for the second step of splicing and for rmint target-site recognition for intron mobility. interestingly, the rmint donor construct in which ibs c+ was replaced by an a residue displayed no significant impairment of excision activity. in addition, replacement of the g+ residue in the 3' exon with an a residue, which has a 40% of reduction on homing efficiency when the target is mutated, resulted in an excision efficiency 87% that of the wild type. one possible interpretation of our data is that the substitution of ibs c+ by an a residue blocks second step of splicing and the nt extension product is generated from lariat-3'exon intermediates. to solve this question, we performed qrt-pcr and measured exon ligation by the a+ mutant. as shown in table the splicing activity of the former mutant was equivalent to that by the wild-type intron. we therefore conclude that neither ibs3-ebs pairing nor 3' exon sequences are required for rmint excision in vivo. a rmint splicing is calculated by 2-ΔΔct method using qrt-pcr primers and conditions specified in methods section. the excision process is a limiting step for rmint intron mobility the above results suggest that the rmint excision in vivo is a highly favored process, with ibs1-ebs pairing the most critical step. as the intron lariat released from the 3'exon remains associated with the iep, forming a ribonucleoprotein particle complex that mediates intron mobility, we investigated whether the observed decrease in excision was correlated with reduced intron mobility. we carried out a homing assay with intron donor and recipient plasmids, in which intron mobility was assessed by dna hybridization. once rmint was excised in vivo from the exon-mutant donor constructs, the rnps produced were wild-type. following introduction of either the wild-type intron or mutant constructs into a s. meliloti rmo strain harboring the target-recipient plasmid pjb as, a broad correspondence was observed between the excision level detected by primer extension and homing efficiency. requirement for δ-δ' interactions in the excision in vivo of the rmint intron the δ-δ' interaction is thought to be required for the splicing reaction and seems to facilitate base pairing between the 5'exon and the intron; disruption of this interaction interferes with exon binding. in the case of rmint this interaction involves residues 266g and 227c. we found that the disruption of this pairing in the rmint intron, through the replacement of the δ residue g nucleotide with a c residue strongly decreased excision, whereas the introduction of a compensatory δ'c to g mutation restored excision to the level of the wild-type intron. consistent with the lower excision efficiency observed following disruption of the δ-δ' interaction, mutant mobility was undetectable at both the wild-type dna target site and a mutant target site at which the ibs nucleotide +1c was replaced with a +1g nucleotide. as expected, the introduction into the δc mutant of the compensatory δ'c to g mutation to restore δ-δ' pairing, re-established mobility at the wild-type target, but not at the mutant target carrying a g nucleotide at position +. these results provide evidence for the occurrence of intradomain di canonical δ-δ' pairing in vivo and its importance for rmint excision.  we show here that the intron-exon interaction ibs1-ebs and δ-δ' interaction in the domain di of the rybozime, play an important role in the rmint excision in vivo. by contrast, however, ibs residue u- in the distal 5' exon and ibs or g+ in the 3' exon are not required for the excision reaction, despite the requirement of these elements for the homing process. nucleotide replacements causing a moderate or severe decrease in the excision efficiency of the intron displayed a similar decrease in homing efficiencies, probably due to a decrease in the production of rnps. thus, rmint has different exon nucleotide sequence requirements in either both forward and reverse splicing reactions. our results contribute to understand how group ii introns spread in nature, and might facilitate the use of rmint in gene targeting. group ii introns can move site-specifically to homologous intronless genes, in a process known as retrohoming or, at much lower frequencies, to novel ectopic sites, in a process known as retrotransposition. it is believed that the latter process has led to the wide dispersal of mobile group ii introns among bacterial species and also may have been used to invade eukaryotic genomes, where mobile group ii intron are thought to have evolved into spliceosomal introns and non-long-terminal-repeat retrotransposons. intron insertion-sites with good ebs/ibs pairings suggest insertion via retrohoming, whereas poorly matched ibs sequences suggest insertion via retrotransposition. it is generally accepted that only those introns inserted by retrohoming at sites with complementary ibs sequences are expected to splice efficiently. however, we show here that rmint excision in vivo is subject to few constraints, the most critical of which is the ebs1-ibs interaction. thus, the spread of group ii introns into bacterial genomes from ectopic sites to homing sites may occur efficiently, increasing their ability to spread in nature. gene targeting technology based on group ii introns requires the modification of ibs ibs and ibs to render these sites complementary to the retargeted ebs ebs and ebs sequences for efficient rna splicing. we have recently demonstrated that the rmint intron could be used for gene targeting, following the design of introns for insertion into different target sites. as the predicted base-pairing interactions with the rmint intron rna -- ebs2-ibs and ebs3-ibs -- have only a small effect on overall intron-splicing efficiency, we hypothesize that the provision of ibs and ibs nucleotide combinations complementary to modified intron ebs and ebs sequences in intron plasmid constructs may not be necessary, potentially facilitating the use of rmint in gene targeting. 
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 rmint in vivo. our results contribute to understand how group ii introns spread in nature, and might facilitate the use of rmint in gene targeting. in addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo. the intron-encoded protein assists the splicing of the intron in vivo and remains bound to the excised intron lariat rna in a ribonucleoprotein particle that promotes intron mobility.
 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 assists the splicing of the intron in vivo and remains bound to the excised intron lariat rna in a ribonucleoprotein particle that promotes intron mobility. exon recognition occurs through base-pairing interactions between two guide sequences on the ribozyme domain di known as ebs and ebs and two stretches of sequence known as ibs and ibs on the 5' exon, whereas the 3' exon is recognized through interaction with the sequence immediately upstream from ebs or with a nucleotide located in the coordination-loop of di. the δ nucleotide is involved in base pairing with another intron residue in subgroup iib introns and this interaction facilitates base pairing between the 5' exon and the intron.  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 rmint in vivo. we found that the ebs1-ibs interaction was required and sufficient for rmint excision. in addition, we provide evidence for the occurrence of canonical δ-δ' pairing and its importance for the intron excision in vivo.  the excision in vivo of the rmint 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 rmint in gene targeting. 