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Transposition and exon shuffling by group II intron RNA molecules in pieces1
doi:10.1006/jmbi.2000.3582 available online at http://www.idealibrary.com on
J. Mol. Biol. (2000) 297, 301±308
COMMUNICATION
Transposition and Exon Shuffling by Group II Intron RNA Molecules in Pieces Reinhard Hiller, Martin Hetzer, Rudolf J. Schweyen and Manfred W. Mueller* Vienna Biocenter (VBC) Institute of Microbiology and Genetics, University of Vienna Austria
In the realms of RNA, transposable elements created by self-inserting introns recombine novel combinations of exon sequences in the background of replicating molecules. Although intermolecular RNA recombination is a wide-spread phenomenon reported for a variety of RNAcontaining viruses, direct evidence to support the theory that modern splicing systems, together with the exon-intron structure, have evolved from the ability of RNA to recombine, is lacking. Here, we used an in vitro deletion-complementation assay to demonstrate trans-activation of forward and reverse self-splicing of a fragmented derivative of the group II intron bI1 from yeast mitochondria. We provide direct evidence for the functional interchangeability of analogous but non-identical domain 1 RNA molecules of group II introns that result in trans-activation of intron transposition and RNA-based exon shuf¯ing. The data extend theories on intron evolution and raise the intriguing possibility that naturally fragmented group III and spliceosomal introns themselves can create transposons, permitting rapid evolution of protein-coding sequences by splicing reactions. # 2000 Academic Press
*Corresponding author
Keywords: RNA recombination; exon shuf¯ing; splicing; transposition; group II intron
Background Group II (Michel & Ferat, 1995), group III (Copertino & Hallick, 1993) and nuclear premRNA introns share features in their primary sequences, structural aspects, reaction modes and splicing intermediates (Sharp, 1991; Jacquier 1990; Moore et al., 1993; Weiner, 1993; Wise 1993; Padgett et al., 1994). RNA processing in these lariat-forming intron classes occurs in two successive transesteri®cation reactions. First, the 20 -OH group of the branch adenosine nucleotide attacks the 50 splice site to release the 50 exon and form a lariat intron intermediate. The released 30 -OH group of the 50 exon then attacks the 30 splice site to join the exons and displace the lariat intron (Michel & Ferat, 1995; Moore, 1993). R.H. and M.H. contributed equally to this work. Abbreviations used: D1, domain 1; EBS, exon binding site; IBS, intron binding site; sn, small nuclear. E-mail address of the corresponding author: [email protected] 0022-2836/00/020301±8 $35.00/0
These recent parallels supported the view that spliceosomal and group III introns are ``introns in pieces'' (Sharp, 1991), fragmented from a selfinserting group II intron ancestor (Cech, 1986; Cavalier-Smith, 1991). The theory presupposes that a similar network of structural RNA molecules form the catalytic core of both cis-acting (group II) and trans-acting (spliceosomal and group III) introns. Evidence for this claim is supported by the detailed resemblance (Madhani & Guthrie, 1994) and functional similarity (Hetzer et al., 1997; O'Keefe et al., 1996) between intramolecular basepairing in the group II core structure with intermolecular interactions of spliceosomal small nuclear (sn) RNA molecules (Madhani & Guthrie, 1994; Hetzer et al., 1997; O'Keefe et al., 1996). Assuming the invasive character of an ancestral group II intron, the frequent insertion of this transposon would generate splice sites and homologous sequences at a variety of positions in the genome (Darnell & Doolittle, 1986; Rogers, 1989). Novel combinations of exons could be generated by intermolecular RNA recombination (Lai, 1992; Chetverin et al., 1997; Gilbert, 1986), by DNA # 2000 Academic Press
302
Transposition and Exon Shuf¯ing
Figure 1 (legend opposite)
Transposition and Exon Shuf¯ing
recombination across two homologous intervening sequences (Darnell & Doolittle, 1986; Gilbert, 1978; Hall et al., 1989; Byrk & Belfort, 1990) or by the trans-splicing of exons (Darnell & Doolittle, 1986). Although exon shuf¯ing by intermolecular RNA recombination may permit rapid evolution of protein-coding sequences, direct evidence to support this theory is lacking. Here, experiments were designed to support the hypothesis that modern splicing systems, together with the exon-intron structure, could have evolved from the ability of RNA to recombine by splicing reactions (Gilbert, 1986). Speci®cally, that fragmented introns themselves could create transposons (Sharp, 1985; Roger & Doolittle, 1993) that assemble novel combinations of Xexon sequences by the interchangeability of analogous but non-identical domain structures. Re-association of homologous group II intron pieces into an active catalytic core in trans promotes forward and reverse self-splicing in vitro We used an in vitro deletion-complementation assay to investigate domain 1 (D1) RNA molecules of group II introns (Figure 1(a)) for trans-activation in splicing a truncated derivative of a group II intron, bI1, from yeast mitochondria. D1, as the major organizing center of group II introns covers almost 50 % of the core structure, and consists of a very complex set of sub-domains (Michel & Ferat, 1995). The terminal loop of sub-domain ID3 contains the exon binding site 1 (EBS1), complementary to the 30 end of the 50 exon (IBS1; intron
303 binding site) (Figure 1(a)). This tertiary interaction contacts the exon-intron boundaries during exon ligation (Hetzer et al., 1997) and de®nes target site speci®city in group II intron homing (Yang et al., 1996) and transposition (Mueller et al., 1993) by reverse splicing (Augustin et al., 1990). In a ®rst step, we analyzed the effect of complete D1 RNA deletion (Figure 1(b)). In contrast to the bI1 wild-type, the mutant bI1D1 pre-mRNA (398 nt) lacking D1bI1 (378 nt), was non-reactive in RNA processing under all conditions tested (Figure 1(b)). Bimolecular complementation assays, using separately transcribed D1bI1 and bI1D1 pre-mRNA, restored processing of the truncated precursor in trans by transesteri®cation reactions, giving rise to splicing end products, excized lariat intron and ligated exons (Figure 1(b)). Complementation assays of bI1D1 in the presence of increasing concentrations of D1bI1 indicated that the domain D1 added in trans acted as an enzyme obeying Michaelis-Menten kinetics (Figure 1(c)). However, the reactivity of the bimolecular reaction (bI1D1 D1bI1; Kd 280 nM, Vmax 0.0016 minÿ1) was at least 100-fold decreased compared to the wild-type intron (kobs 0.12 minÿ1). Nevertheless, precision and orderliness in splicing were conserved. The structure of the bI1D1 lariat was proven by mobilityshift experiments, and the accuracy of branch formation was veri®ed by primer extension analysis (not shown). The ®delity of exon ligation was con®rmed by PCR-mediated sequence analysis (Figure 2(b)).
Figure 1. (a) Secondary structure and tertiary interactions (Michel & Ferat, 1995) of group II intron bI1. Blue boxes, 50 and 30 exon sequences (50 E, cyan; 30 E, dark blue). Continuous lines, six major intron domains (D1-D6); D1 used for trans-complementation, cyan; D2-D6, black. 50 GUGAG...AU, conserved nucleotides at intron boundaries. Individual nucleotides and structures involved in tertiary interactions (EBS-IBS, e-e0 , d-d0 , g-g0 and z-z0 ) (Michel & Ferat, 1995). Note, for bI1, the d-d0 interaction (U-U) is not established. (b) Deletion-complementation assays (bI1D1 D1bI1) compared with the self-splicing of wild-type bI1. Forward self-splicing of bI1D1 precursors (10 nM) in the absence and presence of separately transcribed D1bI1 RNA (500 nM) (bI1D1 D1bI1). Before incubation, RNA molecules were heated to 95 C for two minutes and then cooled to 37 C. The pre-treated samples were incubated in a buffer containing 40 mM Tris-HCl (pH 7.5), 60 mM MgCl2, 5 mM spermidine, 10 % (v/v) EtOH and 1.25 M NH4Cl and samples were taken at speci®c time points as indicated. The reaction products were separated on 5 % polyacrylamide-8 M urea gels. The rate of turnover was quanti®ed with the Phosphor Imager (Molecular Dynamics). Pr and Pr(D1), preRNA molecules; L-30 E and L(D1)-30 E, lariat-30 exon intermediate; L and L(D1), lariat introns; 50 EbI1 ÿ 30 EbI1, ligated exons. BS/bI1DD1 was generated from BS/bI1 (Mueller et al., 1991) using two oligonucleotides (50 - AAA TCT GGT AAC ATG GCT GGG AAC AAA AGG TTA TTG TTG TGT TTA GGA CAG AGT GAG ACT GGT AAG CCC AAT TAT TTC C - 30 and 50 - GTC TGA ATT CCA TGG CTG CAA TTG TGA TAG GTA GAT C - 30 ), thereby deleting domain D1 and inserting an additional thymidine nucleotide at the base of domain D1 in the central intron core element (Hetzer et al., 1997). A second PCR ampli®ed bI1D1, which was subsequently ligated into BSKS through KpnI and EcoRI. Pre-mRNA molecules were synthesized by transcription of EcoRI-digested BS/bI1 and BS/bI1DI with T3 RNA polymerase in the presence of [35S]UTP followed by gel puri®cation. Trans-acting D1 RNA molecules were generated by using primers complementary (12 nt) to 50 and 30 ends (plus primer containing the sequence for T7 promoter) of domain D1 followed by in vitro transcription and gel puri®cation. (c) Splicing of bI1D1 (10 nM) in presence of increasing amounts of D1bI1 RNA molecules (100-1500 nM). Apparent rate constants of the bipartite system were calculated from a log-linear plot of early data-points of the trans-splicing reaction (inset). Fits of the data to a one-side binding curve reveal Kd and kcat values for the trans-splicing system of 280 nM and 0.0016 minÿ1, respectively.
304
Transposition and Exon Shuf¯ing
Figure 2. Recombinant exon ligation by trans-activation of second step catalysis in bI1D1 splicing. (a) Trans-activation of homologous and heterologous exon ligation by D1 RNA molecules. bI1D1 precursors (10 nM) were incubated with D1 RNA molecules (500 nM) derived from intron bI1 (D1bI1) or intron aI5c (D1aI5c) in the presence of their corresponding 50 end-labeled ([32P]ATP) 50 exons (50 EbI1 and 50 EaI5c) (1 mM). For investigation of recombinant forward and reverse splicing reactions, synthesized and 50 terminally [g-32P]ATP-labeled 50 exon RNA molecules (50 EbI1: 50 - GGG AAC AAA GGT TAT TGT TGT GAA GAA GGT TTA TGG ACA GA - 30 and 50 EaI5c: 50 - CTG GTA CCA GCT GAC TTA CTA GCT GGT GGG ACA TTT TC - 30 ) and ligated exon RNA molecules (50 EbI1-TGC ACA TTG CTG GTA ATA TAA - 30 ); 50 EaI5c-ACT TGA TAT TAT CAA TGG TGC - 30 ) or pseudo-ligated exon RNA molecules (Mueller et al., 1991) (50 EbI1-32pCp; 50 EaI5c-32pCp), comprising a single 32P-label at the phosphorus atom that forms the ligation junction, were used. Labeled reaction products: 50 EbI1 ÿ 30 EbI1, homologous system; 50 EaI5c-30 EbI1, heterologous system. (b) PCR-mediated sequence analysis (Hetzer et al., 1997) of the wild-type 50 EbI1 ÿ 30 EbI1 ligation product. (c) PCR-mediated sequence analysis of the recombinant 50 EaI5c ÿ 30 EbI1 ligation product.
Fragmented introns themselves can create transposons D1 activated bI1D1 pre-mRNA splicing suggested that the association of domain structures reconstituted the catalytic core of a group II intron
in trans (Figure 4(a)). Reverse splicing (Augustin et al., 1990) proceeds by the true reversion of both transesteri®cation steps used in forward splicing (Mueller et al., 1991), raising the possibility that fragmented introns themselves can create self-
Transposition and Exon Shuf¯ing
305
Figure 3. (a) Trans-activation of complete reverse splicing by D1 RNA molecules. Internally [35S]UTP-labeled bI1D1 lariat RNAs (10 nM) were co-incubated with 32P-labeled pseudo-ligated exons (50 EbI1-*pCp; 50 EaI5c-*pCp) (1 mM), together with their corresponding D1 RNA molecules (D1bI1; D1aI5c) (500 nM). Samples were removed at the time-points indicated (60, 120, 2400 ). Labeled reaction products: L(D1)-30 E, lariat 30 exon; Bl(D1)-30 E; broken lariat30 exon; 50 EbI1-IVS(D1)-30 E and 50 EaI5c-IVS(D1)-30 E, complete reverse splicing products. (b) PCR-mediated sequence analysis of the recombinant 50 EaI5c-bI1D1 debranching product.
inserting elements by the trans-assembling of separately transcribed domain structures (Figure 4(b)). To provide evidence for this theory, we analyzed trans-activation of reverse splicing by D1 RNA molecules. First, we tested reverse branching as the rate-limiting step in reverse splicing (Augustin et al., 1990; Mueller et al., 1991). In trimolecular splicing assays containing gel-puri®ed bI1D1
lariat RNA molecules, D1bI1 and 50 end-labeled 50 exons (5'EbI1), the product of the self-catalyzed reverse branching reaction (5'EbI1-IVS(D1)), accumulated in the course of the reaction (data not shown). The accuracy of reverse branching was veri®ed by reverse transcriptase (RT) polymerase chain reaction (PCR) followed by dideoxy-sequencing (not shown). When in trimolecular assays, the
306 50 exon RNA molecules were substituted by ligated or pseudo-ligated exon sequences (50 EbI1-*pCp) (Mueller et al., 1991), products characteristic for partial and complete reverse splicing were observed (Figure 3(a)). So far we have provided evidence for re-association of homologous group II intron pieces into an active catalytic core in trans, promoting forward and reverse self-splicing in vitro. Next we analyzed the functional interchangeability of analogous but non-identical D1 RNA molecules. We hypothesized that association of heterologous group II intron domains in trans might result in intron insertions at non-allelic sites (Figure 4(b)), thereby creating novel combinations of exon sequences by intermolecular RNA recombination (Figure 4(c)).
Transposition and Exon Shuf¯ing
To demonstrate this, we separately tested transcribed D1 RNA molecules (D1aI5c) from the yeast mitochondrial group II intron aI5c (subclass IIB) (Michel & Ferat, 1995) for trans-activation of bI1D1 forward and reverse splicing. First, we assayed for trans-complementation of recombinant exon ligation. A trimolecular assay using D1 RNA from intron aI5c (D1aI5c) and its corresponding 50 exon (50 EaI5c) was suf®cient to promote second-step catalysis in bI1D1 preRNA splicing that resulted in recombinant exon ligation (50 EaI5c ÿ 30 EbI1) (Figure 2(a)). As con®rmed by sequence analysis, the 50 exon attack was directed precisely towards the 30 splice site (Figure 2(c)). Next, we investigated trans-activation of recombinant reverse splicing by D1 RNA molecules, i.e.
Figure 4. Trans-activation of bI1DD1 forward splicing, transposition and exon shuf¯ing by D1 RNA molecules. Colored boxes, 50 and 30 exon sequences. D2-D6, black line. D1 RNA molecules (D1bI1; D1X) and EBS1-IBS1 interactions, colored according to their corresponding 50 exons. Small arrows, nucleophilic attacks in forward (a) (1F, branch formation; 2F, exon ligation) and reverse splicing (b) (1R, reversal of exon ligation; 2R, reverse branching). (a) Forward splicing. D1bI1 associates with bI1D1 in trans resulting in lariat formation and precise exon ligation by two successive transesteri®cation reactions. (b) Transposition of bI1D1 into non-cognate ligated exon sequences. D1X selects ligated exons via the IBS1-EBS1 interaction, resulting in the complete insertion of the DD1 lariat RNA by reverse splicing (Augustin et al., 1990). (c) Exon shuf¯ing by the exchange of trans-acting D1 RNA molecules at the intermediate stage in splicing. The model involves trans-activation of reversal of exon ligation (1R) by D1 RNA molecules, followed by a dissociation-association pathway and exon ligation (2F). Note, reverse branching (R2) after the dissociation/association step generates recombinant precursors (50 E(bI1)-IVS(D1)-30 E(X); 50 E(X)-IVS(D1)-30 E(bI1)) which can be activated in splicing generating recombinant exon products in trans (see the text).
307
Transposition and Exon Shuf¯ing
the insertion of the bI1D1 lariat RNA into noncognate exon sequences (Figure 4(b)). In assays containing bI1D1 lariat domain D1 of intron aI5c (D1aI5c) and the corresponding 50 exon (50 EaI5c), the recombinant reverse branching product (50 EaI5c ÿ bI1(D1)) accumulated (data not shown). The use of D1aI5c in the presence of aI5c ligated or pseudo-ligated exons (50 EaI5c ÿ 30 EaI5c) (50 EaI5c ÿ *pCp) promoted the complete and precise insertion of the bI1D1 lariat RNA into the noncognate aI5c target sequences (Figure 3(a)). The accuracy of the recombinant transposition of the truncated bI1D1 intron was veri®ed by RT-PCR followed by dideoxy sequencing (Figure 3(b)). As expected, the recombinant reverse splicing product (50 EaI5c ÿ bI1(D1) ÿ 30 EaI5c), non-reactive by itself, could be re-activated in forward splicing by D1aI5c in trans, giving rise to excised bI1D1 lariat intron and spliced aI5c exons. When D1bI1 RNA was assayed in the presence of the bI1 50 exon (50 EbI1) and the recombinant reverse splicing product (50 EaI5c ÿ bI1(D1)ÿ30 EaI5c), trans-activation of second step catalysis was observed, generating a recombinant ligated exon product (50 EbI1 ÿ 30 EaI5c) (data not shown).
RNA-based exon shuffling by group II intron RNAs in pieces In a ®nal step, exon shuf¯ing by the dynamic interchangeability of non-identical D1 RNA molecules at the intermediate stage of splicing was tested (Figure 4(c)). In experiments involving bI1D1 lariat RNA, D1 RNA molecules of intron aI5c and bI1, together with their corresponding exon sequences, recombinant ligated exon products (50 EbI1 ÿ 30 EaI5c; 50 EaI5c ÿ 30 EbI1) and recombinant complete reverse splicing products 50 EaI5c ÿ bI1(D1) ÿ (50 EbI1 ÿbI1(D1) ÿ 30 EaI5c; 0 3 EbI1) (Figure 4(c)) were observed (data not shown). In conclusion, we have demonstrated an in vitro RNA recombination system catalyzed by group II intron RNA molecules in pieces, resulting in the transposition of fragmented introns and in the creation of novel combinations of exon sequences by splicing reactions. Our data support the hypothesis that modern splicing systems together with their exon-intron structure, may have evolved from the ability of RNA to recombine (Gilbert, 1986) and imply that naturally fragmented group II, group III (Copertino & Hallick, 1993) and spliceosomal introns can create transposons (Sharp, 1985; Roger & Doolittle, 1993). Finally, assembling a functional splicing apparatus by homologous but non-identical intron structures in trans may permit not only intron spreading to non-allelic sites, and thereby the rapid evolution of protein-coding sequences, but might also provide a potential mechanism for RNA-based modulation of gene expression (Robineau et al., 1997; Anderson & Moore, 1997).
Acknowledgements This work was supported by the Austrian Fonds zur FoÈrderung der wissenschaftlichen Forschung (FWF).
References Anderson, K. & Moore, M. J. (1997). Bimolecular exon ligation by the human spliceosome. Science, 276, 1712-1716. Augustin, S., Mueller, M. W. & Schweyen, R. J. (1990). Reverse self-splicing of group II intron RNAs in vitro. Nature, 343, 383-386. Byrk, M. & Belfort, M. (1990). Spontaneous shuf¯ing of domains between introns of phage T4. Nature, 346, 394-396. Cavalier-Smith, T. (1985). Sel®sh DNA and the origin of introns. Nature, 315, 283-284. Cavalier-Smith, T. (1991). Intron phylogeny: a new hypothesis. Trends Genet. 7, 145-148. Cech, T. R. (1986). The generality of self-splicing RNA: relationship to nuclear mRNA splicing. Cell, 44, 207-210. Chetverin, A. B., Chetverina, H. V., Demidenko, A. A. & Ugarov, V. I. (1997). Nonhomologous RNA recombination in a cell-free system: evidence for a transesteri®cation mechanism guided by secondary structure. Cell, 88, 503-513. Copertino, D. W. & Hallick, R. B. (1993). Group II and group III introns of twintrons: potential relationships with nuclear pre-mRNA introns. Trends Biochem. Sci. 18, 467-471. Darnell, J. E. & Doolittle, W. F. (1986). Speculations on the early course of evolution. Proc. Natl Acad. Sci. USA, 83, 1271-1275. Gesteland, R. F. & Atkins, J. F. (1993). The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Gilbert, W. (1978). Why genes in pieces? Nature, 271, 501. Gilbert, W. (1986). The RNA world. Nature, 319, 618. Hall, D. H., Liu, Y. & Shub, D. A. (1989). Exon shuf¯ing by recombination between self-splicing introns of bacteriophage T4. Nature, 340, 574-576. Hetzer, M., Wurzer, G., Schweyen, R. J. & Mueller, M. W. (1997). Trans-activation of group II intron splicing by nuclear U5 snRNA. Nature, 386, 417-420. Jacquier, A. (1990). Self-splicing group II introns and nuclear pre-messenger RNA introns - how similar are they? Trends Bio. Sci. 15, 351-354. Lai, M. M. C. (1992). RNA recombination in animal and plant viruses. Microbiol. Rev. 56, 61-79. Madhani, H. D. & Guthrie, C. (1994). Dynamic RNARNA interactions in the spliceosome. Annu. Rev. Genet. 28, 1-26. Michel, F. & Ferat, J.-L. (1995). Structure and activities of group II introns. Annu. Rev. Biochem. 64, 435-461. Moore, M. J., Query, C. C. & Sharp, P. A. (1993). Splicing of precursor to mRNA by the spliceosome. In The RNA World (Gesteland, R. F. & Atkins, J. F., eds), pp. 303-358, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Moore, M. J. & Sharp, P. A. (1993). Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing. Nature, 365, 364368. Mueller, M. W., Stocker, P., Hetzer, M. & Schweyen, R. J. (1991). Fate of the junction phosphate in alternating
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forward and reverse self-splicing reactions of group II intron RNA. J. Mol. Biol. 222, 145-154. Mueller, M. W., Allmaier, M., Eskes, R. & Schweyen, R. J. (1993). Transposition of group II intron aI1 in yeast and invasion of mitochondrial genes at new locations. Nature, 366, 174-176. O'Keefe, R. T., Norman, C. & Newman, A. J. (1996). The invariant U5 snRNA loop 1 sequence is dispensable for the ®rst catalytic step of pre-mRNA splicing in yeast. Cell, 86, 679-689. Padgett, R. A., Podar, M., Boulanger, S. C. & Perlman, P. S. (1994). The stereochemical course of group II intron self-splicing. Science, 266, 1685-1688. Robineau, S., Bergantino, E., Carignani, G., Michel, F. & Netter, P. (1997). Suppressors of cis-acting splicingde®cient mutations that affect the ribozyme core of a group II intron. J. Mol. Biol. 267, 537-547.
Roger, A. J. & Doolittle, F. W. (1993). Why introns-inpieces? Nature, 364, 289-290. Rogers, J. H. (1989). How were introns inserted into nuclear genes? Trends Genet. 5, 213-216. Sharp, P. A. (1985). On the origin of RNA splicing and introns. Cell, 42, 397-400. Sharp, P. A. (1991). Five easy pieces. Science, 42, 663. Weiner, A. M. (1993). mRNA splicing and autocatalytic introns: distant cousins or the products of chemical determinism? Cell, 72, 161-164. Wise, J. A. (1993). Guides to the heart of the spliceosome. Science, 262, 1978-1979. Yang, J., Zimmerly, S., Perlman, P. S. & Lambowitz, A. M. (1996). Ef®cient integration of an intron RNA into double-stranded DNA by reverse splicing. Nature, 381, 332-335.
Edited by J. Karn (Received 12 October 1999; received in revised form 7 February 2000; accepted 7 February 2000)
J. Mol. Biol. (2000) 297, 301±308
COMMUNICATION
Transposition and Exon Shuffling by Group II Intron RNA Molecules in Pieces Reinhard Hiller, Martin Hetzer, Rudolf J. Schweyen and Manfred W. Mueller* Vienna Biocenter (VBC) Institute of Microbiology and Genetics, University of Vienna Austria
In the realms of RNA, transposable elements created by self-inserting introns recombine novel combinations of exon sequences in the background of replicating molecules. Although intermolecular RNA recombination is a wide-spread phenomenon reported for a variety of RNAcontaining viruses, direct evidence to support the theory that modern splicing systems, together with the exon-intron structure, have evolved from the ability of RNA to recombine, is lacking. Here, we used an in vitro deletion-complementation assay to demonstrate trans-activation of forward and reverse self-splicing of a fragmented derivative of the group II intron bI1 from yeast mitochondria. We provide direct evidence for the functional interchangeability of analogous but non-identical domain 1 RNA molecules of group II introns that result in trans-activation of intron transposition and RNA-based exon shuf¯ing. The data extend theories on intron evolution and raise the intriguing possibility that naturally fragmented group III and spliceosomal introns themselves can create transposons, permitting rapid evolution of protein-coding sequences by splicing reactions. # 2000 Academic Press
*Corresponding author
Keywords: RNA recombination; exon shuf¯ing; splicing; transposition; group II intron
Background Group II (Michel & Ferat, 1995), group III (Copertino & Hallick, 1993) and nuclear premRNA introns share features in their primary sequences, structural aspects, reaction modes and splicing intermediates (Sharp, 1991; Jacquier 1990; Moore et al., 1993; Weiner, 1993; Wise 1993; Padgett et al., 1994). RNA processing in these lariat-forming intron classes occurs in two successive transesteri®cation reactions. First, the 20 -OH group of the branch adenosine nucleotide attacks the 50 splice site to release the 50 exon and form a lariat intron intermediate. The released 30 -OH group of the 50 exon then attacks the 30 splice site to join the exons and displace the lariat intron (Michel & Ferat, 1995; Moore, 1993). R.H. and M.H. contributed equally to this work. Abbreviations used: D1, domain 1; EBS, exon binding site; IBS, intron binding site; sn, small nuclear. E-mail address of the corresponding author: [email protected] 0022-2836/00/020301±8 $35.00/0
These recent parallels supported the view that spliceosomal and group III introns are ``introns in pieces'' (Sharp, 1991), fragmented from a selfinserting group II intron ancestor (Cech, 1986; Cavalier-Smith, 1991). The theory presupposes that a similar network of structural RNA molecules form the catalytic core of both cis-acting (group II) and trans-acting (spliceosomal and group III) introns. Evidence for this claim is supported by the detailed resemblance (Madhani & Guthrie, 1994) and functional similarity (Hetzer et al., 1997; O'Keefe et al., 1996) between intramolecular basepairing in the group II core structure with intermolecular interactions of spliceosomal small nuclear (sn) RNA molecules (Madhani & Guthrie, 1994; Hetzer et al., 1997; O'Keefe et al., 1996). Assuming the invasive character of an ancestral group II intron, the frequent insertion of this transposon would generate splice sites and homologous sequences at a variety of positions in the genome (Darnell & Doolittle, 1986; Rogers, 1989). Novel combinations of exons could be generated by intermolecular RNA recombination (Lai, 1992; Chetverin et al., 1997; Gilbert, 1986), by DNA # 2000 Academic Press
302
Transposition and Exon Shuf¯ing
Figure 1 (legend opposite)
Transposition and Exon Shuf¯ing
recombination across two homologous intervening sequences (Darnell & Doolittle, 1986; Gilbert, 1978; Hall et al., 1989; Byrk & Belfort, 1990) or by the trans-splicing of exons (Darnell & Doolittle, 1986). Although exon shuf¯ing by intermolecular RNA recombination may permit rapid evolution of protein-coding sequences, direct evidence to support this theory is lacking. Here, experiments were designed to support the hypothesis that modern splicing systems, together with the exon-intron structure, could have evolved from the ability of RNA to recombine by splicing reactions (Gilbert, 1986). Speci®cally, that fragmented introns themselves could create transposons (Sharp, 1985; Roger & Doolittle, 1993) that assemble novel combinations of Xexon sequences by the interchangeability of analogous but non-identical domain structures. Re-association of homologous group II intron pieces into an active catalytic core in trans promotes forward and reverse self-splicing in vitro We used an in vitro deletion-complementation assay to investigate domain 1 (D1) RNA molecules of group II introns (Figure 1(a)) for trans-activation in splicing a truncated derivative of a group II intron, bI1, from yeast mitochondria. D1, as the major organizing center of group II introns covers almost 50 % of the core structure, and consists of a very complex set of sub-domains (Michel & Ferat, 1995). The terminal loop of sub-domain ID3 contains the exon binding site 1 (EBS1), complementary to the 30 end of the 50 exon (IBS1; intron
303 binding site) (Figure 1(a)). This tertiary interaction contacts the exon-intron boundaries during exon ligation (Hetzer et al., 1997) and de®nes target site speci®city in group II intron homing (Yang et al., 1996) and transposition (Mueller et al., 1993) by reverse splicing (Augustin et al., 1990). In a ®rst step, we analyzed the effect of complete D1 RNA deletion (Figure 1(b)). In contrast to the bI1 wild-type, the mutant bI1D1 pre-mRNA (398 nt) lacking D1bI1 (378 nt), was non-reactive in RNA processing under all conditions tested (Figure 1(b)). Bimolecular complementation assays, using separately transcribed D1bI1 and bI1D1 pre-mRNA, restored processing of the truncated precursor in trans by transesteri®cation reactions, giving rise to splicing end products, excized lariat intron and ligated exons (Figure 1(b)). Complementation assays of bI1D1 in the presence of increasing concentrations of D1bI1 indicated that the domain D1 added in trans acted as an enzyme obeying Michaelis-Menten kinetics (Figure 1(c)). However, the reactivity of the bimolecular reaction (bI1D1 D1bI1; Kd 280 nM, Vmax 0.0016 minÿ1) was at least 100-fold decreased compared to the wild-type intron (kobs 0.12 minÿ1). Nevertheless, precision and orderliness in splicing were conserved. The structure of the bI1D1 lariat was proven by mobilityshift experiments, and the accuracy of branch formation was veri®ed by primer extension analysis (not shown). The ®delity of exon ligation was con®rmed by PCR-mediated sequence analysis (Figure 2(b)).
Figure 1. (a) Secondary structure and tertiary interactions (Michel & Ferat, 1995) of group II intron bI1. Blue boxes, 50 and 30 exon sequences (50 E, cyan; 30 E, dark blue). Continuous lines, six major intron domains (D1-D6); D1 used for trans-complementation, cyan; D2-D6, black. 50 GUGAG...AU, conserved nucleotides at intron boundaries. Individual nucleotides and structures involved in tertiary interactions (EBS-IBS, e-e0 , d-d0 , g-g0 and z-z0 ) (Michel & Ferat, 1995). Note, for bI1, the d-d0 interaction (U-U) is not established. (b) Deletion-complementation assays (bI1D1 D1bI1) compared with the self-splicing of wild-type bI1. Forward self-splicing of bI1D1 precursors (10 nM) in the absence and presence of separately transcribed D1bI1 RNA (500 nM) (bI1D1 D1bI1). Before incubation, RNA molecules were heated to 95 C for two minutes and then cooled to 37 C. The pre-treated samples were incubated in a buffer containing 40 mM Tris-HCl (pH 7.5), 60 mM MgCl2, 5 mM spermidine, 10 % (v/v) EtOH and 1.25 M NH4Cl and samples were taken at speci®c time points as indicated. The reaction products were separated on 5 % polyacrylamide-8 M urea gels. The rate of turnover was quanti®ed with the Phosphor Imager (Molecular Dynamics). Pr and Pr(D1), preRNA molecules; L-30 E and L(D1)-30 E, lariat-30 exon intermediate; L and L(D1), lariat introns; 50 EbI1 ÿ 30 EbI1, ligated exons. BS/bI1DD1 was generated from BS/bI1 (Mueller et al., 1991) using two oligonucleotides (50 - AAA TCT GGT AAC ATG GCT GGG AAC AAA AGG TTA TTG TTG TGT TTA GGA CAG AGT GAG ACT GGT AAG CCC AAT TAT TTC C - 30 and 50 - GTC TGA ATT CCA TGG CTG CAA TTG TGA TAG GTA GAT C - 30 ), thereby deleting domain D1 and inserting an additional thymidine nucleotide at the base of domain D1 in the central intron core element (Hetzer et al., 1997). A second PCR ampli®ed bI1D1, which was subsequently ligated into BSKS through KpnI and EcoRI. Pre-mRNA molecules were synthesized by transcription of EcoRI-digested BS/bI1 and BS/bI1DI with T3 RNA polymerase in the presence of [35S]UTP followed by gel puri®cation. Trans-acting D1 RNA molecules were generated by using primers complementary (12 nt) to 50 and 30 ends (plus primer containing the sequence for T7 promoter) of domain D1 followed by in vitro transcription and gel puri®cation. (c) Splicing of bI1D1 (10 nM) in presence of increasing amounts of D1bI1 RNA molecules (100-1500 nM). Apparent rate constants of the bipartite system were calculated from a log-linear plot of early data-points of the trans-splicing reaction (inset). Fits of the data to a one-side binding curve reveal Kd and kcat values for the trans-splicing system of 280 nM and 0.0016 minÿ1, respectively.
304
Transposition and Exon Shuf¯ing
Figure 2. Recombinant exon ligation by trans-activation of second step catalysis in bI1D1 splicing. (a) Trans-activation of homologous and heterologous exon ligation by D1 RNA molecules. bI1D1 precursors (10 nM) were incubated with D1 RNA molecules (500 nM) derived from intron bI1 (D1bI1) or intron aI5c (D1aI5c) in the presence of their corresponding 50 end-labeled ([32P]ATP) 50 exons (50 EbI1 and 50 EaI5c) (1 mM). For investigation of recombinant forward and reverse splicing reactions, synthesized and 50 terminally [g-32P]ATP-labeled 50 exon RNA molecules (50 EbI1: 50 - GGG AAC AAA GGT TAT TGT TGT GAA GAA GGT TTA TGG ACA GA - 30 and 50 EaI5c: 50 - CTG GTA CCA GCT GAC TTA CTA GCT GGT GGG ACA TTT TC - 30 ) and ligated exon RNA molecules (50 EbI1-TGC ACA TTG CTG GTA ATA TAA - 30 ); 50 EaI5c-ACT TGA TAT TAT CAA TGG TGC - 30 ) or pseudo-ligated exon RNA molecules (Mueller et al., 1991) (50 EbI1-32pCp; 50 EaI5c-32pCp), comprising a single 32P-label at the phosphorus atom that forms the ligation junction, were used. Labeled reaction products: 50 EbI1 ÿ 30 EbI1, homologous system; 50 EaI5c-30 EbI1, heterologous system. (b) PCR-mediated sequence analysis (Hetzer et al., 1997) of the wild-type 50 EbI1 ÿ 30 EbI1 ligation product. (c) PCR-mediated sequence analysis of the recombinant 50 EaI5c ÿ 30 EbI1 ligation product.
Fragmented introns themselves can create transposons D1 activated bI1D1 pre-mRNA splicing suggested that the association of domain structures reconstituted the catalytic core of a group II intron
in trans (Figure 4(a)). Reverse splicing (Augustin et al., 1990) proceeds by the true reversion of both transesteri®cation steps used in forward splicing (Mueller et al., 1991), raising the possibility that fragmented introns themselves can create self-
Transposition and Exon Shuf¯ing
305
Figure 3. (a) Trans-activation of complete reverse splicing by D1 RNA molecules. Internally [35S]UTP-labeled bI1D1 lariat RNAs (10 nM) were co-incubated with 32P-labeled pseudo-ligated exons (50 EbI1-*pCp; 50 EaI5c-*pCp) (1 mM), together with their corresponding D1 RNA molecules (D1bI1; D1aI5c) (500 nM). Samples were removed at the time-points indicated (60, 120, 2400 ). Labeled reaction products: L(D1)-30 E, lariat 30 exon; Bl(D1)-30 E; broken lariat30 exon; 50 EbI1-IVS(D1)-30 E and 50 EaI5c-IVS(D1)-30 E, complete reverse splicing products. (b) PCR-mediated sequence analysis of the recombinant 50 EaI5c-bI1D1 debranching product.
inserting elements by the trans-assembling of separately transcribed domain structures (Figure 4(b)). To provide evidence for this theory, we analyzed trans-activation of reverse splicing by D1 RNA molecules. First, we tested reverse branching as the rate-limiting step in reverse splicing (Augustin et al., 1990; Mueller et al., 1991). In trimolecular splicing assays containing gel-puri®ed bI1D1
lariat RNA molecules, D1bI1 and 50 end-labeled 50 exons (5'EbI1), the product of the self-catalyzed reverse branching reaction (5'EbI1-IVS(D1)), accumulated in the course of the reaction (data not shown). The accuracy of reverse branching was veri®ed by reverse transcriptase (RT) polymerase chain reaction (PCR) followed by dideoxy-sequencing (not shown). When in trimolecular assays, the
306 50 exon RNA molecules were substituted by ligated or pseudo-ligated exon sequences (50 EbI1-*pCp) (Mueller et al., 1991), products characteristic for partial and complete reverse splicing were observed (Figure 3(a)). So far we have provided evidence for re-association of homologous group II intron pieces into an active catalytic core in trans, promoting forward and reverse self-splicing in vitro. Next we analyzed the functional interchangeability of analogous but non-identical D1 RNA molecules. We hypothesized that association of heterologous group II intron domains in trans might result in intron insertions at non-allelic sites (Figure 4(b)), thereby creating novel combinations of exon sequences by intermolecular RNA recombination (Figure 4(c)).
Transposition and Exon Shuf¯ing
To demonstrate this, we separately tested transcribed D1 RNA molecules (D1aI5c) from the yeast mitochondrial group II intron aI5c (subclass IIB) (Michel & Ferat, 1995) for trans-activation of bI1D1 forward and reverse splicing. First, we assayed for trans-complementation of recombinant exon ligation. A trimolecular assay using D1 RNA from intron aI5c (D1aI5c) and its corresponding 50 exon (50 EaI5c) was suf®cient to promote second-step catalysis in bI1D1 preRNA splicing that resulted in recombinant exon ligation (50 EaI5c ÿ 30 EbI1) (Figure 2(a)). As con®rmed by sequence analysis, the 50 exon attack was directed precisely towards the 30 splice site (Figure 2(c)). Next, we investigated trans-activation of recombinant reverse splicing by D1 RNA molecules, i.e.
Figure 4. Trans-activation of bI1DD1 forward splicing, transposition and exon shuf¯ing by D1 RNA molecules. Colored boxes, 50 and 30 exon sequences. D2-D6, black line. D1 RNA molecules (D1bI1; D1X) and EBS1-IBS1 interactions, colored according to their corresponding 50 exons. Small arrows, nucleophilic attacks in forward (a) (1F, branch formation; 2F, exon ligation) and reverse splicing (b) (1R, reversal of exon ligation; 2R, reverse branching). (a) Forward splicing. D1bI1 associates with bI1D1 in trans resulting in lariat formation and precise exon ligation by two successive transesteri®cation reactions. (b) Transposition of bI1D1 into non-cognate ligated exon sequences. D1X selects ligated exons via the IBS1-EBS1 interaction, resulting in the complete insertion of the DD1 lariat RNA by reverse splicing (Augustin et al., 1990). (c) Exon shuf¯ing by the exchange of trans-acting D1 RNA molecules at the intermediate stage in splicing. The model involves trans-activation of reversal of exon ligation (1R) by D1 RNA molecules, followed by a dissociation-association pathway and exon ligation (2F). Note, reverse branching (R2) after the dissociation/association step generates recombinant precursors (50 E(bI1)-IVS(D1)-30 E(X); 50 E(X)-IVS(D1)-30 E(bI1)) which can be activated in splicing generating recombinant exon products in trans (see the text).
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Transposition and Exon Shuf¯ing
the insertion of the bI1D1 lariat RNA into noncognate exon sequences (Figure 4(b)). In assays containing bI1D1 lariat domain D1 of intron aI5c (D1aI5c) and the corresponding 50 exon (50 EaI5c), the recombinant reverse branching product (50 EaI5c ÿ bI1(D1)) accumulated (data not shown). The use of D1aI5c in the presence of aI5c ligated or pseudo-ligated exons (50 EaI5c ÿ 30 EaI5c) (50 EaI5c ÿ *pCp) promoted the complete and precise insertion of the bI1D1 lariat RNA into the noncognate aI5c target sequences (Figure 3(a)). The accuracy of the recombinant transposition of the truncated bI1D1 intron was veri®ed by RT-PCR followed by dideoxy sequencing (Figure 3(b)). As expected, the recombinant reverse splicing product (50 EaI5c ÿ bI1(D1) ÿ 30 EaI5c), non-reactive by itself, could be re-activated in forward splicing by D1aI5c in trans, giving rise to excised bI1D1 lariat intron and spliced aI5c exons. When D1bI1 RNA was assayed in the presence of the bI1 50 exon (50 EbI1) and the recombinant reverse splicing product (50 EaI5c ÿ bI1(D1)ÿ30 EaI5c), trans-activation of second step catalysis was observed, generating a recombinant ligated exon product (50 EbI1 ÿ 30 EaI5c) (data not shown).
RNA-based exon shuffling by group II intron RNAs in pieces In a ®nal step, exon shuf¯ing by the dynamic interchangeability of non-identical D1 RNA molecules at the intermediate stage of splicing was tested (Figure 4(c)). In experiments involving bI1D1 lariat RNA, D1 RNA molecules of intron aI5c and bI1, together with their corresponding exon sequences, recombinant ligated exon products (50 EbI1 ÿ 30 EaI5c; 50 EaI5c ÿ 30 EbI1) and recombinant complete reverse splicing products 50 EaI5c ÿ bI1(D1) ÿ (50 EbI1 ÿbI1(D1) ÿ 30 EaI5c; 0 3 EbI1) (Figure 4(c)) were observed (data not shown). In conclusion, we have demonstrated an in vitro RNA recombination system catalyzed by group II intron RNA molecules in pieces, resulting in the transposition of fragmented introns and in the creation of novel combinations of exon sequences by splicing reactions. Our data support the hypothesis that modern splicing systems together with their exon-intron structure, may have evolved from the ability of RNA to recombine (Gilbert, 1986) and imply that naturally fragmented group II, group III (Copertino & Hallick, 1993) and spliceosomal introns can create transposons (Sharp, 1985; Roger & Doolittle, 1993). Finally, assembling a functional splicing apparatus by homologous but non-identical intron structures in trans may permit not only intron spreading to non-allelic sites, and thereby the rapid evolution of protein-coding sequences, but might also provide a potential mechanism for RNA-based modulation of gene expression (Robineau et al., 1997; Anderson & Moore, 1997).
Acknowledgements This work was supported by the Austrian Fonds zur FoÈrderung der wissenschaftlichen Forschung (FWF).
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Edited by J. Karn (Received 12 October 1999; received in revised form 7 February 2000; accepted 7 February 2000)