group i & ii intron

8/9/2019 Group I & II intron

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ROLAND SALDANHA, GEORG M OHR MARLENE BELFORT AND A LA N M . LAMBOWIT Z,iD epartm ents of M olecu lar G enetics an d Biochem istry , and the Biotechnology Center, The Ohio State Universi t

Colum bus, O hio 43210, USA ; and tMolar Genetics Program , Wadsworth Center for Laboratories and Research,New York State Department of Health , A lbany, New York 12201-0509, USA

Group I and group II introns

0892 -6 638 /9 3 /0 0 07 -0 015 /$ 0 1. 50 . FASEB 15

ABSTRACT Group I and group II introns ar e tw otypes of RNA enzymes, ribozymes, that catalyze theirown splicing by different mechanism s. In th is review , wesummarize current information about the structures ofgroup I an d group II int rons, their RNA-catalyzed reac-tions, the facilitation of RNA-catalyzed splicing by pro-tein factors, and the ability of the introns to function asm obile elem ents. The RNA-based enzym atic reactionsan d in tron mobility provide a fram ework for consideringthe role of prim ordial catalytic RNAs in evolution and theorigin of introns in higher organism s.- Saldanha, R .,M ohr, G ., Belfort, M ., and Lambowitz, A . M . Group Ian d group I I in tr on s. FASEBJ. 7: 15-24; 1993.Ky Words: catalytic RNA mobile int,vn site-specific endonucleose

re ver se tra nsc rip ta se e vo lu tio n

GROUP I INTRONSGroup I introns are present in rRNA , tRNA, and protein-coding genes. They are particularly abundant in fungal andplant m itochondrial DNAs (m tDNAs),2 but have also beenfound in nuclear rRNA genes of Tetrahymena and other lowereukaryotes, in chloroplast DNAs (ctDNAs), in bacteri-ophage, and recently in several tRNA genes in eubacteria(see refs 1-3). Most group I introns have a highly variabledistribution, even in related organisms, apparently reflectingtheir dispersal as mobile elements. On the other hand, thesame group I intron is present in the tRNA genes ofctDNAs and five different cyanobacterial species, suggestingthat it existed before the evolution of plastids and could be1 to 3.5 billion years old. The finding that some group I in-trons are ancient is consistent w ith the idea that they areremnants of a primordial RNA world. Indeed, the catalyticactivities of group I introns have begun to provide insightinto how prim itive RNAs could have catalyzed their ownreplication an d contributed to the evolution of protein syn-thesis.Splicing mechanism and structureAs first shown for the Tetrahymena large rRNA intron, groupI introns splice by a mechanism involving two transesterifica-tion reactions initiated by nucleophilic attack of guanosine atthe 5 splice site (F ig. 1) (4). The remarkable finding for theTetrahymena in tron was that splicing requires only guanosineand M g. Because bond formation and cleavage are cou-pled, splicing requires no external energy source and is com -pletely reversible. After excision, some group I introns cir-cularize via an additional transesterification, which maycontribute to shifting the equilibrium in favor of splicedproducts (4).

The ability of group I introns to catalyze their own splic-ing is related to their highly conserved secondary and ter-

tiary structures (F ig. 2) (1, 5 , 6). As in protein enzymes, thefolding of the intron results in the formation of an active sitejuxtaposing key residues that are widely separated inprimary sequence. This RNA structure catalyzes splicing bybringing the 5 and 3 splice sites and guanosine into prox-im ity and by activating the phosphodiester bonds at thesplice sites (4). D ifferent group I introns have relatively littlesequence sim ilarity , but all share a series of the short, con-served sequence elements P, Q, R , and S , with parts of P/Qand R /S base pairing in the conserved structure (Fig. 2A).The boundaries of group I introns are marked simply by aU residue at the 3 end of the 5 exon and a G residue at the3 end of the intron (5, 6).The conserved group I intron secondary structure wasdeduced from phylogenetic comparisons, and specific fea-tures have been confirmed by analysis of in vivo and in vitromutations and by structure mapping (1, 5, 6). The structure,shown in Fig. 2A , consists of a series of paired regions,denoted P1-PlO , separated by single-stranded regions(denoted J) or capped by loops (denoted L). P1 and PlO ,which contain the 5 an d 3 splice sites, respectively, areform ed by base pairing between an internal guide sequence(IGS), generally located just downstream of the 5 splice site,and exon sequences flanking the splice sites. G roup I intronshave been classified into four m ajor subgroups, designatedIA to ID (I), based on distinctive structural and sequencefeatures. Group IA introns, for example, contain two extrapairings, P7.l/P7.la or P7.l/P7.2, between P3 and P7,whereas many group lB and IC introns have a large exten-sion of P5, termed P5abc. Individual introns may containadditional sequences, including open reading frames(ORF5), in positions that do not disrupt the conserved corestructure.

The region of the Tetrahymena intron required for en-zymatic activity , the catalytic core, consists of P3, P4, P6,P7, P8, and P9.0 (1). S tudies using Fe(II)-EDTA , a reagentthat cleaves the sugar-phosphate backbone, have shown thatparts of the core are buried in the structure inaccessible tothe solvent, that Mg2 is necessary for folding of the intron,and that individual RNA domains fold in a specific order asM g2 is increased (4, 7). A ll group I introns have fundamen-tally sim ilar core structures, but subgroup-specific structuressuch as P7.1, P7.2, and P5abc appear to participate in addi-

To w hom correspondence should be addressed, at: D epartm entof M olecular Genetics, The Ohio State University, 484 WestTwelfth Ave., Columbus, OH 43210, USA .

2Abbreviations: ctDNA , chioroplast DNA; EBS, exon bindingsite; IBS, intron binding site; IGS, internal guide sequence; LTR,long term inal repeat; m tDNA , mitochondrial DNA; ORF, openreading frame; aaRS, am inoacyl-tRNA synthetase; RT, reversetranscriptase.To accommodate a lim itation of the number of references, wehave cited review s w herever possible.


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5 Ex on Intron 3Exon 5 E xon muon 3ExonU G GUGCG A AY

3 3.j -IIntron 3Exon CExon

5 Exon

I,+5 Exon

, 2w cc cc ?c cl U 3 .O H

I,5 Exon 3ExonU

+G

Group I In tron

16 Vol. 7 January 1993 The FASEBJou rn a l S ALD AN HA E T A L .

5 Exon 3Exonwmnn

+

CLGroup H In tr on

Figure L Splicing mechanism s of group I and group II introns.Conserved nucleotides in introns and flanking exons are indicated.

tional interactions that stabilize the core structure in differ-ent ways (1, 8).

A three-dimensional model of the group I intron catalyticcore has been developed by M ichel and Westhof (1) (Fig. 2B,C). The underlying assumption, first suggested by K im andCech (9), is that adjoining helical segments stack coaxially tocreate two extended helices, P6a-P6-P4-P5 and P8-P3-P7,which form a cleft containing the introns active site (F ig. 2B,C). In the M ichel-W esthof model, the relative orientation ofthe two helices is constrained by a previously proposed triplehelix involving parts ofJ3/4-P4-P6-J6/7 and by potential ter-tiary interactions identified by covariation of nucleotides thatare not accounted for by secondary structure. A number ofthese predicted interactions involve purine-rich loops orbulges engaged in long-range interactions with doublehelices (F ig. 2B ). The active site of the intron, form ed by thecleft between the two helices, contains binding sites for theguanosine cofactor and P1 and PlO containing the 5 and 3splice sites. The model is designed so that the disposition ofthese binding sites accounts for the known splicing mechan-ism , which requires appropriate alignments of guanosineand the 5 an d 3 exons in the first and second steps of splic-ing (4). Deoxynucleotide and phosphorothioate substitutionexperim ents suggest that functionally important M g2 ionsare coordinated at specific positions around the active site(e.g ., P1 and J8/7), where they may function directly in phos-phodiester bond cleavage (1, 10). Basic features of thepredicted three-dim ensional structure have been supportedby mutant analysis in vitro and by the use of specifically posi-tioned photochem ical cross-linking and affinity cleavage rea-gents (1, 11, 12).D efinition of splice sites and binding to the intron coreThe 5 and 3 splice sites of group I introns are substratesthat are acted on by the catalytic core, and they can be recog-

nized and cleaved by the core when added on separate RNAmolecules (4). The 5 splice site is defined by the P1 pairingbetween the lOS and the 5 exon. Neither the sequence norlength of P1 is fixed, but the conserved U at the 3 end of the5 exon always form s a wobble base pair w ith a 0 residue inthe IGS (Fig. 2A). Analysis of in vitro mutants showed thatthe distance of the UG pair from the bottom of the P1 helixis critical for efficient cleavage in the Tetrahymena in tron (4,13) and that Jl/2 and P2 also play a role in the positioningof P1 relative to the core (1, 14, 15).

In the M ichel-W esthof model, P1 is predicted to bind tothe core via tertiary interactions with J8/7 and J4/5 (Fig. 2B )(1). Experiments have shown that the 2 OHs at P1 positions-2 and -3 contribute energetically to binding P1 to the core,and that the 2 OH at -3 interacts w ith a specific residue inJ8/7. The latter interaction is somewhat different from thatproposed by M ichel and Westhof (1), but was readily accom-modated by a local revision of the model (12).

The positioning of the 3 splice site in group I introns de-pends on at least three interactions, whose relative impor-tance differs in different introns (4). These are the PlO pair-ing between the lOS and the 3 exon, binding of theconserved 0 residue at the 3 end of the intron to the 0-binding site in the second step of splicing, and an additionalinteraction, P9.0, which involves base pairing between thetwo nucleotides preceding the term inal 0 of the intron andtwo nucleotides in J7/9 (Fig. 2A ).Guanosine-binding siteG roup I introns have Km values for guanosine that are aslow as 1 eM and readily discrim inate between guanosineand other nucleosides (4). The major component of theguanosine-binding site corresponds to a universally con-served GC pair in P7 (Fig. 2A) (1, 4). Guanosine was initiallyproposed to interact w ith this base pair via formation of abase triple, but the contribution of neighboring nucleotidesand the binding of analogs are also consistent w ith a modelin which guanosine binds axially to the conserved 0 andflanking nucleotides (16). The guanosine-binding site ofgroup I introns can also be occupied by the guanidinogroups of arginine or antibiotics, such as streptomycin,which act as competitive inhibitors of splicing (17). Yarus (18)noted that the three nucleotides that constitute theguanosine-binding site in different introns, AGA /G andCGA /G, correspond to A rg codons, and speculated thatrecognition of am ino acids by this and other functionally im-portant sites in catalytic RNAs could have played a role inthe evolution of the genetic code.Other reactions catalyzed by group I intronsIn addition to splicing, group I ribozymes can catalyze a var-iety of intermolecular reactions, including endonucleolyticcleavage of RNA and DNA , RNA polymerization, nucleo-tide transfer, templated RNA ligation, and am inoacyl-estercleavage (4, 19). A ll these reactions use the same active siteas the splicing reaction, and they can be generalized asshown in Fig. 3A . In the forward direction, equivalent to thefirst step of splicing, the reaction is thi nucleophilic attack ofguanosine on the phosphodiester b nd succeeding XXU(where XX denote exon nucleotides that pair w ith the IGS[XX ] to form P1). In the reverse dire tion, equivalent to thesecond step of splicing, the reaction is nucleophilic attack ofthe 3OH of XXU on the phosphodiester bond 3 of the ter-m inal 0 of the intron. In the intermolecular reactions, thesubstrates are oligonucleotides that are either cognates of P1


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LI L2

P1

IllL8

P8 S

B C

A L5

GROUP I AND GROUP II INTRONS 17

Figure 2. Group I i nt ro n s tru ctu re . A) Conserved secondary structure. A rrows indicate splice sites. Conserved sequences P , Q, R, andS (5, 6) are indicated by heavy lines. Asterisk denotes guanosine-binding site. D ashes indicate base pairs. Dot indicates wobble base pairat the 5 splice site. Nucleotides involved in P9.0 pairing are indicated as NN and NN. Nucleotides involved in PlO pairing are indicatedas MMM and MMM. IGS, internal guide sequence. B) Schem atic illustrating some proposed tertiary interactions in the yeast w intron(1, 8). Interacting nucleotides are connected by dashed lines. Circled G shows free guanosine interacting w ith conserved GC pair of theguanosine-binding site. C) Three-dim ensional structural model of the Tetrahymena large rRNA intron from M ichel and W esthof (1),reproduced by perm ission of the J ou rn al o f M olec ula r B io lo gy .

or base pair w ith the lOS to reconstitute analogs of P1 orPlO.

In the site-specific endonuclease reaction, an RNA sub-strate that resembles the 5 exon and base pairs to the IGSis cleaved by guanosine (Fig. 3B ) or OH- (not shown) in aprocess analogous to the first step of splicing. This reactionhas been modified in various ways that illustrate the versatil-ity of the ribozyme. The rate-lim iting step is the release ofthe cleaved RNA products, so that mutations in the lOS thatweaken RNA binding increase the rate of reaction (20). Thesubstrate specificity of the reaction is determ ined by the se-quence of the IGS, which can be changed to enable the ribo-zyme to cleave other RNA substrates. DNA is cleavedinefficiently , but ribozymes having enhanced DNA cleavage

activity have been obtained by iterative selection (21, 22).Recently, the Tel rahymena ribozyme was shown to catalyze thereverse of an aminoacylation reaction, hydrolysis of anaminoacyl-ester, N-formyl-L-methionine, attached to theCCA end of an oligonucleotide that base pairs to an ap-propriately modified IGS sequence (Fig. 3E ) ( 19 ). A lt ho ug hinefficient, this reaction demonstrates that the ribozyme canact on carbon centers, an d supports the hypothesis that an-cient catalytic RNAs could have functioned as am inoacyl-tRNA synthetases in the early evolution of protein synthesis.

In support of the idea that prim itive RNAs could havebeen the first self-replicating molecules, group I ribozymeshave been shown to act as RNA polymerases. Initially , non-templated addition of nucleotides to oligonucleotides bound


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XXUpN + G-OH XXU-OH + GpN

B) Sequence-Specif icEndonuclease-CUCUpN- + G-OH--- -CUCU-OH + GpN-

C) Nuc leo ti dy l T rans fe raseCCCCC-OH + GpN -. CCCCCpN + G-OH

0 ) T em pla te -D ep en de nt L ig atio n-M + GpN- - -MpN- + G

LrXX U-OHmumXX GTs Reverse

+

Lr aCCCC C-OH1m mmm

rGGAGGI O S

- M OH N -I I I II

+CA AC CA IM etmmrG U U0003$

18 Vol. 7 January 1993 The FASEBJou rn a l SALDANHA ET AL.

Activ e S iteActivity Interactions

X.X.GEG SA) Genera lized Forward

E ) Am in oa cy l E ste ra seCAACCAfMe t + OH - CAACCA-OH + fMetFigure 3. Reactions catalyzed by group I ribozymes. Theguanosine-binding site of the intron core is indicated by an indenta-tion. This site m ay be occupied by a free guanosine in the first stepof splicing or by the conserved guanosine at the 3 end of the intronin the second step of splicing. The figure is based on Cech (4).

to the introns lOS was dem onstrated by intron-catalyzeddisproportionation, nucleotidyl transferase, or ligation reac-tions (4). The latter are analogous to the second step of splic-ing in that short oligonucleotides bound to the lOS can at-tack dinucleotides GpN or oligonucleotides OpN(N ),where the 0 residue of the di- or oligonucleotide is analogousto the conserved 0 at the 3 end of the intron (Fig. 3C). Sub-sequently , template-dependent addition of oligonucleotideswas demonstrated in a reaction analogous to reversal of thefirst step of splicing (Fig. 3D ) (23). Fortuitously, the additionof sperm idine suppressed the need for the UG pair ordinar-ily required at the ligation junction, enabling the reaction totolerate a Watson-Crick pair at this position (23). A recentbreakthrough toward achieving self-replicating RNA was thedemonstration that different segments of a group I ribozymecould assemble to form a multisubunit ribozyme that repli-cated one of its segments by template-directed ligation ofoligonucleotides (24).

GROUP II INTRONSThus far, group II introns have been found only in fungaland plant m itochondria and in chloroplasts. M ost of theseintrons are in protein-coding genes, w ith a few in tRNA and

rRNA genes (25). A s in the case of group I introns, the varia-ble distribution of group II introns suggests that they weredispersed recently as mobile elements, but unlike group I in-trons, group II introns have not been found outside of or-ganelles (2, 25). G roup II introns are of particular interestbecause of their possible evolutionary relationship to nuclearmRNA introns, which was suggested initially by sim ilaritiesin their splicing mechanisms (25-28).Splicing mechanism and structureThe splicing mechanism of group II introns is shown in Fig.1. As in nuclear mRNA introns, splicing is initiated by theformation of an intron lariat in which the 5 end of the intronis linked by a 2-5 phosphodiester bond to a nucleotideresidue, usually an A , near the 3 end of the intron (25). Afew group II introns have been shown to self-splice in vitro,but only at slow rates (11,2 = 10 mm) u nd er n on ph ys io lo gi-ca l conditions (e.g ., 45#{176}C ,00 mM Mg), which suggeststhat additional trans-acting components are ordinarily re-quired for efficient splicing. In contrast to the situation fornuclear mRNA introns, branching is not obligatory for thesplicing of group II introns, and at least under in vitro condi-tions H2O or OH- are able to substitute for the branch point2 OH in the initial nucleophiic attack at the 5 splice site(29).L ike group I in trons, group II introns have little sequencesim ilarity, but share a conserved secondary structure re-quired for catalytic activity. This structure is generallydepicted as six helical domains (I to V I) radiating from acentral w heel (Fig. 4) (25). Two major subclasses of group IIintrons (IIA and IIB) have been distinguished based onstructural features, and like group I introns, individualgroup II introns may contain additional sequence, includingORFs, in positions that do not disrupt the conserved struc-ture. G roup II introns have conserved 5- and 3-boundarysequences (GUGYG and AY), which somewhat resemblethose in nuclear mRNA introns (25).

A lthough there is no three-dim ensional model for group IIintrons, the function of some domains has been established(25, 30, 31). Domain I contains binding sites for the 5 and3 exons (see below ). Domain VI is a helix containing thebranch site, usually a bulged A residue. Domain V , the mosthighly conserved substructure, is required for catalytic ac-tivity and binds to domain I to form the catalytic core. Aderivative of yeast intron aI5-y containing domains I, III,and V splices, albeit poorly , and a derivative containing onlydomains I and V cleaves at the 5 splice site (29). Consistentw ith the view that group II intron domains could haveevolved into trans-acting snRNAs, domain V and subdomainIC I can function in t rans to facilitate in vitro splicing of mu-tant introns lacking these domains (31, 32).

D efinition of splice sitesA s in group I introns, the definition and binding of the 5splice site in group II introns depends on interaction be-tween the intron and sequences in the 5 exon (25, 28, 30).The most critical interaction is base pairing of exon bindingsite I (EBS1), a short sequence in domain I, w ith exon se-quences immediately upstream of the 5 splice site (intronbinding site 1 [IBS1J; Fig. 4). Like the P1 pairing in groupI introns, the EBSI-IBS1 pairing is not conserved in se-quence, but the 5 splice site is always after a specific basepair in the structure. Two additional base-pairing interac-tions, EBS2-IBS2 and c-c, also contribute to positioning the5 splice site.


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vI5_ 3


Figure 4. Group II intron conserved secondary structure. Thedrawing is based on yeast intron coxl-15y (28). I to VI representgroup II intron domains. Dotted lines indicate interactionsdescribed in the text. EBS, exon binding site; IBS, intron bindingsite.

The definition of the 3 splice site in group II introns in-volves contributions of at least four interactions: 1) dockingof domain VI to the core, 2) base pairing of the term inalnucleotide of the intron and a nucleotide between domainsII and III (-y--y), 3) base pairing of the first nucleotide of the3 exon and the nucleotide preceding EBS1 in a guide inter-action, and 4) another, undefined interaction, also involvingthe first nucleotide of the 3 exon (25, 30). Under in vitroconditions some of the 5 and 3 splice site interactions ingroup II introns are dispensable, but they may all be re-quired for efficient splicing in vivo (30).

D egenerate and trans-spliced group II int ronsDegenerate group II introns that are functional despite lack-ing some domains have been found in plant m itochondriaand chloroplasts (25). Euglena ctDNA , for example, containsa large number of relatively short group II introns (277-618nt compared with 1 kb), which sometimes lack recog-nizable cognates of domains I, II, III, or IV , and it also con-tains a potentially related class of short (- 100 nt) A T-richintrons termed group III introns (25, 33). The latter have 5boundary sequences that are degenerate versions of the con-served group II intron sequence, and some have potentialcognates of domains I and V I. The degenerate group II andgroup III introns may be akin to evolutionary interm ediatesbetween group II introns and nuclear pre-mRNA introns(34, 35). They presumably require trans-acting factors forsplicing, and it is possible that m issing RNA domains are en-coded elsewhere and function in trans.

The ability of group II intron domains to reassociatespecifically in vivo is evidenced by trans-spliced group II in-trons, which have been found in the rps-12 gene of higherplant ctDNA , the psaA gene in C hia my do mo na s re in ha rd tiictDNA, and the nadi an d nad5 genes in higher plant m tDNA

(25, 26). These genes consist of w idely separated exonsflanked by 5- or 3-segments of group II introns split ineither domains III or IV . The exons at different loci are tran-scribed into separate precursor RNAs, which are trans-spliced, presumably after the association of the two segmentsof the group II intron. In all these cases, continuous versionsof the same gene or closely related genes are present in bac-

teria or in organelles of other species, suggesting that t rans-spliced genes reflect genom ic rearrangements that occurredw ithin group II introns.

Genetic analysis of trans-splicing of the C hia myd om ona s rein - /z ar dtii p sa A gene showed that a number of nuclear gene

products, presumably proteins, are required. Additionally ,intron 1 of this gene is split into three segments. The 5 exonis flanked by parts of domain I and the 3 exon by parts ofdomains IV to V I, respectively. The m iddle segment of theintron is encoded at a remote locus, t scA, and consists of theremainder of domains I to IV . This tscA segment can appar-ently associate with the other two intron segments to recon-stitute the intron (36). The findings for tscA RNA bolster theidea that group II intron domains m ight have evolved intosnRNAs in the evolution of nuclear pre-mRNA introns.An evolutionary relationship between group II intronsan d nuclear mRNA introns?The sim ilar splicing mechanisms of group II and nuclearmRNA introns immediately suggested a possible evolution-ary relationship, and this belief has been reinforced byfindings of degenerate group II introns in some organismsand the ability of group II intron domains to function intrans. The evolution of group Il-like introns into nuclearmRNA introns is thought to have occurred by the progres-sive loss of internal RNA structures, which were assim ilatedby the host organism s and evolved into snRNAs (26-28). In-deed, it seem s difficult to imagine anything other than anevolutionary rationale for the existence of snRNAs, as splic-ing could be carried out simply by using protein enzymes,as for nuclear tRNA introns.

Cavalier-Sm ith (37) and Palmer and Logsdon (2) notedthat nuclear mRNA introns are lim ited to recently evolvedeukaryotes and thus far appear to be lacking in Giardia an dother prim itive eukaryotes. Because of this restrictedphylogenetic distribution, they argue that nuclear mRNAintrons arose late in eukaryotic evolution and suggest thatthis m ight have involved transfer of organellar group II in-trons to the nucleus. The required process of DNA transferfrom organelles to nudear genomes is well documented.Once in the nucleus, the separation of transcription fromtranslation would lessen the selective pressure for rapid splic-ing and perm it evolution of the slower spliceosomal mechan-ism . Nuclear mRNA introns could then disperse by a varietyof processes, including exon shuffling, insertion into proto-splice sites, e.g. via reverse splicing, and molecular m im icryby certain transposable elements containing splice sites neartheir boundaries (2).

A lthough the structures and functions of group II introndomains and snRNAs ar e not as yet sufficiently defined toperm it strong conclusions about evolutionary relationships,a number of potential correspondences have been noted.T hese include 1) the EBS1-IBS1, guide and c-c interactionsinvolved in defining the 5 and 3 splice sites, which appearanalogous to interactions involving U i and U5 snRNAs(38), 2) domain VI, whose structure resembles that form edby binding of U2 snRNA to nuclear mRNA introns in thatthe branch point nucleotide is bulged from a helix (28), and3) domain V, whose structure and disposition relative to the


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Exon 5

G ro up I:S.c. cob-14

GroupI :T4 td-11

G ro up II:S .c. c oxl-I1

1 6 a a

fl \IS V VV G

Exon I

20 Vol. 7 January 1993 The FASEB Journal SA LD AN HA ET A L.

branchpoint may resemble those resulting from a newlydescribed interaction between U2 and U6 snRNAs (39).Further biochemical analysis should provide insight into theextent to which the group II intron and nuclear mRNA in-tron splicing mechanism s ar e related.

INVOLVEMENT OF PROTEINS IN SPLICINGGROUP I AND GROUP II INTRONSGroup I and group II introns are presumed to have beenself-splicing initially , but many of these introns now requireproteins for efficient splicing in vivo, presumably in order tocompensate for structural defects that have accumulatedduring evolution (1, 6, 40). G enetic analysis of m itochondrialRNA splicing in Neurospora and yeast has shown that some ofthe proteins required for splicing group I and group II in-trons are encoded by host chromosomal genes, whereasothers are encoded by the introns them selves.MaturasesSeveral group I and group II introns in yeast m tDNA encodematurases that function in splicing the intron that encodesthem . These include group I introns cob-12, -13, and -14, andgroup II introns coxl-I1 and -12 (6, 40). The cob-I4 an dcoxl-I1 proteins are shown schematically in Fig. 5. In all thecases indicated above, m aturase function has been demon-strated genetically by show ing that mutations in the intronsORF result in defective splicing, which can be com-plemented by the wild-type protein in vivo. Thus far, thereare no biochem ical assays for m aturases, and althoughrelated ORFs are present in other organisms, the onlyconfirmed maturases remain those defined genetically inyeast .

Characterization of mutants has shown that all the yeastm tDNA maturases primarily function only in splicing the in-tron that encodes them , except for the cob-14 maturase,which splices both cob-14 and another closely related groupI in tr on , coxl-14 (6 , 40). The coxl-14 intron encodes a proteinthat is structurally related to the cob-14 maturase and has alatent maturase activity that can be activated by mutation.However, the coxl-14 protein does not ordinarily function insplicing and instead has a site-specific endonuclease activitythat functions in intron mobility (40) (see below). The intronspecificity of maturases suggests that they function in splic-ing by recognizing unique structural features of the intronsthat encode them .

All the yeast m tDNA group I and group II maturases arein frame w ith the upstream exons, and the active maturasemay be generated by proteolytic cleavage downstream of the5 splice site (6, 40). This mode of synthesis presumablyresults in a feedback regulation in which a decreased rate ofsplicing leads to an increased amount of m aturase and viceversa. The maturases encoded by group I introns are charac-terized by two repeats of a sequence motif variously referredto as P1 and P2, dodecapeptide, or LAGLI-DADG (Fig. 5).This same motif is characteristic of a larger fam ily of pro-teins, which have site-specific endonuclease activities thatm ediate group I intron mobility (41). Group II intronmaturases, on the other hand, are structurally related toreverse transcriptases, which may also function in intron mo-bility (F ig. 5) (42). A s discussed elsewhere, it seems likelythat the mobility functions of group I and group IIm aturases evolved first and the splicing function evolvedsecondarily as a result of ability of the proteins to recognizespecific sequences or structures w ithin the intron or flankingexons (40, 43).

N uclear-en cod ed p rote in sNuclear-encoded proteins required for splicing m itochon-drial introns have been identified by screening ofcytochrom e-deficient m utants (per in yeast or cy t in Neu-rospora) or by isolating nuclear suppressors of splicing mu-tants (6, 40). In Neurospora, the products of three nucleargenes ( cy t- 18 , c yl- 19 , an d cyt-4) have been implicated in splic-ing the m t large rRNA intron and a number of other groupI m itochondrial introns. By contrast, most of the yeast pro-teins function in splicing only a single intron (e.g., CBP2functions in splicing cob-I5 and MSS18 functions in splicingof coxl-I513).

As reviewed elsewhere (40), the proteins required for splic-ing group I introns include aminoacyl-tRNA synthetases(aaRSs) and other proteins that have some additional func-tion in their host cells. The Neurospora cyt-18 gene, for exam -ple, has been shown to encode the m t TyrRS. Likew ise, theyeast m t LeuRS, which is encoded by nuclear gene NAM2,functions in splicing the two closely related group I intronscob-14 an d coxl-14 (see previous text), apparently by acting inconcert w ith one or both of the intron-encoded proteins.

S tudies w ith protein-dependent in vitro splicing systemshave shown that the group I intron splicing reactionspromoted by the Neurospora CYT-18 and the yeast CBP2 pro-teins proceed by the same guanosine-initiated transesterifica-tion mechanism used by self-splicing group I introns and re-main dependent on the conserved group I intron structure,suggesting that they are still essentially RNA catalyzed (40).The CYT-18 protein, which functions in splicing many differ-ent group I introns, was shown to suppress structural muta-tions in different regions of the phage T4 Id or yeast a in-trons. From the spectrum of mutations that could besuppressed, it was inferred that the CYT-18 functions insplicing by stabilizing the catalytically active structure of thegroup I intron core (44). The CYT-18 protein binds toP4-P6, a highly conserved structure of the group I introncatalytic core, and may additionally contact P7-P9 to stabi-lize the two major helices of the core in the correct relativeorientation to form the introns active site (45). The abilityof the CYT-18 protein , the m t TyrRS, to bind specifically tothe group I intron catalytic core suggests that the core mayhave structural features that resemble those in tRNAs, which

In tro n-Encoded P ro te in s3 86 a a

#{149}l5aa,

Exon4 rLAGLIDGDG FIGFFDADG

. Exoni

- 2 45 a x -

Exon2

260aa 75aa 53aaI I::: ..Z RI-Homology DomainX Zn l inger E x on 2Figure 5. R epresentatives of three types of intron-encoded proteins.D emarcated areas are those containing conserved am ino acid se-quences shared by other members of the same protein fam ily. Pro-tein sequences that m atch consensus sequences described in the textare show n below each protein . RT, reverse transcriptase.


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could reflect convergent evolution or an ancestral relation-ship between group I introns and tRNAs (45). The yeastCBP2 protein also requires an intact catalytic core for bind-ing, but in this case, binding is dependent on structural fea-tures that ar e specific to cob-15 (46).

A s discussed elsewhere, synthetases and other pre-existingcellular RNA-binding proteins may have adapted to functionin splicing by fortuitously recognizing sequences or struc-tures in group I introns that resemble their normal cellularRNA targets (40). The finding that m any of the proteins re-quired for splicing group I introns differ between Neurosporaand yeast suggests that this adaptation occurred after thedivergence of fungal species, possibly reflecting the relativelyrecent dispersal of the introns as mobile elem ents (40). Insome cases, protein-dependent splicing may provide a meansof regulating RNA catalyzed reactions. The NeurosporaCYT-4 protein, for example, shows significant sim ilarity toS ac ch ar om yce s ce rev is ia e an d S ch iz os acc ha ro my ces p om be geneproducts involved in the function of cell cycle protein phos-phatases, and may play a role in coordinating mitochondrialRNA splicing and processing reactions w ith cell cycle andother aspects of cellular m etabolism (47).

If group II introns ar e evolutionarily related to nuclearmRNA introns, then one might expect sim ilar proteins to beinvolved in their splicing. Unfortunately, no protein-dependent in vitro splicing system s have been developed forgroup II introns. The number of group II intron splicing fac-tors identified genetically is sm all, and some of these mayaffect splicing indirectly. For example, the MRS3 an d MRS4genes, which were identified by suppression of a group II in-tron splicing defect when expressed from a multi-copy plas-m id, are homologous to ion-carrier proteins and may affectsplicing by altering the ionic environment in m itochondria(48). Two genetically identified proteins that are likely tofunction directly in group II intron splicing are MRS2 an dMSSJJ6 (49, 50). Analyses of mutants suggest that MRS2functions in splicing all group II introns (coxl-II, -12, -ISyan d cob-Il) and is relatively specific for these introns, whereasMSS1I6 is involved in splicing group II introns (coxl-II an dcob-Il) and some group I introns. Both MRS2 and MSSII6appear to have some additional function besides splicing, asgene disruptions result in a respiratory-deficient phenotypein yeast strains whose m tDNA contains no introns. MRS2has no reported sim ilarity to other proteins, but the MSS1I6protein has a DEAD-box motif characteristic of RNA heli-cases, which do indeed function in splicing nuclear mRNAintrons (49, 50).

GROUP I AND GROUP II INTRONS ARE MOBILEGENETIC ELEMENTSBoth group I and group II introns have been shown to bemobile genetic elements that have developed mechanisms forinserting into intronless genes (41, 43, 51, 52). Because groupI an d group II introns carry their own splicing apparatus,they could in principle spread to different cellular compart-ments or organisms, w ith their insertion having minim aleffects on gene expression as long as they can be splicedefficiently. A s discussed previously, the splicing of both groupI and group II introns is dependent on specific pairings be-tween the intron and flanking exons (e.g., P1 andEBS1/IBS1). Because the sequences involved in these pair-ings differ in individual introns, mobile introns must be in-serted in the appropriate sequence context in order to spliceefficiently.

Group I intron mobility via site -sp ecific en don uclea sesA number of group I introns achieve such site-specific inser-tion by using site-specific endonucleases encoded within theintron (41, 43, 51, 52). Remarkably, each intron-encoded en-donuclease cleaves at a different asymmetric target sequence,gen erally sp an nin g - 20 bp, which is located at or near thesite of intron insertion. In crosses between strains containingintron and intron alleles, the endonuclease promotes highfrequency transfer of the intron or hom ing by generatinga double-stranded break in the intron allele. The resultingDNA ends invade the intron allele to prim e replicativetransfer of the intron by a double-stranded break repairprocess. Because form ation of the initial heteroduplex de-pends on homology of flanking exons and there is nucleolyticdegradation of the cleaved recipient, transfer of the intron isaccompanied by coconversion of flanking genetic m arkers, ahallmark of this m echanism .

Sequence comparisons show that group I intron-encodedendonucleases include two major structural classes, onecharacterized by the LAGLI-DADG motif also found ingroup I intron maturases an d the other by some variation ofthe motif G IY-(1O /ll aa)-YIG (41, 51) (F ig. 5). Membersof both the LAGLI-DADG and OIYYIG classes have beenfound outside of introns, and the former include the well-known HO endonuclease, which is involved in yeast matingtype switching (41, 53). In two cases, LAGLI-DADO poly-peptides, which are not associated with conventional introns,have their coding sequences inserted directly in those ofother proteins - the yeast vacuolar W -ATPase and the ar-chaebacterial T he rr no co ccu s lito ra lis DNA polymerase.Remarkably, these inserted LAGLI-DADG polypeptides notonly have site-specific endonuclease activity, which couldpromote mobility of the insertion, but are also associatedwith protein-splicing events that excise them and join theflanking protein sequences (53, 54). The finding of mobile,non-intron-encoded endonucleases strongly supports theproposal that ORFs encoding such endonucleases are in-dependent genetic elem ents that inserted into previously ex-isting group I introns (51, 55). Further, the association ofsome LAGLI-DADO proteins w ith protein splicing eventsraises the possibility that the related group I intron proteins,which are translated in-frame w ith upstream exons, catalyzetheir own proteolytic processing (53).Reverse sp licing and other possib le m echanism s forin tro n m ob ilityBecause the double-stranded break repair process dependson exon homology, it does not favor transposition of intronsto other locations, and there are indications that other typesof processes contribute to the mobility of group II introns(see below). A second possible mechanism for intron inser-tion, reverse splicing, has been demonstrated in vitro forboth group I an d group II introns (56). For both types of in-trons, reverse splicing requires only a short RNA target se-quence, which corresponds to the 5 exon and suffices to formthe P1 pairing in the case of group I introns and theEBS1/IBS1 pairing in the case of group II introns. Therecombined RNA could in principle reintegrate into the ge-nome after reverse transcription (57, 58). A lthough reversesplicing is inefficient in vitro, proteins that function in splic-ing of the intron should also accelerate reverse splicing andmay thus contribute to intron mobility in vivo (59).Group I and group II introns both have some activity withDNA substrates (21, 22, 60) and, in principle, the excised in-


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22 Vol. 7 January 1993 The FASEB Journa l SALDANHA FT AL.

tron RNAs could integrate directly by reverse splicing intosingle-stranded DNA or into RNA prim ers at replicationforks (57, 58). In addition, autonomous DNA elements havebeen described, which may either be circular cDNA copiesof excised group I or group II introns or evolved from them(43). cDNA copies of excised introns could conceivably con-tribute to mobility by integrating directly into genomicDNA , perhaps facilitated by an integration activity as forretroviral proviruses. Possible evolutionary relationships be-tween autonomous forms of introns and infectious elem ents,such as RNA viruses or retroviruses, have been discussedelsew here (43).Group II introns are m obile and encode reversetranscriptase-like proteinsThe first indication that group II introns m ight be mobileelem ents was the finding that a number of these introns con-tain ORFs w ith significant sim ilarity to reverse transcrip-tases, particularly those of the non-LTR class of retroid ele-ments, i.e., those lacking long term inal repeats (LTR5) (42,61, 62). The yeast coxl-Il intron, which encodes such anORF, is shown in Fig. 5. A lthough reverse transcriptase ac-tivity has not been demonstrated biochemically for anygroup II intron-protein, most ORFs have good matches forthe seven conserved sequence blocks found in all functionalreverse transcriptases. The group II ORFs generally containan additional upstream conserved sequence (Z) characteris-tic of non-LTR reverse transcriptases; some also have a Zn2finger-like motif at their COOH-term inus, and a few , e.g .,the yeast coxl-I1 and -12 ORFs, contain weak matches toretroviral protease domains. Another conserved domain,which we denoted X , is always found between the reversetranscriptase and Zn2 finger domains, but is also found ingroup II proteins that lack the latter domains. As discussedpreviously, the reverse transcriptase-like proteins encoded byyeast coxl-I1 and -12 function as maturases in splicing the in-trons that encode them .

Three group II introns that enco de rev erse transcrip tase-like proteins, S. c e re v is iae coxl -I I and -12 and K lu yv er om yce s In s-t is c o xl -I 1, a cognate of S . c e re v is iae coxl -12 , have been shownto transfer at frequencies approaching 100% to intronless al-leles during crosses (63, 64). T ransfer of the yeast coxl-I1 an d-12 introns is accompanied by coconversion of distal groupI introns but is not dependent on group I introns, as it occursin strains from which all group I introns have been deleted.Significantly, the transfer of group II introns was shown tobe inhibited in two splicing-defective mutants; one, a cis mu-tation that affects intron structure, and the other a trans mu-tation that affects maturase activity of the intron ORF (63).

The requirem ent for splicing is clearly different from thesituation for group I intron mobility and strongly suggeststhat excision of the intron is required. In general, such a re-quirem ent could reflect either that the excised intron is anintermediate in mobility (e.g., via reverse splicing or directintegration) or that the excised intron acts as a mRNA orcofactor for an activity that functions in mobility (e.g.,reverse transcriptase or site-specific endonuclease). Thecoconversion of flanking markers, on the other hand, couldresult either from recombination with reverse transcripts ofall or part of the donor pre-mRNA or from double-strandedbreak repair promoted by an endonuclease. The R2 elementof Bombyx mon encodes a reverse transcriptase-like proteinthat is related to the group II intron proteins and has a site-specific endonuclease activity , which cleaves at the insertionsite of the element (65).

Instances where group II introns have transposed toanother location have been found in EuIena ctDNA . These

are composite introns, term ed twintrons, in which a groupII intron has inserted into another group II or group III in-tron (34, 35). In two cases described in detail, the insertionappears to have occurred into a region essential for splicing,so that the internal intron must be spliced first to reconstitutethe external intron. For both tw introns, it was possible toidentify potential EBS1-IBS1 and EBS2-IBS2 interactionsbetween the internal intron an d the external intron, leadingto the suggestion that insertion occurring by reverse splicingfollowed by reverse transcription and reintegration into thegenome.The reverse transcriptase-like proteins encoded by groupII introns may also contribute to various site-specific dele-tions resulting from recombination of genomic DNA withcDNA copies of spliced or m isspliced RNAs (52). A cogentexample is the phenomenon of precise intron deletion inyeast m tDNA , which presumably results from recombina-tion with a cDNA copy of spliced mRNA. The phenomenonwas reported first by Slonimski and co-workers (reviewed inref 52), who found that a number of splicing defective mu-tants revert by precise deletion of the impaired intron frommtDNA . Both group I and group II introns could be deletedin this way, and the deletion of the impaired intron was fre-quently accompanied by deletions of neighboring upstreamor downstream introns, which were not under selection. Inaddition, it w as found that the initial intron mutation had tobe somewhat leaky, presumably in order to generate thespliced mRNA interm ediate. By using yeast strains w ithdifferent combinations of m tDNA introns, it was shown thatprecise intron deletion was dependent on the presence ofcoxl-I1 and/or coxl-12 in the m tDNA , consistent w ith a re-quirement for a reverse transcriptase activity encoded by oneof these introns. The ability to precisely delete introns fromyeast m tDNA is presumably counterbalanced by efficientmechanism s for intron insertion, but this process could havecontributed significantly to intron loss in the course of evo-lut ion.

PROSPECTS FOR FUTURE RESEARCHResearch on group I and group II introns should continueto provide fundamental insights into RNA chem istry and theevolution of proteins to assist and regulate RNA-catalyzedsplicing reactions, as well as into the amazing adaptations ofthe introns to function as mobile genetic elements. Theresearch also seems poised to address a number of long-standing evolutionary questions about the feasibility of self-replicating RNAs, the evolution or protein synthesis, and theorigin of introns. Yet it is worth keeping in m ind that con-temporary group I and group II introns are them selveshighly evolved. They have a demonstrated propensity forrapid evolutionary change and may be only distantly or notat all related to molecules in the primordial world. S tudiesof group I and group II introns, as well as other catalyticRNAs, w ill undoubtedly provide insights into evolutionarymechanisms, leading to logically compelling scenarios.However, if what we have seen so far is any indication, wew ill probably still be surprised by the real answers.

W e thank our laboratory colleagues and Drs. Donald Copertino(U . A rizona), D aniel Herschlag (Stanford), and Philip Perlm an (U .T exas) fo r their comments on the manuscript. W ork in the authorslaboratories was supported by grants GM39422 and GM44844 toM. B . and grants GM37949 and GM 37951 to A . M . L.


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group i & ii intron

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