©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mechanisms of Intron Mobility (*)

Marlene Belfort (1) Philip S. Perlman (2)

From the  (1)Molecular Genetics Program, Wadsworth Center, and School of Public Health, State University of New York, New York State Department of Health, Albany, New York 12201-2002 and (2)Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9038

INTRODUCTION
Intron Homing
Intron Transposition
Proteins That Promote Intron Mobility
Perspectives
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


INTRODUCTION

Group I and group II introns, which splice via RNA-catalyzed pathways, can invade DNA sequences by virtue of proteins expressed from open reading frames (ORFs) (^1)contained within them. These intron products are endonucleases in the case of group I introns and reverse transcriptases (RTs) with associated endonuclease activity in the case of the group II introns. Two kinds of mobility reactions will be considered for each intron type: homing into cognate intronless alleles and transposition to non-allelic sites.


Intron Homing

Intron homing is a process whereby the intron moves from an intron-containing allele to an intronless allele in a homologydependent gene conversion event. Coconversion of flanking exon sequences often accompanies intron homing (reviewed in (1) and (2) ).

Group I Intron Homing

Homing is initiated by the intron-encoded endonuclease, which catalyzes a double strand break (DSB) in the intron-minus (recipient) allele (Fig. 1A). Invasion of the homologous intron donor duplex by a cleaved 3`-end primes repair synthesis that results in copying of the intron into the recipient DNA. This process is thought to proceed via the DSB repair (DSBR) pathway, wherein a D-loop formed as the result of repair synthesis by the invading strand serves as a template for repair synthesis of the opposite strand (Fig. 1B). Two resulting Holliday junctions must be resolved to yield two intron-containing alleles, which would either have an exchange of flanking markers (crossovers), or not (non-crossovers), depending upon the relative direction of resolution of the two junctions. Because of nucleolytic degradation of the cleaved recipient and branch migration, co-conversion of exon sequences flanking the intron is common.


Figure 1: DNA-mediated group I intron homing. A, overview of mobility pathway. Cleavage of the recipient by the endonuclease (ENDO) results in intron inheritance via gene conversion. B, the DSBR pathway. Subsequent to endonuclease cleavage the recipient allele undergoes exonucleolytic degradation and homologous sequence alignment with an intron-containing donor (a). A 3`-end of the recipient invades the donor, which serves as a template for repair synthesis (b). During DSBR (c-g), DNA synthesis through the intron results in expansion of a D-loop (c), which then serves as a substrate for repair synthesis of the non-invading strand (d). Holliday junctions formed during this process (e) are resolved to produce either non-crossover (f) or crossover (g) products. Activities involved in the T4 intron homing pathway are indicated, with Polymerase plus signifying the requirements for polymerase accessory functions (3) ^2; presumably similar activities participate in homing events in other systems. C, the SDSA (BM) model (6, 7) .^2 As DNA synthesis proceeds, the replication bubble migrates with the replicative end (c`-e`). The newly synthesized strand is released from the donor (f`) and serves as template for repair of the noninvading strand (f`-g`) to generate non-crossover products only (g`).^2Half-arrows indicate 3`-ends of DNA strands.



Experiments in the T4 phage system have implicated exonucleolytic, synaptic, and DNA-synthetic functions of the phage in the homing process (3) (^2)(Fig. 1B). A role for DNA ligase and resolvase was also established. However, a reduced level of homing can occur in the absence of known resolvases, implying the existence of alternative resolution enzymes or additional pathways for intron homing. The latter possibility is favored by the underrepresentation of crossover events among the homing products.^2 One such alternative pathway is the synthesis-dependent strand annealing (SDSA) model (reviewed in (5) ), which has been invoked as an alternative to the DSBR pathway to explain gene conversion from ectopic sites in P-element-induced gap repair in Drosophila (Fig. 1C)(6) . This pathway is similar to the bubble migration (BM) pathway for T4 phage replication(7) . The initial steps of the SDSA (and BM) pathway involving cleavage and strand invasion are the same as those of DSBR. Unlike DSBR, however, Holliday junctions are not formed, obviating the need for resolvase function and resulting only in non-crossover products(5) .^2 Alternative homing pathways in both pro- and eukaryotic systems require further investigation.

Group II Intron Homing

Recent studies on the mobility of the group II introns aI1 and aI2 of the COX1 gene of yeast mtDNA establish a number of key features of group II intron homing(8, 9, 10) . Like homing of group I introns, the process is highly efficient and site-specific. However, there are three important differences from group I intron mobility. First, group II homing depends on an intron-encoded protein with RT activity; second, the process requires a splicing proficient intron, whose RNA processing function in turn relies on maturase activity of the intron-encoded protein; and third, the extent and symmetry of co-conversion are more limited for group II than for group I introns. In group I intron homing co-conversion appears, on average over multiple events, to be roughly symmetric, whereas co-conversion associated with group II intron homing appears asymmetric, extending further upstream than downstream of the intron insertion site.

The group II homing pathway (Fig. 2A), which has been elucidated recently for the aI2 intron, is remarkable in that it depends on three activities of the aI2-encoded protein: endonuclease, maturase, and RT(9, 10) . The endonuclease activity, which also requires the intron RNA, makes a staggered cut in the recipient DNA, cleaving the antisense strand at a specific site in the downstream exon and the sense strand at the junction between the two exons. The 3`-OH of the cleaved antisense strand is used as a primer for first-strand cDNA synthesis by RT, using the pre-mRNA precursor as template(10) . This sequence of events is rather analogous to the priming of cDNA synthesis by the site-specific non-long terminal repeat retroelement R2Bm(11) , consistent with the group II introns representing a type of site-specific retrotransposon.


Figure 2: RNA-mediated mobility events. Wavy lines, RNA; straight lines, DNA; thin lines, exons; thick lines, intron. A, group II intron homing. The pathway, worked out for the yeast mitochondrial aI2 intron (8, 9, 10) involves the maturase (M), endonuclease (E), and RT activities of the aI2-encoded protein. The activity required for each step is indicated as white lettering on a black background. Endonuclease activity requires intron RNA, which is excised as a lariat but is depicted as a linear molecule. Cleavage sites on the DNA recipient are shown as dots, with the exon junction represented as a line. RT-directed cDNA synthesis is primed from the downstream 3`-OH of the cleaved recipient DNA, with the pre-mRNA as template. Details on completion of the integration step remain to be determined. B, group I and group II intron transposition. Reverse splicing into a foreign RNA, shown to occur for both intron types, yields an RNA that, when reverse-transcribed and recombined with the genome, results in transposition of the intron to an ectopic site. The latter step has some experimental support for group II introns only (22, 23, 24) . C, group I and group II intron loss. A cDNA copy of the spliced mRNA is proposed to recombine with the genome to render it intron-minus.



This model is in accord with several unexplained observations. First, inhibition of splicing blocks intron homing(9, 12) . The finding that specific defects of the cis-acting intron RNA substructure abolish homing (9) can be reconciled with the role of intron RNA in endonuclease function as either cofactor or catalyst(10, 13) . Second, the model can explain the relatively inefficient co-conversion downstream of the cleavage site as resulting from the limited exonucleolytic degradation that can occur before priming of cDNA synthesis ensues from the downstream 3`-OH of the cleaved recipient. One issue that remains unclear is the manner in which the complement to the first strand cDNA is made. Another is the possibility that group II homing may occur by more than one mechanism. For example, an RT-independent group I-type pathway was suggested by the finding that a mutant aI2 protein that lacks RT activity but retains endonuclease function supports 40% homing activity(9, 10) . However, further study is needed to determine whether that pathway occurs in wild-type crosses.


Intron Transposition

The sporadic distribution of the conserved group I and group II introns suggests that each arose from ancestral introns that transposed to heterologous sites. One possibility is that the introns transpose by a degenerate homing event with relaxed homology requirements, resulting in illegitimate recombination. Although no true transposition events by this pathway have yet been documented, evidence for transposition via reverse splicing into foreign RNAs is accumulating for group I and particularly for group II introns. The pathway involves reverse splicing of an excised intron into non-allelic RNA, followed by transfer into the genome at the heterologous site, most likely via a cDNA copy of the recombinant RNA (Fig. 2B).

Group I Intron Transposition

Although RNA-mediated group I intron transposition has not yet been observed in its entirety, partial reactions have been noted both in vitro and in vivo. Reverse splicing of the Tetrahymena thermophila group I intron has been demonstrated into both its natural target site and into foreign RNAs that contain short sequences homologous to the normal ligation junction(14, 15) . The target sequence can be as small as 4 nucleotides so long as it can pair with an internal guide sequence in the intron to direct the integration reaction. Proteins that normally promote splicing of specific introns can also facilitate reverse splicing in vitro, as was demonstrated for the Neurospora LSU intron and its splicing effector CYT-18 protein(16) .

The first demonstration of a partial reverse splicing reaction in vivo was with the Cr.LSU intron, a chloroplast group I intron from Chlamydomonas reinhardtii(17) . This intron has been shown to undergo the first step of reverse splicing into the cytoplasmic 5.8 S rRNA of its host in vivo and in vitro. Minor changes in the 5.8 S sequence would allow complete integration of the intron. Nevertheless, how cDNA synthesis would ensue for introns that do not encode their own RT activity remains unclear, although a role for trans-acting cellular RTs can be readily envisaged.

The preferential occurrence of group I introns in rRNA and tRNA genes may reflect the abundance of these RNAs, which could provide copious targets for reverse splicing in vivo(17) . Although integration into these targets may be a common phenomenon(14) , maintenance of stable RNA function and trapping of the intron insertion event through capture by the genome (e.g. via a cDNA intermediate) are likely to be rare. Such infrequent events would be most likely to occur with abundant RNAs.

Group II Intron Transposition

The discovery of twintrons in the chloroplast DNA of Euglena gracilis was the first indication that group II introns can transpose to other genomic sites (18) . In the simplest twintrons, the internal intron disrupts splicing of the external intron so that the splicing pathway is ordered. The formation of twintrons has been explained by an intron reverse splicing into another intron by the pathway proposed in Fig. 2B. This pathway is further supported by the demonstration of the reversal of the self-splicing reaction in vitro and by integration of a group II intron into foreign RNA and DNA(19, 20, 21) .

Several recent studies aimed at understanding site-specific deletions of fungal mtDNAs led to the discovery that group II introns that encode RT-like proteins can transpose to ectopic sites in mtDNAs. For example, intron 1 of the COX1 gene of yeast mtDNA, a group II intron that can also carry out site-specific homing(8) , has been inferred to reverse splice into several sites in a group I intron of the COX1 gene, aI5beta, to form twintrons with an internal group II intron and an external group I intron(22) . A similar intron in Podospora mtDNA was found to transpose to a site in mtDNA near a tRNA gene(23) , whereas the group II intron of Schizosaccharomyces pombe mtDNA was found to transpose to multiple sites(24) . In each case, an RNA-mediated event involving RT was inferred (Fig. 2B). After ectopic insertion, the genome would contain two copies of the intron, so that homologous recombination would result in the deletion of one intron copy plus sequences between them. This type of deletion event could explain the circular alphaSen DNA, which contains a group II intron and is involved in the senescence phenomenon in Podospora(23) (reviewed in (2) ).

Although no cDNA intermediate for these transposition events has yet been demonstrated, other studies in yeast mitochondria support the inference that such cDNAs can be made. For example, the RT activity overproduced in a mutant of the aI2 intron deleted for a catalytic domain (domain 5) is much less specific for aI2 and, instead, uses other mitochondrial RNAs as a template(9) . Furthermore, reverse transcription and cDNA synthesis are strongly implicated in the intron loss phenomenon (Fig. 2C), which has been reported for both group I and group II introns of the COB and COX1 genes of yeast mtDNA. These events are detected among revertants of some intron mutants and always involve loss of the mutant intron. Frequently adjacent (and unmutated) introns are lost simultaneously (for example, see (25) ). Since the exons separating the lost introns are retained, it was proposed that spliced mRNAs are reverse transcribed and recombined into mtDNA, resulting in loss of the introns. The RT-encoding group II introns are the likely source of the RT activity since strains lacking both aI1 and aI2 do not undergo intron loss(25) .


Proteins That Promote Intron Mobility

The proteins encoded by the group I introns comprise four families of endonucleases, whereas the group II intron proteins form one fairly homogenous class of RT-like proteins. Interestingly, a relationship has been established between one of the group I intron endonuclease families and a Zn finger-like motif in some group II intron-encoded proteins(26, 27) . The recently discovered group II intron endonuclease activity is likely to be associated with this motif(10) . Additionally, several of the proteins encoded by both group I and group II introns have maturase function to promote splicing of their cognate intron (reviewed in Refs. 2 and 28). The reading frames of the group I and group II introns may occur in freestanding form within the intron or in-frame with the upstream exon (reviewed in (28) ). In the latter case the precursor protein appears to be processed proteolytically to release the active intron-encoded protein(2, 28, 29, 30) .

The LAGLIDADG Proteins

The LAGLIDADG consensus sequence, which occurs as repeats (P1 and P2) flanking a region of 110 ± 40 amino acids(31) , is present in the majority of homing endonucleases (2, 28) (Fig. 3A). The motif is phylogenetically widespread, occurring in all three kingdoms, and is present in endonucleases of group I introns, archaeal introns and inteins, as well as in all four known group I intron-encoded maturases. The number of highly conserved residues (>66.7%) among the 16 endonucleases is limited (see consensus in Fig. 3A). However, DNA cleavage activity has been related to the conserved aspartate residues of both P1 and P2(32, 33) . This observation is interesting, since some endonucleases have only P1 (Fig. 3A). Missense mutants of the two conserved glycines of P2 block maturase function, implicating that motif in maturase function as well(34) . The protein encoded by aI4alpha of yeast mtDNA has both endonuclease (I-SceII) and latent maturase function(35) . Mutations of the conserved glycines of P1 of the aI4alpha protein specifically blocked endonuclease activity, but equivalent P2 mutations inhibited maturase function(34) . Together these findings suggest that P1 is more important for endonuclease activity while P2 has a role in maturase function.


Figure 3: Conserved endonuclease motifs in intron-encoded and related proteins. Numbers in brackets indicate position in protein, counting from the beginning of the intron reading frame; the last bracket contains the number of amino acids to the end of the protein, when known. Dots correspond to gaps in the alignment. In each case, the amino acids for which the motif is named are indicated in black boxes above the compilations. Highly conserved amino acids are indicated below the compilations (Consensus). Uppercase, invariant; lowercase, conserved in >66.7% of cases. Letters in parentheses indicate acidic residues (D or E) or hydrophobic residues (I, L, or V) in upper- or lowercase depending on whether they collectively represent 100% or >66.7% of residues at a particular position, respectively. A, the LAGLIDADG motif. The consensus was derived from the depicted sequences, which represent proteins with demonstrated endonuclease activity. I, intron-encoded; PI, intein; HO Endo and Endo.SceI, non-intron endonucleases of Saccharomyces cerevisiae. The compilation is modified from (28) , which identifies all endonucleases except I-PorI (51) and PI-PspI(52) . B, the GIY-YIG motif. Consensus-17 was derived by the Pileup program of the GCG sequence analysis package and visual examination of 17 aligned sequences ((53-55); Mary E. Bryk, personal communication). Consensus-3 was derived from the three GIY-YIG proteins with demonstrated endonuclease activity. Dashes correspond to a non-conserved 19-31-amino acid block with each sequence containing an arginine residue. C, the H-N-H motif. The consensus motif was derived from 40 proteins as described(26, 27) . Only the group II bacterial ORFs and those proteins with demonstrated endonuclease activity are listed: colicins and endonuclease McrA from Escherichia coli (Eco), bacteriophage group I intron endonucleases I-HmuI and I-TevIII(28) , bacterial group II intron ORFs from Calothrix PCC7601 (Cpc) and Azotobacter vinelandii (Avi)(4) , chloroplast group II intron ORF from the blue-green alga Scenedesmus obliquus (Sob), and the S. cerevisiae mitochondrial group II protein from the aI2 intron (Sce). The Cs of a CXXC component of a putative Zn finger located upstream of the H-N-H motif and (H/C)XC immediately preceding HX(3)H of the H-N-H motif are boxed. D, the His-Cys box. Alignment is from (38) .



The GIY-YIG Proteins

The two components of the GIY-YIG motif are separated by 10-11 amino acids (36, 37) and occur upstream of a conserved sequence block of 15 amino acids (Fig. 3B). This is the second most common consensus sequence found in intron-encoded proteins (reviewed in (2) and (28) ). It occurs in both intergenic and group I intron-encoded endonucleases of phage T4 and is prevalent in group I intron proteins of fungal mitochondria. Unlike the LAGLIDADG motif, no functional studies have yet been reported with the GIY-YIG motif and neither has the motif yet been associated with either inteins or maturases.

The H-N-H Proteins and the ZnFinger-like Motif

The H-N-H proteins contain a consensus sequence spanning 30-33 amino acids with two highly conserved pairs of histidine residues contained within a less conserved domain of 50-80 amino acids (26, 27) (Fig. 3C). The H-N-H motif occurs in group I intron endonucleases of both Bacillus subtilis phage and coliphage and in group I intron proteins of algal chloroplasts. In addition to its presence in a number of other bacterial endonucleases, McrA, and colicins, the H-N-H motif appears in a Zn finger-like domain of group II intron ORFs. The occurrence of this endonuclease motif in group I and group II intron proteins likely reflects the coincidence of endonuclease function in both intron mobility pathways. Consistent with a role for this domain as an endonuclease involved in the target-site selection, a missense mutant in the Zn domain of the aI2 intron protein inhibits endonuclease activity and mobility(9, 10) .

The His-Cys Box Proteins

The His-Cys box is a conserved region identified in three nuclear homing endonucleases of myxomycetes and an amoeba flagellate(38) . Among the three known examples, the motif contains three cysteine and two histidine residues within a generally conserved region of about 30 amino acids (Fig. 3D). The His-Cys box, like the above mentioned Zn finger-like motif, is likely to be a metal coordination site within the DNA-binding domain of these endonucleases.

The RT-like Proteins

Group II intron ORFs contain five discrete domains including the aforementioned RT domain(39, 40) , maturase domain (domain X), and Zn domain (containing the H-N-H motif and probably encoding the endonuclease activity)(9, 41, 42, 43) . These domains are each characterized by conserved motifs that have been previously reviewed as follows: RT(44) , domain X(42) , and H-N-H (26, 27) (Fig. 3C). For the aI2 intron of yeast mitochondria, the three domains have been functionally distinguished by mutation(9, 43) . However, not all group II intron ORFs have all of these domains(42) . Whereas domain X is present in nearly all such ORFs, some have a highly divergent or partially deleted RT domain, and a number lack the Zn domain.

The Zn domains of group II intron ORFs were previously proposed to resemble the Zn finger of retroviral integrases (45) ; that similarity, however, is probably superseded by the definition of the H-N-H family of proteins. Furthermore, the Zn finger is the most amino-terminal of three integrase domains, whereas in group II introns the Zn domain is the carboxyl-terminal domain, and there are no cognates in group II ORFs for the other integrase domains. The relationship between the Zn domain with its two conserved CXXC or HXXC motifs and the H-N-H motif in the group II introns is shown in Fig. 3C.


Perspectives

What might be the unifying feature behind two different types of introns being capable of movement by at least two disparate pathways driven by distinct intron-encoded enzymatic activities? The underpinnings of this situation likely reside in two basic premises. First, the intron-encoded functions originated from non-intronic sources. Presumably they were capable of mobilizing their own coding sequences, and that ability eventually led to their colonizing introns. Second, self-splicing introns might provide convenient havens for invasive genetic elements, as insertion into non-essential regions of introns would not be expected to have catastrophic consequences. Thereby, both intron types, rather than fall victim to invasion, acquired the potential for mobility. The intron-ORF invasion hypothesis (46, 47) has gained credence with the discovery of LAGLIDADG endonucleases in the structurally and mechanistically distinct archaeal introns(48) . Further support for the hypothesis derives from lengthy endonuclease recognition sequences flanking corresponding intron ORFs (49, 50) . Nevertheless, the reasons specific endonuclease families are confined to group I introns whereas RT activity is associated strictly with group II introns remain obscure.


FOOTNOTES

*
This minireview will be reprinted in the 1995 Minireview Compendium, which will be available in December, 1995. Work in our laboratories is supported by National Institutes of Health Grants GM39422 and GM44844 (to M. B.) and GM31480 and GM35510 (to P. S. P.).

(^1)
The abbreviations used are: ORF, open reading frame; RT, reverse transcriptase; DSB, double strand break; DSBR, DSB repair; SDSA, synthesis-dependent strand annealing; BM, bubble migration.

(^2)
Mueller, J. E., Clyman, J., Huang, Y., Parker, M. M., and Belfort, M.(1996) Genes & Dev., in press.


ACKNOWLEDGEMENTS

We thank Maryellen Carl and Maureen Belisle for preparing the manuscript and illustrations, Mary Bryk for refining the compilations in Fig. 3, and members of our laboratories for their comments on the manuscript.


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