Transcript
Page 1: Origin of genes encoding multi-enzymatic proteins in eukaryotes

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M ulti-enzymatic proteins are defined here as proteins that have two or more enzymatic activities, representing independent domains, physically linked on a single polypeptide. There are numerous examples of multi- enzymatic proteins in the amino acid, folate, purine, pyrimidine and fatty acid biosynthetic pathways of eukaryotes. The possible forces behind the evolution of eukaryotic multi-enzymatic proteins are described else- where’. in most cases, the orthologous enzymes in prokaryotes are found on separate, mono-enzymatic proteins. Looking at the structures of mammalian genes encoding multi-enzymatic proteins, one often finds that the set of exons encoding one enzymatic domain are separated by an intron from the set of exons encoding another enzymatic domain. We believe that these special introns represent evolutionary footprints of the recom- bination events that brought together genes encoding mono-enzymatic pro:eins to create the genes encoding muiti-enzymatic proteins. Because the presence and placement of these introns are often conserved across species, such introns probably Ate back to a common ancestor (e.g. Ref. 2).

In the initial model1 for these recombination events, we extended the Gilbert mode13.4 of intron-intron- mediated rearrangement to the evolution of multi- enzymatic proteins. The idea proposed was that the final intron of one gene would rearrange with the first intron of another gene; the genes need not be on the Same chromosome. Four major consequences result from this rearrangement: II) the promoter for the _wcond gene would be lost; (2) if the frame was maintained across the intron containing the newly introduced sequences, a multi-enzymatic protein would be created; (3) the entire process would have taken place in one step; and (4) the intron where the rearrangement took place might remain as evidence of the origin of the fusion gene. This model is supported by the creation of new genes in certain cancers, such as the BCR-ABL gene found in chronic myelogenous leukemia?. However, because of recent discoveries made with the GARTgene and with certain genes showing alternative processing, this model must be revised. As discussed here, the GARTgene possesses an intronic cleavage-polyadenylation signal whose exist- ence might reflect the recombinational events giving rise to a gene encoding a multi-enzymatic protein.

The multi-enzymatic GART protein Glycinamide ribonucleotide synthetase (CARS), gly-

cinamide ribonucleotide formyltransferase (GART) and aminoimidazole ribonucieotide synthetase (AIRS) are the second, third and ffih enzymatic steps, respectively, in the de 12000 synthesis of purines. Each of these three en- zymes are encoded by separate cistrons in Edm-ichia colib-9 and Bacillus subfilisl~. In the yeasts, Scbizosac- cbarOrn~~cespotnbe~l and Succharornyces cerwisiuelz, a gene encoding a bi-enzymatic GARS-AIRS protein” is found (Fig. 1). In chicken’s, human13e14 and mouselj a single gene locus encodes the hi-enzymatic GART pro- tein with the arrangement GARS-AIRS-CART. Thus, the gene arrangements for CARS, AIRS and CART suggest that two independent gene fusion events have taken place over time to create the GARTgene seen in mouse. Drosophila tnclatzogasm 10 and LX psettdmhctrra~7 also

Origin of genes encoding multi-enzymatic proteins in eukaryotes JEFFHEY N. DAVlDSON @[email protected]&)

MARTHA L PETERWN ([email protected])

genese7tcudfng~protehrsrettrbe~& to the genes encoding mn&i~ptv#i* bow the gemfksed mnains an open quest&m However, the recent dliicovery of a ckavage*&@ation sig8al witlrin an intmn of tbe GMTgene pm&h clues to tbfs ptmcess and nugbt also baue more general imp#cntfons for the &gin of gems tbat contain alternative RNA processing reactionsattbeirSor3’ends.

have a multi-enzymatic GART protein similar to mouse, but an intragenic duplication of the AIRS region has led to a protein with two tandem AIRS domains. Two unex- pected observations were made with the GARTgene of D. me/anogaster(Ref. 16) and mouset5? (1) two tran- scripts of different sizes are produced from the CART gene, a finding also made with chicken and human CART (Ref. 16); and (21 there is a cleavage-polyadenylation signal within the intron that follows the last exon en- coding the GARS domain (Fig. 2). In l3. melanogaste# and mouseIS, the smaller of the two GART transcripts is cleaved and polyadenylated at the intronic po&CA) signal, and encodes a functional mono-enzymatic GARS protein, while the larger transcript (in which the

Mouse GARS AIRS GART 1

Drosophila GARS AIRS AtAS GART

Yeast 1 GARS AIRS 1 [GARTj Bacteria -Ij -1

F~GUXE 1. Gene and domain organization for GARS, AIRSand GARTin ~~rious species. In !Mmicbia coli, AIRS and GART are encoded by seplnte cistrons (h&fand Puny respectively) of one operon”,-. and GARS (AND) is encoded by a gne at another Iocu.W. In Bacifhrssrrbtilis’~, 12 enzymes of the pwine biosynthetic pathway are amxird by cistrms da single operon with the relative order similar to that in E. coli(i.e. AIRS, CART, CAR.!& CARS and AIRS are pan: of 3. bi-enzymatic protein encoded by the Ade5,7gene. and GART is part of a mono-enzymatic protein encoded by the A&?gene in Sc~izosrrccbarotr!,Kcs~llj~t’ and in Succhatwtym cetwisiael~. In Dmsophila~6J’, chickeN. mouselj and humarW~, all thrw enzymes SF domains of a tri-enqmatic protein when a full-length tnnscript is synthesized from the CARTgene. The Drosophila protein differs by having an internal duplirztion of the AIRS dorrmin. The mou@ and Drosophilalh CARTgenes also give rise to 3 short tnnscn’pt encoding only a mono-enzymatic GARS.

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FKAJRE 2. Comparison of the amino acid sequences at the end of CARS and the beginning of AIRS in various species. Identities in comparison with the molise sequence are shown $ bold letters. The stop codon at the end of CARS found in the bacterial spcies is shown by an asterisk. The ~LLU CARS-AIRSgene does not conwin an intron (dashes) that split the sequences encoding the CARS and AIRS domains. The plscement of the intron in the GART gene of Dmsophilcr W. nrelmugus/er and D. psemkmbscurd. Chimnonm (C. terJtms1 and mouse is shown by slashes. and the intron (dotted line) divides the sequence at the =cond ha.% of an isoleucine or xginine codon zs shown (tower ca.se letters in three-letter code). The intron contains a cleavage-polyadenylation sequence shown as [c/p1 that, if used. can lead to the .synthesis of a monc-enqmatic protein with CARS activity in the Drosq%ilnspecies and in the mouse.

cleavage-polyadenylation signal was removed by splic- ing) encodes the &i-enzymatic CART protein. A monc- enzymatic CARS is synthesized from the GAR7’gene of D. pseudoobscwa~7 as well, but not from the GART gene of the more distant Dmsopbilu relative, Cbimnomrls tentmd9, nor from the CARS-AIRS gene of yeast.

While the location of the intron between the CARS and AIRS domains has remained conse,ved in mouse and D@~H+LI, the intron size, tile location of the cleavage- polyadenylation signal sequences within the intron and the number of cleavage-polyadenylation signal sequences have not (Table 1). The existence and location of the cleavage-polyadenylation site within an intron of the GARTgene provides a tantalizing clue to the origin of the CARTgene and suggests two related models for the

evolution of genes encoding multi-enzymatic proteins+

Models for gene evoh~tion The model in Fig. 3(a) show5 the intron-intron-

mediated rearrangement event proposed previously’. This model cannot explain the presence of the &wage- polyadenylation signal found in the intron at the hound- ary of the exons encoding the GARS and the AIRS domains. The models in Fig. 30~) and 3(c) are proposed to account for the presence of this intronic cleavage- polyadenylation signal. The model in Fig. 309 shows a rearrangement event that occurs downstream of the cleavage-polyadenylation signal of the CARS gene and within the first intron of the AIRSgene. If either a cryptic 5’ splice junction, located upstream of the stop codon of the CARSgene, or the donor splice site of the penultimate

GARSexon, were spliced to AIRScoding sequences, then this new fusion transcript might encode a hi-enzymatic GARS-AIRS protein similar to that found in yeast. The activation of a cryptic splice site is not an unlikely scenario because there are numerous examples of the use of cryptic splice sites due to minor changes in RNA sequence20. To account for the current yeast GAB- AIRS gene, the entire intron containing the cleavage- polyadenylation signal must have been lost; in the C. tentans gene, the intronic sequence has drifted such that a functional cleavage-polyadenylation signal no longer exists, although an APAAA sequence is present. In the initial gene rearrangement event, it is possible that spiicing across the newly created intron would compete with cleavage-polyadenylation. This is, in fact, what is seen currently in the CARTgenes of Drosophila and mouse, which yield a shorter transcript encoding a mono-enzymatic CARS and a longer transcript encoding a tri-enzymatic GARS-AH&CART protein.

In the model shown in Fig. 3(c), the rearrangement event takes place downstream of the CARS gene and upstream of the AIRS gene. In this model, both genes remain intact and preserve their separate promoters. If there was not a strong transcriptional stop signal between the two genes, transcription from the CARS promoter might extend through the AIRS gene. Then. if a cryptic splice site upstream of the CARS translational stop codon, or a splice donor site in the penultimate CARS exon, were spliced to the AZRS coding sequences, a transcript that encodes a hi-enzymatic GARS-AIRS pro- tein might be produced. As in the previous model, it

would be expected that there would T~l.-of~in~nthatseparatestheGARSandAlRSdomains be competition between splicing

and cleavage-polyadenylation at Total number the intron following the last CARS

Iat.ro~ c/P sign;rl C/P SW of imrons exon. Eventually, the AIRS promoter _@P) pos~@P~ v fngeae would have heen lost. Then, a gene

Mouse 3100 257, 314,347, AATAAA (41 21 encoding a mono-enzymatic GARS

443,447 A’ITAAA (1) protein and a b&enzymatic GARS-

D. pseU&obscuru 525 470 AATUA 6 AIRS protein would result. With this

D. melanognsrer 183 42 AATAAA G model, a deletion removing the

C. tentans 81 6 A’ITAAA 3 CARS-AIRS intergenic region would have had to occur in an ancestor of

aDistan= of ckavage-p&&ny&on (c/p> si@ from 5’ splice junction. yeast to explain the current gene structure in yeast.

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Page 3: Origin of genes encoding multi-enzymatic proteins in eukaryotes

The size of the GMTintron con- taining the cleavagqx$&enylation signal varies dramatically across species of Dip&m as shown in Table 1. Studies of an amylase intron in hundreds of Dmsopbilu species has also shown broad size vari- ation&l. Some species do not have an intron. Most have an intron vary- ing in size from 54 to 757 hp, with the majority being in the range of Go to 80 hp. Studies in our lahora- tory have shown that, as a cleavage- polyadenylation signal-containing intron is shortened, the splice re- action becomes more efficient rela- tive to cleavage-polyadenylation~~. Thus, it is possible that the reason the C. Ie?ztans GARTgtme does not yield a shorter transcript encoding a mono-enzymatic GART protein, even though a potential polyCA) sig- nal exists within the intron, is be- cause the inlron is so small that splic- ing simply out-competes cleavage- polyadenylation.

(azEL - lb) 1

Molecular biolugicd fossils? A prediction of these recomhi-

national pathways is that the mono- enzymatic GARS protein founti in Drosopbi/u and mouse is a relic of the genetic events that brought two genes together and does not have a significant function [Or MMViVd today. This prediction is testable. The intron and its cleavage- polyadenylation signal could IX re- moved in a mouse model. Then, one could see if any phenotype appears when a mono-enzymatic GARS can- not be synthesized. The same could be done in Drosophila In this case, population genetics could be ap- _. . . .

FIGURE 3. Three models that can account for the original fusion of the GARYand AIRS genes, (a) A rearrangement event takes place within the last intron of the CARSgene and within rh< first intron of the AIRSgene. tb) A reamngement event trtkti place after tlte cl~v3~e-Ix)l~~densl~ticln signal (reprexnteci its AATAAA) of the CARSgene and within the first intron cjf the illR,‘g~ne. RNA processing mrght involve splicing to existing splice sites (dashed line). to cyptic splice sites Casxrisk and dotted line) or a combination of the CA’O. (cl In 3 variation t)f the mtxlel shtnvn in (h). the rexrangement event takes place dosvnstream <If the cl~v~~e-polyadmyIarion sign4 of the CARSgene and upstream of the promoter (shown as an xrow marked PI of the AIRSgene. Possible RNA processing. is shtnvn as in (b). Boxes reprrsent cxcms: light and dark shaded hoses have cctiing .squence while open boons repre.sent 5’ or 3’ untmnslated .sequences. In the models shown in (h) and Cc), the u.se of cryptic splice bites might not seem to be neccc‘ssaty. Howyever. within the new tmnscrtpt predicted from the fusion gene, a cvptic splice site might be u.sed preferentially over the existing splice site.

plied to determine whether the normal allele provides even a small advantage over the deletion allele. It should be noted that there is no tissue-spcific difference in the ratio of GARS:GARTtranscripts seen in either Rmsophi/a or mouse’s, indicating that the synthesis of a mono- enzymatic GARS is not regulated. However, if the mono-enzymatic CARS is not crucial to survival in Drosophila and mouse, why has the intronic cleavage- polyadenylation signal persisted over millions of years? There is no current answer to this question. Neverthe- less, it is clear from Chironorn~s where the intron remains but the cleavage-polyadenylation signal is nonfunc- tional”), that a mono-enzymatic GARS need not be essen- tial. This also appears to be true in yeast where not only the intronic cleavage-polyadenylation signal is lost, hut the intron as well. If a mono-enzymatic CARS is irrelevant in animals, then the intron and its cleavag+ polyadenylation signal in the GARTgenes of Dtvsophii~ and mouse might represent a molecular biological fossil that is a footprint for an important mechanism whereby

AIRS

* 7

*

genes encoding multi-enzymatic proteins are created. Moreover, even if a mont>-functional CARS is crucial to mouse and Drosophilcl growth or development (thus explaining its preservation in these species over evolu- tion), the location and contents of this intron still might reflect the recombinational origin of the CARTgene.

Tht: recombination event shown in Fig. 3(b) xould create a gene encoding a multi-enzymatic protein in a single step, because the AIRS promoter would be lost in the process. However, the G#?S-AMSreanzngement can potentially produce an out-of-frame translation product that would result in the loss of e.ssential AIKS activity. DuplicSion of the AIRSgene before oue copy paniripates in the rearrangement would protect against this possi- bility. Evidence of gene duplication kfote gene fusion appears to explain, in part, the pathway in the evoiution of the multi-enzymatic CADgene’, a gene involved in the de 110~ synthesis of pyPnidines. No similar evidence currently exists to suggest that gene duplication was involved in the origin of the tri-enzymatic CARTgene.

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CzdcitoninKGRP

fl-CGRP

L

CGRP

(cc) Exons p Spectrin I Zl 30 31 32 lm 2m 3m 4m

cz gene exon 18

Factor 13 exoll 1

P

16kb ,L 4z1bp +- : 6kb --

FCGUW 4. Genes that might have arisen by the pathways su~ested in Fig. 3. Open boxes represent common exon~: light and dark .shaded boxes represent exonb that are specihc to an alternative tranmipt: thin fines are introm: black cl&s are the cieava~t+pc$adenylation sites, anp,ied lines above and below the figures are the splice rwctions as detailed below; and the arrow repre.sents the tnt-wriptional initiation sites. (a) The immunoglobulin p-6

locus. The VLIJCp and VDJ-CS alternative splice reactions are shown hy heavy splice lines; other ahemative splice reactions known to occur are shown hy dashed or dotted splice lines. (h) The cnlri!oonDz/CGfPgene and the related &CGfPgene. The splice to

produce calcitonin tnRiiA and that to produce CGRP mRNA are shown by heavy lines; the dashed line is the alternative splice reaction that is Seen in the humian gene. The grey box

in the /3-CGRPgene is the ‘VeStigkIi cakitonin exon. tc) The 3’ end of the p S$&i~z f gene, starting from exon 30. The splice to exons lm-4m. which encode a pieckstrin

homology domain, produces the z2 mRP4A and is shown by the heavy splice line: the alternative processing reaction to produce 21 mRNA is cleavagrt-polyadenylarion at the

end of exon 32. (d) The intergenic region of the C2 and Fuctor B complement AUKS, The 3’ end of the CZgene, which spans 18 kh. and the i’ end of the B~for Bg~nr. whch spans 6 kh, is shown. Trantiption termmates within the 421 hp that separates these nw genes.

The model in Fig. J(c) would not require dupli- comparing features of these genes provides some clues cation of an AIRS gene nor the immediate one-step to changes that might have occurred over time. The creation of a gene encoding a bi-enzymatic protein (i.e. fl-CGRF’gene encodes only CGRP, but contains a ‘vestigial’ the process could rake several steps before the loss of calcitonin exon, one that has lost the calcitonin cleavage- the AIRS promoter function). However, this model does polyadenylation signal, as well as a complete calcitonin require that at least some transcription from the GMS coding sequence 26 (Fig. 4b). This type of change is promoter must continue through the AIRS gene. consistent with the models proposed here: following Because transcription terminates at a variable distance a recombination or duplication event, mutations in downstream from cleavage-polyadenylation sites, from the DNA sequence can occur that will activate cryptic about 600 bp to 4 kb in some gene&s, this is not a splice sites, remove unnecessary promoter sequences serious obstacle ro this model. Indeed, read-through and/or cleavage--polyadenylation sites. The p-CGP transcription is observed in the expression of the heavy gene would be somewhat analogous to the GARTgene of chain immunoglobuiin p-6 gene2” (Fig. 4a). To form an C. tentans, which no longer has a functional cleavage- mRNA encoding IgD, the exons encoding the VDJ polyadenylation signal at the end of the GARS exons antigen-recognition domain are spliced to the exons (Fig. 2). AIso consistent with predictions of the above encoding the constant region of the 6 immunoglobulin models, the human calcitonin/CGtfP gene contains an protein; the p constant region exons, along with two alternative 5’ splice site within the calcitonin exon 4 that cleavagepolyadenylation signals, are spliced out as removes the cleavage-polyadenylation site and joins part of an intron. Thus, the immunoglobulin heavy the CGRP and calcitonin exonsl’ (the dashed splice line chain gene is an excellent example of a gene that might in Fig. 4b). Thii alternative splice product from the

have arisen along a pathway similar to that shown in Fig. 3011) or Xc>. It is believed that all the immune globulii constant-region gene seg- ments are derived from an ancestral p-like gene by duplication even@. In the case of p and 8, these gene segments have become fused into a single transcriptional unit; the 6 exons do not contain an independ- ent promoter, but are transcribed from the promoter upstream of the VDJ exons. In fact, transcriptional termination between the JJ, and 6 exons serves to downregulate the expression of IgD (Ref. 24).

The models in Fig. 3(b) and 3(c) might explain the origin of altema- tive RNA processing at the 3’ ends of genes, and the origins of genes that utilize alternative promoters. One example of a gene that is altema- tively processed at its 3’ end, and whose gene suucture is consistent with an origin predicted by the above models, is the gene that encodes the two neuropeptides calcitonin and CGRP, the culcifonirdCGRP gene26 (Fig. 4b). The cornmon first three exons, which encode a pep- tide sequence that is removed from the mature peptide hormones, are either spliced to exon 4 (which encodes calcitonin) or to exons 5 and 6 (which encode CGRP); there are cleavage-polyadenylation signals at the ends of exon 4 and exon 6. This gene structure is conserved in fish, rats and humans. Another gene exists in rats and humans, p-CGRP, which is related to culcitonin/CGRP;

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human gene has not been detected for the rat gene; there are nucleotide sequence differences between the rat and human genes at this location.

Mother gene that could have been derived by the proposed pathways is the j? spectrin Igene that is alterna- tively processed in etythroid and non-erythroid cells to produce the xl and 22 forms, respectively& (Fig. 4~). The Zl mRNA is produced by use of a cleavage- polyadenylation signal at the end of exon 32, while the X2 mRNA is spliced from a 5’ splice site within exon 32 to four exons located about 13 kb downstream. These four exons add an additional 21.4 kDa to the p spectrin protein and encode a pleckstrin homology domain. Pleckstrin homology domains are protein modules that have been identied in more than 60 proteins to date, and are related to the N- and C-terminal domains of pleck- strin, the major protein kinase C substrate in platelet@. Thus, p +~crtin Icould be an example of a gene that has gained an additional functional domain by gene fusion. Moreover, these models might accourt for how a domain, the pleckstrin homology domain in this case, could be added to the 5’ or 3’ ends of a diverse set of genes.

concludiig reIna& While the models proposed here suggest ways in

which an organism can generate additional diversity from its genetic material, it is clear that such recombination and gene fusion events could also be deleterious if expression of an essential gene were lost. As mentioned above, one mechanism by which deleterious events could be prevented is to duplicate the essential genes before the recombination event. Another mechanism would be to prevent read-through transcription, which would eliminate the possibility that the downstream promoter would be occluded, and would also prevent production of a fusion transcript. An example of where this might have occurred is between the related and closely linked complement genes CZ and Factor B (Ref. 30). The gene encoding C2 is located just upstream of the gene encod- ing Factor B; the cleavage-polyadenylation signal for C2 is only 421 bp from the Fnc!oorB gene trarsriptional initiation site (Fig. 4d). Transcripts that contain se- quences from C2 and from Factor B have not been identified. In fact, the 421 bp region between these two genes has been shown to contain a very efficient tmn- scriptional termination sequence to prevent read-through transcriptions*. Thus, these genes represent an ancient gene duplication event that, instead of resulting in for- mation of a multi-functional protein, evolved a strategy to ensure that the transcription units remain separate.

It has been proposed (e.g. Ref. 32) that, by tracing the history of introns, insight might be gained into the events that lead to the genes seen today. The CART gene and other examples provided here might provide supporting evidence for this proposal. Additional evi- dence for the models proposed here may well come from examining the volumes of sequence data coming from the human genome program and from sequencing the GARTgene in other species. It is necessary to note that, for any of these models to be correct with regard to the origin of genes encoding multi-enzymatic pro- teins, there is an underlying assumption that at least some introns must Ix very old. Recently, one group argued that some introns are over a billion years oldas.

This work is supported by grants MC&9418413 (J.N.D.1 and MCB-9507513 (M.L.P) from the National Science Foundation and by grant GM47644 (J.N.D.) from the National Institutes of Heakh, and is dedicated to the pioneering work of Walter Gilbert.

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