©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Analysis of a Mouse Gene Encoding Three Steps of Purine Synthesis Reveals Use of an Intronic Polyadenylation Signal without Alternative Exon Usage (*)

(Received for publication, September 28, 1994; and in revised form, November 18, 1994)

Julie L. C. Kan Richard G. Moran (§)

From the Department of Pharmacology and Toxicology and the Massey Cancer Center, Medical College of Virginia, Richmond, Virginia 23298

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A single mouse genomic locus encodes proteins catalyzing three steps of purine synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide formyltransferase (GART). This gene has 22 exons and spans 28 kilobases. The existence of a second genetic locus and closely related pseudogenes was ruled out by Southern analysis. Mouse tissues express two related classes of messages encoded by this single locus: a trifunctional GARS-AIRS-GART mRNA and a monofunctional GARS mRNA. These transcripts used the same set of multiple transcriptional start sites, and both used the same first 10 exons. CCAAT and TATA elements were not found for this locus. Exon 11, which represented the last coding sequence of the GARS domain, was differentially utilized for the two messages. The trifunctional mRNA was generated by splicing exon 11 to exon 12, the first coding sequence for the AIRS domain with subsequent use of a polyadenylation signal at the end of exon 22. Genomic sequence corresponding to the 3`-UTR of the monofunctional GARS mRNA was contiguous with exon 11, so that the smaller message arose from the recognition of one of the multiple polyadenylation signals present within the intron between exons 11 and 12. Hence, polyadenylation of the primary transcript at a position corresponding to an intron of the genomic locus was responsible for the generation of the monofunctional GARS class of mRNAs. This utilization of an intronic polyadenylation site without alternative exon usage is comparable to the mechanism whereby both secreted and membrane-bound forms of the immunoglobulin µ heavy chain are made from a single genetic locus.


INTRODUCTION

The enzyme activities catalyzing the second, third, and fifth steps in de novo purine synthesis, i.e. glycinamide ribonucleotide synthetase (GARS), (^1)glycinamide ribonucleotide formyltransferase (GART), and aminoimidazole ribonucleotide synthetase (AIRS), respectively, are present as a single 110-kDa trifunctional protein in those vertebrate species studied to date(1, 2) . The mouse cDNA for the trifunctional GARS-AIRS-GART was previously isolated in this laboratory by initial screening of a mouse expression library using a polyclonal antibody generated against the 110-kDa protein(3) . Using one of the cDNA clones obtained from this initial screen as a probe, a second class of mouse cDNAs encoding a monofunctional GARS whose coding region had the identical sequence with that of the GARS domain of the mouse trifunctional cDNA, but a completely different 3`-untranslated region (UTR) was isolated. In agreement with the sequence of this GARS cDNA, a protein with the molecular weight predicted for a monofunctional GARS, in addition to the trifunctional GARS-AIRS-GART, was detected in mouse L1210 cells by Western blot. These two proteins were subsequently separated, and both protein fractions were shown to have GARS enzymatic activity. Two cDNAs for monofunctional GARS and trifunctional GARS-AIRS-GART have also been reported in chicken and human tissue(4) . These enzymatic activities are present in bacteria as three single-domain proteins(5, 6, 7, 8) . Drosophila melanogaster and Drosophila pseudoobscura also express two mRNAs encompassing these three enzyme activities, one encoding a trifunctional GARS-AIRS-AIRS-GART, with an apparent endoduplication of the AIRS domain, and the other corresponding to only the GARS domain(9) . These two mRNAs have been reported to be encoded by a single gene in Drosophila(10) . Hence, although higher eukaryotes have evolved a presumably more efficient or more favorable trifunctional protein combining a GARS active site with peptides that form the active sites for two subsequent pathway reactions, a catalytically active monofunctional GARS has been shown to be present in mouse(3) ; all three vertebrate species studied to date express the monofunctional GARS mRNA(3, 4) . The reason for this apparent functional redundancy remains unclear.

We now report the organization of the region of the mouse genome corresponding to these mRNAs. Our evidence demonstrates that both the trifunctional GARS-AIRS-GART and the monofunctional GARS are encoded by a single 28-kb genomic locus. The detailed structure of the gene indicates that the monofunctional GARS mRNA is generated by alternative processing of the intron separating the last coding domain for GARS and the first exon of the AIRS domain of the trifunctional transcript, and that this occurs without the use of alternative exons. Multiple polyadenylation signals in intron 11 are used to generate the monofunctional GARS mRNA, and both transcripts used the same set of multiple transcriptional start sites.


MATERIALS AND METHODS

Library Screening

Clones representing the genomic locus encoding trifunctional GARS-AIRS-GART were isolated from a FIX II genomic library (Stratagene) constructed from spleen DNA of a (C57 BL6 times CBA) F1 hybrid female mouse. This library was screened using a mixed cDNA probe consisting of the 5`-EcoRI fragment of pJKMF containing most of the 5`-UTR and the 2.4-kb 3`-EcoRI fragment of pWTF which comprises the entire coding region for GARS-AIRS-GART and also contained the 3`-UTR of the trifunctional message(3) . The probes were labeled to a specific activity of 1-5 times 10^9 dpm/µg DNA by priming DNA synthesis with random hexamers. Hybridization of the nitrocellulose filters was performed at 63 °C using 0.5 M sodium phosphate, pH 7.0, 7% SDS, 1% bovine serum albumin, and 1 mM EDTA(11) . The filters were washed at 50 °C in 2 times SSC containing 0.2% SDS until the background was below 100 cpm/cm^2.

RNA Analysis

Total RNA from cultured mouse L1210 leukemic cells and from mouse tissues was isolated using the guanidinium thiocyanate/acidic phenol/chloroform method (12) with the addition of a LiCl extraction step for the mouse tissues(13) . Poly(A) RNA was isolated by applying total RNA to a poly(dT) resin using the Fastrack system (Invitrogen). RNA was fractionated on a 1% agarose gel following denaturation with glyoxal/dimethyl sulfoxide (14) and was subsequently transferred to nylon membranes (Biotrans, ICN) by capillary action in 20 times SSC. The RNA was cross-linked to the membrane using 0.12 J of 254 nm uv irradiation in a Stratalinker apparatus (Stratagene). The two probes used, a GARS specific probe (pQ 0.8) and an AIRS-GART specific probe (pW1.15T) (Fig. 2A), were labeled using Klenow fragment to extend random primers, incorporating both [alpha-P]dATP and [alpha-P]dCTP (3000 Ci/mmol) to a specific activity of 1-3 times 10^9 dpm/µg DNA. The filter hybridization and wash conditions were those described above.


Figure 2: Schematic representation of overlapping mouse genomic clones for GARS-AIRS-GART, restriction map and exon location. A, four cDNA probes, pK4MF0.8, pK4MF1.0, pYTF1.0, and pYTF0.7, corresponding to the monofunctional GARS and the trifunctional GARS-AIRS-GART cDNA, were used to determine the overlap between the four genomic clones. The positions of oligonucleotide and cDNA probes that are described in the text are indicated. B, the EcoRI, HindIII, and BamHI sites were mapped by Southern analysis using four cDNA probes (pK4MF0.8, pK4MF1.0, pYTF1.0, and pYTF0.7 in A) and four oligonucleotides (P(R), JK9, JK20, and JK1R). The HindIII site indicated by the asterisk (*) is an RFLP for the mouse strains, CBA and C57 BL6. The genomic fragments that were subcloned into pBluescript SK II(+) are noted below the restriction map. These subclones were used for sequencing analysis to determine exon size and location within the genomic locus.



Analysis of the Genomic Locus for GARS-AIRS-GART by Southern Blotting

An apparent RFLP present in genomic clone LC6 was analyzed by Southern blotting. DNAs from the livers of C57 BL6 male, CBA female, and hybrid C57 BL6 times CBA female mice were digested with restriction endonuclease HindIII. The digested DNAs were separated on a 1% agarose gel in TBE buffer, capillary-transferred to nylon membrane in 20 times SSC, and subsequently probed using a random-primed cDNA corresponding to the AIRS-GART region (pYTF)(3) . In other experiments, Southern blots were used to determine whether other closely related genes were present in the mouse genome. DNA from the genomic clones spanning the GARS-AIRS-GART locus (10 ng of DNA) and mouse liver genomic DNA (C57 BL6 times CBA female) (10 µg of DNA) were digested with EcoRI or BamHI, fractionated on a 1% agarose gel, then transferred to nylon membranes; these blots were probed with the 5`-EcoRI fragment of pJKMF and with the 3`-EcoRI fragment of pWTF, which, together, represented the full-length cDNA for trifunctional GARS-AIRS-GART(3) . Hybridization and washing of these filters was performed as described above.

Restriction Mapping, Subcloning, and Exon Mapping

The genomic clones were mapped using slot blots and Southern blots of insert DNA digested with endonucleases HindIII, EcoRI, and BamHI and analyzed with several radiolabeled cDNA probes and with oligonucleotides previously used for sequencing the GARS-AIRS-GART cDNA(3) . The alignment of these probes with the cDNAs for the trifunctional GARS-AIRS-GART and the monofunctional GARS are indicated in Fig. 2A. The cDNA probes used were: 1) the 5`-EcoRI fragment of pJKMF(3) , i.e. pK4MF0.8, corresponding to the 5`-coding region for the GARS domain and the 5`-UTR of the monofunctional GARS cDNA, 2) the KpnI-XhoI fragment from pJKMF corresponding to the 3` domain of GARS and the 3`-UTR of the monofunctional GARS mRNA, i.e. pK4MF1.0; 3) the 1.0-kb EcoRI-EcoRV restriction fragment of pYTF (3) which corresponded to most of the AIRS domain, i.e. pYTF1.0; and 4) a 0.7-kb EcoRV-EcoRI restriction fragment of pYTF consisting mainly of the GART domain, including most of the 3`-UTR of the trifunctional cDNA, i.e. pYTF0.7. The oligonucleotide probes used were 20 to 25 nucleotides in length and served to identify genomic restriction fragments corresponding to the 5` and 3` ends of the GARS domain, the beginning of the AIRS domain, and the 3` end of the GART domain (Fig. 2A). Seventeen restriction fragments (some of which were overlapping), representing the entire genomic locus encoding GARS-AIRS-GART, were subcloned into pBluescript II SK(+) for mapping of intron-exon junctions (Fig. 2B). Sequence analysis was performed using double-stranded plasmid DNA and Sequenase 2.0 (U. S. Biochemical Corp.) to determine the exon sizes and the sequences at exon-intron junctions.

Placing the Location of Exons on the Genomic Map

The positions of the exons in the genomic locus were initially localized relative to the restriction sites that flanked subcloned genomic fragments, using the polymerase chain reaction (PCR). A specific gene primer within an exon or at a known distance into the adjacent intron and the corresponding pBluescript SK(+) M13 Forward or Reverse vector primer (Stratagene) were used to amplify the sequence extending to each restriction site; the lengths of these PCR products were then measured on 1% agarose gels. The reactions were performed using 10 pg/µl denatured plasmid containing subcloned inserts, a 3 ng/µl concentration of each primer, 2.5 milliunits/µl of Taq polymerase (Promega), and 2 mM MgCl(2) using 30 cycles of 92 °C for 60 s, 55-60 °C for 30-60 s, and 72 °C for 2-5 min in a MJ Research thermocycler in a total volume of 10-20 µl. Three of the small exons were mapped relative to the restriction sites defined by subcloning using direct sequence analysis. Subsequently, PCR was used to amplify the length of each entire intron by using pairs of exon-specific primers known to be on adjacent exons and using genomic clones LC1, LC6, and LC5 as templates. These PCR reactions were performed using 1.5 µg/ml denatured genomic cloned DNA and, otherwise, the same conditions as used for PCR amplification of plasmid subclones. This experimental design allowed an independent check on both intron lengths and the accuracy of the restriction map generated for these clones.

Analysis of the Use of the Multiple Polyadenylation Signals in Intron 11

The utilization of the multiple polyadenylation signals in intron 11 was determined by the method of 3`-RACE (Rapid Amplification of cDNA ends)(15) . A poly(T)-containing primer with several restriction site sequences at its 5` terminus (GACTGAGCTCGGTACCAAGCTTGATGC(T)) was used for first strand cDNA synthesis. Approximately 0.5 µg of poly(A)-selected RNA from mouse L1210 leukemic cells was annealed with 50 ng of the poly(T) restriction site primer, and the mixture was incubated in a total volume of 20 µl with 1 unit of Superscript reverse transcriptase (Life Technologies, Inc.) in 20 mM Tris-Cl, pH 8.4, 50 mM KCl, 2.5 mM MgCl(2), 100 µg/ml bovine serum albumin, 10 mM dithiothreitol at 42 °C for 1 h. The RNA was subsequently destroyed by incubation of the sample at 55 °C for 30 min with 1 unit of RNase H. The cDNA and the poly(T) restriction site primer was separated by binding of the cDNA to a Glassmax column (Life Technologies, Inc.). The PCR was performed using 2.5 µl of cDNA, a 3 ng/µl concentration of the restriction site primer (above primer without the poly(T) region), 1.5 ng/µl gene-specific primer corresponding to the 3`-UTR of monofunctional GARS and present 5` to the first poly(A) signal, 2 mM MgCl(2), 2.5 milliunits/µl Taq polymerase (Promega) using 40 cycles of 94 °C for 60 s, 60 °C for 60 s, and 70 °C for 3 min in an MJ Research thermocycler in a total volume of 50 µl. The PCR product obtained was again amplified with another nested gene-specific primer, and the reamplified product(s) were subcloned directly into the pCRII vector (Invitrogen).

Determination of the Transcriptional Start Sites Used by the GARS-AIRS-GART and GARS Messages

The transcriptional start sites for both messages were determined by the method of 5`-RACE(15) . This was performed essentially as described for 3`-RACE except that first strand cDNA synthesis used a gene specific primer corresponding either to the AIRS domain (nt 1601-1625 and a second nested primer to the AIRS region, nt 1500-1522) or to the 3`-UTR of monofunctional GARS (nt 1495 to 1519 and 1450 to 1474 of the monofunctional GARS cDNA). A poly(C) tail was added to the 5` end of the cDNA using terminal deoxynucleotidyltransferase. This dC-tailed DNA was used as the template for PCR with a nested gene-specific primer and a second universal primer containing a poly(dG-dI) tract at the 3` end of the oligonucleotide.


RESULTS

Mouse Tissues Contain Two Classes of mRNAs Coding for GARS

Although mammals have evolved a trifunctional protein that catalyzes all three of the GARS, AIRS, and GART reactions, our previous studies (3) have indicated the presence of a second protein with GARS activity in mouse L1210 cells. We used probes corresponding to the functional domains of our previously isolated mouse GARS-AIRS-GART cDNA (3) to examine the mRNA expression pattern in various mouse tissues as an approach to understanding this apparent duplication in function. Northern analysis of poly(A) RNA with a cDNA probe specific for the GARS domain detected a distinct 3.4-kb band in all of the mouse tissues and in mouse L1210 leukemia cells as well as a broader zone of hybridization to a smaller transcript(s) in L1210, kidney, and brain (Fig. 1A). Interestingly, two clearly distinguishable smaller mRNAs of 1.7 and 1.9 kb were found in poly(A) RNA from adult mouse liver. Using a cDNA probe corresponding to AIRS and GART domains, hybridization was observed only to the 3.4-kb mRNA (Fig. 1B). The presence of these two mRNA species for a monofunctional GARS and a trifunctional GARS-AIRS-GART concurs with the two classes of cDNAs previously isolated from mouse T cell libraries(3) . The broad hybridization seen for the smaller message along with the pattern in mouse liver suggest that multiple monofunctional GARS mRNAs are present that are similar in size (1.7-1.9 kb). This agrees with the presence of multiple potential polyadenylation signals we have previously reported at the 3` end of the monofunctional GARS cDNA(3) . It was noted that both mRNA species were more highly expressed in leukemic L1210 cells compared to the level of expression in the three normal mouse tissues examined and that the trifunctional GARS-AIRS-GART was substantially more abundant than the monofunctional GARS, regardless of the level of expression in a particular tissue. This coordinate expression suggested to us that the two messages were transcribed from a single gene.


Figure 1: Northern analysis of monofunctional GARS and trifunctional GARS-AIRS-GART in mouse tissues and in mouse L1210 leukemic cells. The blots were hybridized with a radiolabeled probe corresponding to the GARS domain (probe pQ0.8, Fig. 2A) (A) and to the AIRS and GART domains (probe pW1.15T, Fig. 2A) (B). The GARS domain probe hybridized to two messages of 3.4 and 1.7-1.9 kb, and the AIRS-GART probe only hybridized with the 3.4-kb message. In mouse liver, two distinct messages of approximately 1.7 to 1.9 kb are detected. Note the different amounts of poly(A) RNA per lane; the film was exposed for 36 h.



Isolation, Alignment, and Intron-Exon Organization of the Mouse GARS-AIRS-GART/GARS Genomic Locus

Mouse genomic DNAs corresponding to both messages were isolated from a genomic library in FIX II. Seven genomic clones were isolated from a screen of 1.5 times 10 plaques using radiolabeled probes spanning all three domains of the mouse trifunctional GARS-AIRS-GART cDNA. These seven clones were initially aligned using cDNA fragments corresponding to each of the three domains and several oligonucleotides as probes against DNA on slot blots; the alignment of these probes with the cDNA for transcripts from this gene and with the genomic clones is noted in Fig. 2A. Two clones (LC1 and LC7) hybridized to cDNA sequence for the GARS domain, one clone (LC6) contained sequence for all three domains, and the other four clones hybridized to a probe corresponding to part of AIRS and to the GART domain.

Restriction maps were constructed using endonucleases EcoRI, BamHI, and HindIII for four of the genomic clones (Fig. 2B), but three clones (LC1, LC6, and LC5) were sufficient to encompass the entire genomic locus (Fig. 2A). LC1 contained sequence corresponding to most of the GARS domain, the first domain of the trifunctional GARS-AIRS-GART cDNA, and had a common restriction map with LC7 over 13 kb. LC6 had a 3.0-kb overlap with LC1 and had a sequence corresponding to the most 3` end of the GARS domain, all of the AIRS domain, and most of the GART domain. LC5 had a 6-kb overlap with LC6 and contained coding sequence corresponding to the most 3` end of the AIRS domain and all of the GART domain. The genomic locus was found to span 28 kb.

Seventeen genomic restriction fragments that spanned the entire genomic locus were subcloned into pBluescript SK II(+) to allow a more detailed analysis of the intron/exon organization of this gene. The sizes of the exons and junctional sequences were determined by limited sequence analysis of the subcloned fragments first using oligonucleotides from the GARS-AIRS-GART sequence known to hybridize with the various subcloned fragments, followed by a second round of sequencing extending intron primers adjacent to the exon back into and through each exon (see ``Materials and Methods''). The sequence obtained was compared with that of the previously isolated mouse cDNAs(3) ; divergence from the cDNA and match to consensus splice junction sequence (16) were used as criteria for identification of the 5` and the 3` splice sites of each exon. The exons ranged in size from 69 to 342 nt (Table 1), and the genomic locus was comprised of 22 exons. Exon 2 contained the sequence for the start methionine for both classes of RNAs and, along with exons 3 to 11, encoded the GARS domain for both GARS-AIRS-GART and the monofunctional GARS. Exons 12 to 17 encoded AIRS with exon 18 having a sequence corresponding to the end of the AIRS domain and the beginning of the GART domain. Exons 19 to 22 encoded GART and the 3`-UTR of GARS-AIRS-GART. The beginning of the genomic GART domain was clearly identifiable from the high conservation of the N-terminal amino acid sequence of yeast and Escherichia coli monofunctional GART cDNAs with the mouse cDNA GART domain(3, 8, 17) . Exon 1 in the genomic map (Fig. 2B) had sequence identity with that of the initial 5`-UTR region for the monofunctional GARS cDNA isolated from cDNA libraries(3) . Because the cDNA for the 5`-UTR of GARS-AIRS-GART was not found in cDNA libraries, it was not clear whether this first exon was utilized by both classes of mRNAs. Alternative transcriptional start sites and different first exons might be responsible for the generation of these two classes of messages from the same gene. This was not the case (see below).



Intron Sizing

Intron sizes were determined by amplification of the genomic subclones by PCR using either a gene-specific primer or an intron primer and the corresponding vector primer from pBluescript SK II(+). This allowed the exon to be initially mapped relative to the restriction sites used to generate each subcloned fragment. The intron sizes determined by this method concurred with that obtained by PCR amplification of intronic DNA in the genomic clones using gene-specific primer pairs from adjacent exons ( Fig. 3and Table 1). The use of two different DNA substrates for PCR, namely, subcloned genomic fragments and genomic clones, allowed verfication of the position of the exons and the sizes of the introns. In addition, only PCR reactions which gave a single well-defined product (Fig. 3) were used for estimation of intron sizes. The intron between exons 12 and 13, exons 19 and 20, and exons 21 and 22 were completely sequenced and found to be 263, 363, and 98 nt in length, respectively. The exon sizes and estimated intron sizes are summarized in Table 1, and the positions of the exons are mapped relative to the known restriction sites (Fig. 2B). The position of the exons and the sizes of the introns verified the genomic map deduced from Southern analysis and restriction mapping.


Figure 3: Use of PCR for sizing the mouse GARS-AIRS-GART gene. Primer pairs between adjacent exons were used for PCR against the genomic clones. Lanes 1 and 2 represent primer pairs between exon 1 to exon 2 and exon 2 to exon 3, respectively; lane 3, exons 2-4; lane 4, exons 4-5; lane 5, exons 5-6; lane 6, exons 6-7; lane 7, exons 7-8; lane 8, exons 8-9; lane 9, exons 8-10; lane 10, exons 10-11; lane 11, exons 11-12; lane 12, exons 14-15; lane 13, exons 15-17; lane 14, exons 17-18; lane 15, exons 18-19; lane 16, exons 19-20; and lane 17, exons 19-21. The standards are DNA restricted with HindIII and ØX restricted with HaeIII (M) (Life Technologies, Inc.).



Evidence That a Single Genomic Locus Encodes Both Classes of mRNAs

We previously showed that LC1 and LC6 were overlapping clones by limited sequencing of the common restriction fragment 1RR0.9 and 6RR0.9(3) . To rule out the presence of other genomic loci containing nucleotide sequences for GARS, AIRS, or GART, Southern blots of the three isolated overlapping clones were compared with that of mouse genomic DNA. The three clones LC1, LC6, and LC5 along with mouse genomic DNA were cleaved with either BamHI or EcoRI, and Southern blots of these DNAs were hybridized with probes spanning the entire length of the GARS-AIRS-GART cDNA, including the 5`- and 3`-UTRs. If other loci containing any of these cDNA sequences were present in mouse DNA, not all of the hybridizing bands in mouse genomic DNA would be present in the clones isolated. The pattern of hybridization obtained (Fig. 4) indicated that all restriction fragments found in mouse genomic DNA were also present in at least one of the genomic clones. Thus, the absence of additional bands in mouse genomic DNA rules out the presence of a separate locus encoding the monofunctional GARS cDNA as well as loci for any one of the three domains, redundant GARS-AIRS-GART genes, or related pseudogenes.


Figure 4: Restriction enzyme analysis of mouse genomic DNA and isolated genomic clones defining the GARS-AIRS-GART locus. Cloned DNAs (0.01 µg) (LC1, lanes 1 and 5; LC6, lanes 2 and 6; and LC5, lanes 3 and 7) or CBA mouse genomic DNA (10 µg, lanes 4 and 8) were cleaved with EcoRI (lanes 1-4) or HindIII (lanes 5-8), Southern-blotted, and hybridized with a mixed probe corresponding to the entire cDNA for trifunctional GARS-AIRS-GART. The hybridizing EcoRI and HindIII restriction fragments found in the genomic DNA are also found in the overlapping clones. Note that there were no restriction fragments found in the CBA genomic DNA that were not represented in these three clones either as an identically sized fragment or as a fragment attached to the arms, specifically the 2.3-kb fragment in the EcoRI digest and the 4.4-kb band in the HindIII digest.



Intronic Sequence Immediately Adjacent to the last GARS Coding Region Indicates That Both mRNAs Are Transcribed from This Gene

Our previous analysis of the sequence at the junction between the stop codon of the monofunctional GARS cDNA and the 3`-UTR of this message indicated close homology to the 5` splice donor site consensus(3) . If the sequence surrounding this stop codon was capable of acting as a splice donor, then it seemed reasonable that the 3`-UTR sequence of the monofunctional GARS mRNA represents adjacent intronic sequence which is spliced out during the maturation of the trifunctional GARS-AIRS-GART mRNA (Fig. 5A). Two other possible mechanisms which could explain the generation of the two classes of mRNAs would be alternative use of exons which code for the most downstream GARS coding domain, one exon (exon 11A, Fig. 5B) with immediately adjacent sequence which corresponds to the 3`-UTR of monofunctional GARS (exon 11B, Fig. 5B), and one with intronic sequence which allows the splicing with the first AIRS exon, or usage of alternative exons, one containing the 3`-UTR of monofunctional GARS and the other containing the first AIRS coding sequence (Fig. 5C).


Figure 5: Schematic representation of three models for the generation of both the monofunctional GARS and trifunctional GARS-AIRS-GART transcripts from a single mouse locus. The boxes represent exons which are separated by introns (line). The splicing used to generate the trifunctional GARS-AIRS-GART (solid line above the intron) and the monofunctional GARS (dashed line below the intron) are shown. Exon 11 (solid box) represents the exon containing the last of the GARS coding domain, and exon 12 (striped box) represents the beginning of the AIRS coding domain. The 3`-UTR for the monofunctional transcript is indicated by the shaded box. A, both transcripts utilize the same exon 11 with the trifunctional transcript generated by splicing of exon 11 to exon 12 and the monofunctional transcript by the recognition that polyadenylation signals in intronic sequence are contiguous with exon 11. B, the two transcripts are generated by alternative splicing of two exons 11; 11A for monofunctional transcript that contains a contiguous 3`-UTR and 11B for trifunctional transcript. C, the two transcripts are produced by usage of the same exon 11 but alternative splicing to either the 3`-UTR of monofunctional or to exon 12 for the trifunctional.



To determine the mechanism for generation of the two classes of mRNAs, two oligonucleotides, JK9 corresponding to the GARS coding domain (amino acids 410-416) and JK10R corresponding to the antisense strand of the 3`-UTR of monofunctional GARS (nt 1724-1743) (see Fig. 2A), were used for Southern blot analysis of LC6. Both oligonucleotides hybridized to the same restriction fragments for DNA cut with BamHI, HindIII, and EcoRI. The smallest hybridizing fragments, a 3.9-kb HindIII-EcoRI and a 4.4-kb EcoRI fragment, were subcloned into pBluescript SK II(+) (Fig. 2B). Sequence analysis was performed on the two subclones using oligonucleotides JK9 and JK11R to ascertain their relative positions with respect to each other and for comparison of genomic sequence with the 3`-UTR of monofunctional GARS cDNA. Double-stranded DNA sequencing of these genomic subclones primed from oligonucleotide JK9 gave clear sequence of the last GARS coding domain (amino acids 419-433) along with the consensus 5` splice donor site which contained the stop codon of monofunctional GARS and which ran immediately into a sequence identical with the 3`-UTR of monofunctional GARS including that representing the oligonucleotide JK10R (Fig. 6). Using JK10R as a primer, the confirmatory antisense sequence was obtained including a sequence representing oligo JK9. These results indicated that the 3`-UTR of monofunctional GARS was immediately adjacent to the last coding region for GARS in the mouse genome. Thus, splicing of an exon containing only the 3`-UTR of monofunctional GARS (Fig. 5C) was not the case, but two alternative mechanisms could not be definitively distinguished (Fig. 5, A and B). Because the coding region of GARS for both classes of messages was identical, it was unlikely that there would be two alternative exons (11A and 11B in Fig. 5B) representing the last 232 nt of identical GARS coding domain, as evolutionary drift should have resulted in some sequence differences between the two classes of mRNAs. Detailed Southern analysis of the region of LC6 which separates the penultimate exon of the GARS domain (exon 10) and the first coding region of the AIRS domain (exon 12) demonstrated that both the oligonucleotide probes JK9 and JK10R hybridized to the same restriction fragments. Sequence analysis of the smallest hybridizing fragment, a 1.0-kb RsaI-StyI fragment, indicated the presence of only one copy of exon 11 (data not shown). Therefore, we rule out the possibility of two alternative nearly identical exons and conclude that the monofunctional GARS mRNA results from alternative processing of the 5` splice junction (Fig. 5A).


Figure 6: Nucleotide sequence of the GARS-AIRS-GART exon 11 and adjacent genomic intron sequences. Sequence of exon 11 obtained from double-stranded sequencing of subcloned restriction fragments, 6RR4.4 and 6RR3.9 (see Fig. 2B). The taa stop codon for the monofunctional GARS transcript and the five potential polyadenylation signals are underlined. The three polyadenylation signals that are used for polyadenylation as identified by 3`-RACE are indicated by roman numerals (I, II, III), and the sites of cleavage for addition of the polyadenylation tail are noted (). For polyadenylation site I (3/19 clones analyzed), three different sites for cleavage were identified; for site II (11/19 clones), the major class of clones was identified; for site III (2/19 clones), the cleavage was identified by both 3`-RACE and by the monofunctional GARS cDNA previously isolated. The putative pausing signal is boxed.



Utilization of 3` Polyadenylation Signals

Five potential polyadenylation signals were found within the 3`-UTR of monofunctional GARS cDNA ( Fig. 6and (3) ). If more than one of these are recognized and used, then this would explain the two distinct monofunctional GARS transcripts present in adult mouse liver and the broad zone of hybridization seen in the other adult mouse tissues and in L1210 mouse leukemic cells (see Fig. 1). The possibility of whether all or just a subset of these potential polyadenylation signals could be used to generate the monofunctional GARS transcript was investigated. First strand cDNA synthesis was performed using a poly(T) restriction site primer (see ``Materials and Methods'') against poly(A)-selected RNA from mouse leukemic L1210 cells. PCR of this cDNA population was performed with a gene-specific primer corresponding to the 3`-UTR of the monofunctional GARS and an anchor primer (the restriction site portion of the poly(T) restriction site primer).

Of the 19 clones analyzed, three polyadenylation signals were found to be utilized, two of which had the more common consensus polyadenylation signal sequence of AATAAA (which has been reported to be used in 86% of genes surveyed, (18) ), and one of which had the less common consensus sequence of ATTAAA (present in 12% of genes previously surveyed) (Fig. 6). Of the two AATAAA sequences recognized, one (site III in Fig. 6) was already known to be used in the cDNA previously isolated (3) and the other (site I) allowed cleavage of the hnRNA at one of three closely spaced positions for subsequent addition of the poly(A) tail. Only one cleavage site was found immediately downstream of two overlapping polyadenylation signals identified jointly as site III in Fig. 6. The predominant polyadenylation signal that was used and identified by this 3`-RACE experiment was the ATTAAA (site II). The presence of more than one polyadenylation signal and the ability to use several of these polyadenylation signals appears to represent a redundant mechanism. This suggests that the monofunctional GARS plays an important role which cannot be sufficed by the GARS domain of the trifunctional GARS-AIRS-GART. The site of cleavage for poly(A) addition for all three polyadenylation signals used, a GT-like tract was found in proximity to all of the utilized poly(A) sites; such a motif is important for 3` end formation and cleavage (reviewed in (19) ). In addition, examination of the sequence in the intron downstream of the last polyadenylation site for monofunctional GARS mRNA revealed a 29-nucleotide poly(T) tract (Fig. 6). This poly(T) tract was separate from the GT-like tract. Similar poly(T) tracts present in the first intron of c-myc, c-myb, and c-fos(20, 21, 22) have been implicated in premature transcript arrest, subsequent generation of abbreviated transcripts, and control of message levels for those genes. There is a precedent that such a mechanism would allow intronic polyadenylation: a synthetic poly(A) site placed in intron 2 of the beta-globin gene could be recognized efficiently only if the upstream 5` splice donor site was deleted or when a pause site was placed downstream of the synthetic poly(A) site (23) . However, we note that a functional mRNA is generated, by utilization of an intronic polyadenylation signal in the mouse GARS-AIRS-GART gene (Fig. 6), whereas this is not the case for c-myc, c-myb, and c-fos.

Both Classes of mRNAs Use the Same Set of Transcriptional Start Sites

One possible mechanism of obtaining two different messages from a single gene is by the use of alternative start sites, suggestive of the use of different transcriptional control elements. This was first investigated by primer extension using a primer corresponding to the GARS coding domain. This primer extension analysis did not give a single start site but, rather, indicated multiple start sites (Fig. 7). Hence, it was not possible to distinguish which of these start sites was responsible for the GARS-AIRS-GART mRNA and which corresponded to transcriptional starts for the monofunctional GARS mRNA. The presence of multiple start sites was consistent with the structure of the immediate promoter region of this gene. Sequence analysis of the genomic region upstream to the start sites indicated the absence of both the TATA and CCAAT boxes and the presence of five Sp1 binding sites (Fig. 7). The other classical means of investigating transcriptional start sites, that of ribonuclease protection, also would not allow us to distinguish which of the multiple start sites corresponded to the two classes of mRNAs. Hence, 5`-RACE was used which would allow the start site(s) for the trifunctional GARS-AIRS-GART mRNA and the monofunctional GARS mRNA to be determined independently. This technique relied on the use of a gene-specific primer for first strand cDNA synthesis from mRNA followed by PCR. By choosing a gene-specific primer which corresponded to the AIRS coding domain, the start sites obtained would only be from the trifunctional GARS-AIRS-GART mRNA. Using a gene-specific primer corresponding to the 3`-UTR of the monofunctional GARS mRNA, the start sites of this set of messages would be determined. The reverse-transcribed cDNAs obtained from these two reactions were tailed with a poly(dC) tract using terminal deoxynucleotidyltransferase. The extended cDNAs were then amplified by PCR using a nested gene-specific primer and a poly(dG-dI) primer which had linked to it several restriction sites (Life Technologies, Inc.), and the amplified products were cloned into the pCRII vector. DNA sequencing of several representative clones from the two PCR reactions indicated that both classes of mRNAs used the same set of multiple transcriptional start sites (Fig. 7). In addition, the major start site was identical for both classes of transcripts. We note that the sequence surrounding this major start site has similarity to two initiator (Inr) elements, that of terminal deoxynucleotidyltransferase (24) and p5(25) . In all three cases, transcription intiation occurs at an A, but the major difference in our case is the presence of 2 G residues at positions -1 and -2. In the previously identified consensus sequence for an Inr element, these two positions are usually occupied by pyrimidines.


Figure 7: Nucleotide sequence of exon 1 of GARS-AIRS-GART and adjacent genomic sequence. The sequence of this exon was obtained by double stranded sequencing of subcloned restriction fragment, 1BB3.0 (see Fig. 2B). The start sites identified by 5`-RACE for the monofunctional and trifunctional transcripts () and by primer extension analysis (bullet) are noted. The potential GC boxes are underlined. The prevalent start site identified by 5`-RACE was used to denote nucleotide position +1 for numbering of the exons (upper case) with the promoter region and a portion of intron 1 shown (lower case letters).



An Enzyme Site Absent from an Overlap Region on One Clone Represented a Restriction Fragment Length Polymorphism (RFLP)

The genomic clones LC6 and LC5 had 6 kb of overlap which fit an overlapping map constructed using three endonucleases. However, there was one HindIII site present in LC6 that was not found in LC5 (see Fig. 2B). Because the library used for the genomic screen was generated from a hybrid C57 BL6 times CBA mouse, it was likely that this HindIII site represented allelic variation. Southern analysis was performed using HindIII-digested genomic DNA from the two parental mouse strains and the F1 hybrid. The HindIII site in LC6 was present in the parental CBA mouse and absent in the C57 BL6 mouse (Fig. 8). Thus, the genomic clone LC6 represents the allele from the CBA mouse, and clone LC5 represents that from the C57 BL6 mouse.


Figure 8: Southern analysis of the HindIII RFLP. Mouse genomic DNA (10 µg) from CBA, C57 BL6, and the F1 hybrid were restricted with HindIII. The DNA was separated on a 1% agarose gel, blotted, and probed with the cDNAs pYTF1.0 and pYTF0.7 (see Fig. 2A). The additional HindIII site which resulted in restriction fragments of 3.9 and 2.2 kb is seen in both the CBA strain and the F1 hybrid, but not in the C57 BL6 mouse strain.



The exonic sequence for this gene differed from the sequence of the cDNA that we previously published (3) by several single nucleotide changes. Almost all of these polymorphisms were in the third nucleotide of the codon and did not result in an amino acid change. One of these polymorphisms (an A in the cDNA to a G transition in the genomic DNA sequence at the second nucleotide of the codon) resulted in Gly instead of the previously reported Asp present in the mouse cDNA. All of the polymorphisms identified are noted in Genbank.


DISCUSSION

We show that there is only one mouse genomic locus, spanning 28 kb, responsible for the generation of two related mRNAs, a monofunctional GARS mRNA and a trifunctional GARS-AIRS-GART. The sequence and relative location of all 22 exons of this gene and the intron sequence adjacent to these exons was determined. The first 11 exons of this gene encode the GARS domain and are used by both mRNAs. Exons 12 to 17 contain sequence for the AIRS domain of the trifunctional mRNA, exon 18 contains sequence for the end of the AIRS domain and the beginning of the GART domain, and exons 19 to 22 comprise the rest of the GART domain and the 3`-UTR of the trifunctional transcript. The mechanism used to generate the mature GARS and GARS-AIRS-GART transcripts does not involve alternative exon usage. Instead, both mature transcripts use the same terminal GARS exon (exon 11), with splicing to the next downstream exon (exon 12) generating the trifunctional GARS-AIRS-GART transcript, while 3` end formation and cleavage-polyadenylation within intron 11 of this gene generates the monofunctional transcript (Fig. 5A). The sequence of both the 3`-UTR of the monofunctional GARS cDNA and the corresponding location within intron 11 indicated the presence of multiple polyadenylation signals (four AATAAAs and one ATTAAA); at least three of the five can be recognized and used for polyadenylation. In addition, both transcripts use the same set of multiple transcriptional start sites, and, hence, the generation of the two classes of transcripts is unlikely to be regulated by different promoter elements in mouse L1210 cells.

Whereas both transcripts use the same first 10 exons, removal of intron 11 by splicing competes directly with the recognition of the polyadenylation signals in intron 11. The ratio of the trifunctional to monofunctional mRNAs indicated that splicing predominated over polyadenylation in this alternative processing in spite of some substantial differences in the level of total expression between, for instance, L1210 cells and mouse liver (Fig. 1). This ratio also did not change appreciably, even when the levels of the trifunctional mRNA were down-regulated by contact inhibition of the growth of 10 T1/2 mouse embryo fibroblasts (data not shown). In all mouse tissues examined to date, the smaller message was always found. Hence, it appears that the GARS and the GARS-AIRS-GART mRNAs are coordinately expressed regardless of the level of transcription of this gene, at least in the adult tissues and cell lines we have examined.

A few other cases are known in which an intronic polyadenylation signal is used without alternative exon usage: the immunoglobulin heavy chain genes(26) , the thyroid hormone receptor alpha (c-erb-A-1)(27) , beta spectrin(28) , and the 2`-5`-oligo(A) synthetase gene(29, 30) . In these cases, the production of the different forms of mRNA by controlled use of an intronic polyadenylation site constitutes a mechanism of tissue-specific or developmentally regulated gene expression. In the well-characterized immunoglobulin µ heavy chain gene, the recognition and use of the intronic polyadenylation signal is developmentally regulated as a part of the process of B cell maturation(31) . Thus, in plasma cells, the prevalent form is the secreted (µ(s)) form produced by recognition of the polyadenylation signal in intron 4, and the membrane form (µ(m)), predominant in B cells, is generated by splicing out intron 4 and use of the polyadenylation signal at the end of exon 6. Several studies on regulation of µ(s) and µ(m) forms of immunoglobulin genes indicate that developmentally regulated trans-acting factors are involved (31, 32, 33) . The similarity of this aspect of alternative processing of the immunoglobulin heavy chain gene to that of the GART locus is striking, yet, so far, we do not have evidence for the modulation of the intronic polyadenylation mechanism as a control feature to regulate the two transcripts from the GART gene.

An analysis of the common features between the immunoglobulin heavy chain and the GART gene might permit insight into the underlying mechanism responsible for recognition of intronic polyadenylation sites. In previous studies on the immunoglobulin µ heavy chain gene, it was found that the sequence at the 5` splice donor site immediately preceding the site of intronic polyadenylation differed from the consensus sequence usually found at this position in that the nucleotide found at the +5 position of the intron was an A. In an extensive compilation of sequence homology around splice sites(16) , the nucleotide normally found at this position is a G, which is present in 87% of cases; A is present at this position in only 7% of analyzed genes. The centrality of this A to the mechanism of intronic polyadenylation is further suggested by its conservation in six surveyed mammalian and vertebrate species(31) . When this A was changed to a G in the mouse heavy chain gene, splicing predominated over intronic polyadenylation, even in cells that would have otherwise preferentially used the intronic polyadenylation signal(31) . The nucleotide used at this position of the 5` splice site of the 11th intron in the mouse GART gene is also an A (Fig. 6), suggesting that the immunoglobulin heavy chain and the GART genes have common steps in the mechanism used to generate a transcript by intronic polyadenylation.

Both a monofunctional GARS mRNA and a trifunctional message occur in chicken, human, D. melanogaster, and D. pseudoobscura (Fig. 1, Refs. 4, 9, and 34). The Drosophila GART locus has been cloned and sequenced in its entirety. Even though the Drosophila GART locus contains 7 exons compared to the 22 exons comprising the mouse locus, the position of three of the introns found in the Drosophila GART gene are exactly conserved in the mouse GART gene. Thus, introns 2, 3, and 5 in the Drosophila GART locus correspond to the position of introns 4, 10, and 14 in the mouse gene, respectively (Fig. 9). Intron 2 in Drosophila and intron 4 in the mouse gene interrupt the codon for a homologous serine (amino acid 142 in Drosophila and 149 in the mouse protein) both between the second and third nucleotides of the codon. Introns 3 and 5 in the Drosophila gene and introns 10 and 14 in the mouse GART locus interrupt the codons for homologous glycines; both sets of homologous introns again interrupt the codon between the second and third nucleotides. Hence, the position of these ancestral introns has been precisely conserved during the substantial divergent evolution between these two species. In addition, the intron involved in generation of the monofunctional GARS (Drosophila intron 4 and mouse intron 11) was conserved and interrupted the codon between the second and third nucleotides, occurring before the first AIRS coding region in both cases. However, the amino acid that is interrupted has diverged (arginine in mouse and isoleucine in Drosophila).


Figure 9: Conservation of the positioning of three introns between Drosophila and mouse GART loci. The sequence of the exons (upper case letters) surrounding the intron position (lower case letters) are shown. The amino acid interrupted by the intron is conserved between the two species. Note that the position of interruption within the codon is also the same between the two species.



In both Drosophila and mouse, the monofunctional GARS message contains an in-frame stop codon consisting of the TAA present as part of the 5` splice donor site. Thus, there are no additional amino acids which are added to the monofunctional GARS protein relative to the GARS domain present in the trifunctional GARS-AIRS-GART protein in both species. We have previously presented evidence that the monofunctional mouse GARS mRNA is translated and that this protein has GARS catalytic activity. The role of this monofunctional GARS protein is unknown, and its presence in mouse tissues is somewhat surprising in view of the development of a fused trifunctional GARS-AIRS-GART during evolution, presumably as a result of a selective advantage lent by substrate channeling or some related effect. It would seem that, if the monofunctional GARS did not have a function and was merely an evolutionary remnant, the intronic sequences causative of the origin of the monofunctional GARS mRNA would have been lost. The opposite is the case: the mouse 3`-UTR for the monofunctional message contains five polyadenylation signals, of which at least three are used, whereas the Drosophila GART gene contains only one polyadenylation signal in the intron immediately downstream from the GARS domains. Hence, not only was the Drosophila polyadenylation signal maintained during evolution, but these additional features appeared in the mouse gene, apparently to ensure the generation of a monofunctional mRNA and a catalytically active GARS protein. At the moment, we conclude only that the monofunctional GARS has an essential function which cannot be fulfilled by the GARS domain of the trifunctional protein.

We conclude that several aspects of the GARS-AIRS-GART gene would define it as a typical housekeeping gene, in particular, a ubiquitous expression pattern, and the patterns of promoter elements and multiple start sites found. However, the polyfunctionality of one of the encoded proteins, the careful conservation of this function during evolution, the use of multiple intronic polyadenylation sites as a means of generating an alternate transcript, and the coordinate up-regulation of both sets of transcripts in dividing cells all suggest it to be an intriguing example of higher order control of gene expression. This gene could serve as a valuable model system for the study of the mechanism of competition between splicing and cleavage/polyadenylation in an endogenous transcript in mammalian cells and aid in identification of protein factors required for these two processes. Unlike the immunoglobulin heavy chain gene, the mouse GARS-AIRS-GART locus does not have the added complications of complex gene rearrangements.

Note Added in Proof-A polymorphism was found in half of the 5`-RACE clones in which an additional CAG was present in these cDNAs at the position equivalent to the end of exon 1, resulting in a trinucleotide repeat (not shown in Fig. 7).


FOOTNOTES

*
This work was supported by Grant CA 27605 from the National Institutes of Health, DHHS, and was performed in part while the authors were affiliated with the Department of Biochemistry and Molecular Biology of the University of Southern California, Los Angeles, CA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U18418[GenBank].

§
To whom correspondence and reprint requests should be addressed: Massey Cancer Center, Medical College of Virginia, MCV Box 980230, Richmond, VA 23298. Tel.: 804-828-9645; Fax: 804-828-8079; rmoran{at}gems.vcu.edu.

(^1)
The abbreviations used are: GARS, glycinamide ribonucleotide synthetase; AIRS, aminoimidazole ribonucleotide synthetase; GART, glycinamide ribonucleotide formyltransferase; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RFLP, restriction fragment length polymorphism; UTR, untranslated region; kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Drs. Deborah Lebman, Peter Melera, and Shirley Taylor for their critiques of this manuscript. We also are grateful for the continuous help and advice of Drs. Taylor and Eric Westin during this work and for the excellent technical assistance of Mehrdad Jannatipour.


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