THE Joumw OF BIOL.OGICA~ CHEMWW @ 1990 by The American Society for Biochemistry and MolecuIar Biology, Inc.

Vol. 265, No. 32, Issue of November 15, pp. 19716-19720, 1990 Printed in US. A.

Analysis of a Splice Acceptor Site Mutation Which Produces Multiple Splicing Abnormalities in the Human Argininosuccinate Synthetase Locus*

(Received for publication, April 4, 1990)

Tsung-Sheng Su$$!l~~ and Ling-Huang Lin$ From the .$Department of Medical Research, Veterans General Hospital, Graduate Znstitutes of §Microbiology & Zmmunology and of lfGenetics, National Yang-Ming Medical College, Taipei, Taiwun, Republic of China

The cloned argininosuccinate synthetase gene from a citrullinemia patient’s fibroblast cell line revealed a single base substitution (G to C) within the splice ac- ceptor site of the last intron. The mutation abolished normal RNA splicing, and, by cDNA analysis, three abnormal splicing pathways were demonstrated. The major pathway involved the activation of a cryptic acceptor site in the last exon that resulted in a deletion of seven nucleotides in the mature RNA. Another path- way involved a downstream cryptic acceptor site, that is 388 nucleotides downstream from the first cryptic site. Northern blot analysis showed that this second cryptic site is present on the minor 2.7-kilobase mRNA, but not on the major species of argininosuccin- ate synthetase mRNA, which is 1.7-kilobases in length. Using this aberrant cDNA as a probe, the cDNA of the 2.7-kilobase mRNA was isolated and studied. Sequence analysis suggests that this species of RNA is the one that bypasses the polyadenylation signal employed by the 1.7-kilobase RNA. Since both transcripts encoun- ter the same translation termination codon, both RNAs should encode identical protein. Furthermore, a tract of 22 repeats of d(CA)*(GT) is found at the 3’ end of the gene and this repeat sequence is present on the 2.7- kilobase RNA. The third pathway of the abnormal splicing revealed a rare class of transcript that has the last intron retained in the mature RNA. This study shows that in human the intron inclusion can occur through a naturally occurring point mutation. All these abnormally spliced RNAs resulted in a protein reading frame shift.

Argininosuccinate synthetase catalyzes the conversion of citrulline and aspartate to argininosuccinate (1). The enzyme is present in all tissues and cultured cells studied, but the highest enzyme activity is in the liver where the enzyme functions in the urea cycle to eliminate ammonia (1). Analysis of genomic DNA clones indicates the presence of multiple- processed dispersed pseudogenes and of a single large ex- pressed gene for argininosuccinate synthetase (1, 2). The

* This work was supported in part by grant NSC78-0412-BO75-11 from the National Science Council and a grant from the Veterans General Hospital, Taipei, Taiwan, Republic of China. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “udver- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addresse& Dept. of Medical Research, Veterans General Hospital, Taipei, Taiwan 11217.

expressed gene spans 63 kilobase pairs (kb)l and is composed of at least 16 exons (1).

Deficiency of this enzyme results in an autosomal recessive disorder, citrullinemia (3). Patients with this disorder exhibit profound elevations of ammonia and citrulline in the blood. The majority of such patients present in the neonatal period with hyperammonemic coma. The molecular basis of disease has been studied and shown to be heterogenous (1, 4). Sl nuclease analysis of patients’ RNA suggests one class of mutation producing RNA with Sl nuclease-detectable defects (1, 4). To elucidate the molecular nature of these mutations, a study was undertaken of an argininosuccinate synthetase mutation in a fibroblast cell line of a citrullinemia patient, CG; the mutation was known to be homozygous for an Sl nuclease-detectable defect (4).

EXPERIMENTAL PROCEDURES

Cell Line-Fibroblast cell line CG was established from citrullines mia patient CG, who is the offspring of a first cousin mating (4). Fibroblast cell line AC was established from another citrullinemia patient (4). Cells were cultured in Dulbecco’s modified Eagle’s me- dium with 10% fetal calf serum.

Zsolation of DNA and RNA-High molecular weight DNA was Drenared bv the method of Blin and Stafford (5). The total RNA was Lx&acted iy the guanidinium/cesium chloridi ‘method (6). Poly(A+) RNA was purified by fractionation using oligodeoxythymidylate cel- lulose chromatography as described by Aviv and Leder (7).

Electrophoresis and Detection of RNA-Poly(A’) RNA isolated from culiured cell line was denatured with glyoxal and applied to a 1.2% azarose gel for electronhoresis (8). The RNA was transferred to nitrocejlulose-paper and hibridized with nick-translated ‘*P-labeled DNA probes under the conditions described (9). The blots were washed in 15 mM NaCl, 1.5 mM sodium citrate plus 0.1% sodium dodecyl sulfate at 50-55 OC and exposed to Fuji x-ray film at -70 ‘C using a Kyokko intensifying screen.

Genomic DNA Cloning of Both Normal and Patient’s Argininosuc- cinate Synth&ase ,!,ocu.-High molecular weight DNAs isolated from a normal individual and from a citrullinemia patient CG’s fibroblast line were partially digested with restriction inzyme MboI to obtain 15-20 kb (9). The sucrose gradient purified DNA was then cloned into the BumHI site of AEMBL3 vector. The clones with arginino- succinate synthetase sequence were isolated by plaque hybridization (9) to a unique sequence probe within intron 14 of the gene. The subgenomic fragments were cloned into pBluescript II vector (Stra- tagene, La Jolla, CA) for DNA sequence analysis.

Zsolation of Argininosuccinate Synthetase cDNA from Citrullinemia Fibroblast Cell Z,ine CG-Poly(A+) RNA isolated from citrullinemia fibroblast CG was used for cDNA library construction with vector Qtll as described by Huynh et ul. (10). The cDNA library was screened bv nlaaue hvbridization using “P-labeled nick-translated wild type c%A,-pASi, as a probe (10, il). The cDNA insert excised from vector &$ll by digestion with restriction enzyme EcoRl was subcloned into pBluescript II vector. The resulting clones were des- ignated as pCGc1, pCGc5, and pCGcl3.

’ The abbreviation used is: kb, kilobase pair(s).

19716

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A Splice Acceptor Site Mutation in Human Argininosuccinate Synthetase

DNA Sequence Analysis-The single-stranded DNA prepared from the pB1uescript II clone was sequenced by the dideoxy chain termi- nation method (12).

Sl NucLease Mapping-The appropriate restriction fragment from the argininosuccinate synthetase gene was labeled at the 5’ end with T4 polynucleotide kinase (13). Sl nuclease analysis was performed with poly(A+) RNA isolated from fibroblast lines (14). Also included in Sl analysis were RNAs transcribed in uitro from the mutant cDNA templates pCGc1, pCGc5, pCGcl3, and wild type cDNA template pbAS-1 which is the pAS1 cDNA insert cloned into pB1uescript II vector (15). The protected products were displayed in 8 M urea- polyacrylamide gel.

RESULTS

Comparison of Nucleotide Sequences of Argininosuccinate Synthetase Gene Between Normal and Mutant Loci-Previous study suggested that citrullinemia patient CG has an Sl nuclease detectable abnormality toward the 3’ end of the RNA (4). Therefore, effort has focused on the study of the 3’ end of the mutant gene by screening the CG genomic library with a unique sequence probe inside intron 14 of the gene. The use of the intron probe also eliminated the isolation of processed pseudogenes (1, 2). By sequencing the intron-exon junctions of the isolated genomic clones, a single base mis- match from G- to C- was found to occur at the splice acceptor site of intron 15, the last intron of this gene. (Fig. 1). The conserved sequence of the 3’ splice site was changed from AG to AC.

Analysis of cDNA, pCGc5 from Citrullinemia CG-To study the effect of the splice acceptor site mutation on RNA proc- essing, the cDNAs of argininosuccinate synthetase from pa- tient CG were analyzed. The partial restriction mapping of cDNA clones pCGc1, pCGc5, and pCGcl3 suggested that they were reversely transcribed from different species of arginino- succinate synthetase RNAs. To study their identities, DNA sequences of these cDNAs were determined. Fig. 2 compares the sequence of wild type cDNA, pAS1, and mutant cDNA pCGc5. The sequence was identical until the end of exon 15. After that, there was a deletion of seven nucleotides at the beginning of exon 16 in pCGc5 as compared with the wild type cDNA. The 3’ end of the deleted sequence was the AG dinucleotide, the same sequence as in the normal splice ac- ceptor site. Apparently a cryptic splice site, seven nucleotides away from the authentic site, was activated, resulting in a deletion of seven nucleotides on mutant RNA.

Analysis of Argininosuccinate Synthetase cDNA pCGc1 from Citrullinemia CG-When cDNA, designated as “pCGc1” was analyzed, the length was found to be about 3 kb long. This is longer than the size of the mature RNA (11). However, when its sequence was analyzed, it was clear that this cDNA re-

Normal mutant

AQ G AJCG

FIG. 1. DNA sequence in the vicinity of intron 15 (115) and exon 16 (E16) junction. A comparison of the normal and mutant sequence illustrated a single base G- to C- substitution at the 3’ end of intron 15.

Normal CG c5

FIG. 2. A, splicing pattern of normal and mutant. RNA CCGcc5). Box represents exon, thin line represents intron. The highly conserved sequences GU--AG at the 5’ and 3’ ends of intron, respectively, are indicated. The mutation G- to C- in the highly conserved AG sequence is marked. B, comparison of normal cDNA (pAS1) and mutant cDNA (pCGc5) sequence at the exons 15 and 16 junction. The nucleotides missing in pCGc5 are dotted.

s-

FIG. 3. A, splicing pattern of mutant RNA (CGcl). The mutation G- to C- in the highly conserved AG sequence is marked. The thick line representing intron remains unspliced. B, a comparison of the normal genomic DNA (& panel), mutant genomic DNA (rnicldle punel), and mutant cDNA, CGcl (rightpcmel) sequence in the vicinity of the exon 16. The mutation G- to C- in the highly conserved AG sequence is circled.

tained the mutated splice acceptor site and intron 15 sequence (Fig. 3). Besides, the 5’ end of intron 15 which includes the splice donor site was also present on this cDNA (data not shown). Thus, it became clear that the large size of this cDNA was due to the inclusion of intron 15 in the mature RNA (Fig. 3). To examine the percentage of the RNA that retains this intron, an Sl nuclease mapping analysis was carried out. The probe was a 5’ end-labeled genomic DNA fragment that covered part of intron 14 to exon 16 (Fig. 4). The size of the protected fragment in this analysis can be predicted from the cDNA sequence and was demonstrated in Fig. 4. Therefore, if wild type RNA (pbAS1 as template for in uitro RNA synthesis) was used in the Sl analysis, the protected fragment would be 222 nucleotides in length (Fig. 4, lane 4). In mutant RNA, if the splicing pattern is as in pCGc5, the protected fragment would be 215 nucleotides (Fig. 4, lane 5), and if the entire intron 15 remained on RNA as in pCGc1, the protected fragment will be about 1800 nucleotides in length (Fig. 4, lane 6). When poly(A+) RNA from a citrullinemia cell line AC (1), that is known to be normal in this region of the argininosuc- cinate synthetase gene was employed as a control in Sl nuclease analysis, a fragment of 222 nucleotides in length was protected as would be expected (Fig. 4, lane 1). However, when

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1971s A Splice Acwptor Site Mutation in Human Argininosuccinate Synthetase

Ml 234567M , , , , , 1 ,

“t

1800- *%* -' --,353 - - ,078

- 872

. - - 603

222--m=> @ -- 234 215- V SI

FI(;. 4. Sl nuclease mapping of argininosuccinate synthe- tase transcripts. A, the relationship of a 5’ end-labeled (Toy1 site) prohe to a portion of transcription units is schematically indicated, along \vith the expected nucleotides (~1 of the fragments protected hy RXA generated from the respective cDNA templates. .S~or.s indi- cate “‘I’ -S-end labels; t/rick line at one end of the labeled probe indicates vector sequences that distinguish a reannealed probe from a probe that is protected by hybridization to RNA. The drawing is not to scale. B, polytA+) RNAs prepared from control human fibro- blast AC (/atic 1). citrullinemia fihroblast CC (lane 2), pbAS1 tem- plate t/anr 4, pCGc5 template ~/CUE 51, and pCGc1 template (LWW f3) Lvere hybridized to a 5’ end-labeled genomic probe, subjected to Sl nuclease digestion, and the products were electrophoresed through a 4’; polyacrylamide gel containing 8 M urea. As controls, tRNA was substituted for experimental RNAs tlanc 31, whereas Lane 7 was with probe only. DNA marker tA4) is the .5’ end-laheled Hue111 digested q~x1Y-t DXA in nucleotides.

RNA from citrullinemia patient CG was used, a major frag- ment, 215 nucleotides, and a minor one, 1800 nucleotides, were detected (Fig. 4, lane 2). By densitometer scanning, the ratio of these two fragments is about 50 to 1. Clearly, most of the mutant RNA was processed to a cryptic acceptor site on exon 16 as in pCGc5, whereas a small percentage of RNA was processed with intron 15 unspliced. In this study, a 222- nucleotide fragment presumed to be the protected fragment by the wild type RNA was also detected in lane 2 where RNA was from citrullinemia CG. This was due to incomplete diges- tion by Sl nuclease, since this fragment was also detected in lane 5 where the in uitro synthesized RNA from pCGc5 template was employed. No other extra protected fragment was observed when comparing the pattern by control RNA (lane 1) and by mutant RNA (lane Z), suggesting there was no detectable cryptic acceptor site inside intron 15.

Analysis of cDNA pCGcl3 from Citrullinemia CG and of the 2.7.kb RNA from Argininosuccinate Synthetase Locus-DNA sequence analysis of cDNA, pCGcl3, showed that the se- quence 3’ from the exon 15 is different from the sequence in pCGc1 and pCGc5 and does not correspond to any of the known cDNA sequence. From the result of Sl nuclease analy-

sis in Fig. 4, it was known that there is no cryptic acceptor site inside intron 15. Therefore, the sequence at the 3’ end of the cDNA of pCGcl3 could not have come from the intron 15 sequence. Sequence comparison between wild type cDNA and genomic DNA of argininosuccinate synthetase indicates that exon 16 is the last, exon in mature RNA. However, previous study has shown that there are two species of argininosuccin- ate synthetase RNA (11): a major one, about 1.7 kb in size, and a minor one, about 2.7 kb in size (Ref. 11 and Fig. 5A). The ratio of these two species of RNA is about 50 to 1 (11). The exact relationship of these two species of RNA is not known, but preliminary results suggest that the size difference is at the 3’ end of the RNA. Therefore the possibility was explored that RNA of pCGcl3 was produced by aberrant splicing to a cryptic site, the sequence of which is only present on the 2.7-kb mRNA. A DNA fragment at the 3’ end of the pCGcl3 that differed from the wild type cDNA sequence was used as a probe for Northern blot analysis with RNA from a human hepatoma cell line and two citrullinemia cell lines AC and CG (Fig. 5). When using pAS1 cDNA as a probe (Fig. 5A), both 2.7- and 1.7-kb transcripts from a human hepatoma cell line were hybridized as expected (Fig. 5A, lane I), whereas a much weaker signal was observed in citrullinemia cell lines (Fig. 5A, lanes 2 and 3). With Northern blot analysis using a probe that was derived from the 3’ end of the pCGcl3, it is clear that this probe hybridized preferentially to the 2.7-kb RNA (Fig. 5B, lane I), suggesting that the 3’ end of pCGcl3 was from this species of RNA. Thus, in RNA of pCGcl3, the 3’ end of the exon 15 was joined to a sequence that was only present in the 2.7.kb RNA. The hybridization signal is much weaker in RNAs from citrullinemia cell lines than that from hepatoma (Fig. 5). This is because liver is known to express much higher argininosuccinate synthetase mRNA than any other organ studied (1). The citrullinemia cell line AC used in this study is known to involve an RNA negative allele (1). The finding that the hybridization signal in CG RNA (Fig. 5A, lane 3) is about twice of that in AC RNA (Fig. 5A, lane 2) suggests that the splicing defect in the CG cell line does not significantly affect its RNA stability. It is of interest to note that although the major species of 1.7.kb RNA is about the same size in both hepatoma and citrullinemia CG (Fig. 5A, lanes 1 and 3), the minor species of RNA in CG is about 2.3 kb in size which is smaller than the 2.7.kb RNA detected in human hepatoma (Fig. 5, lanes I and 3).

To elucidate the relationship between 2.7. and 1.7-kb RNAs that are both transcribed from the argininosuccinate synthe- tase locus, the cDNA specific to 2.7-kb RNA was isolated

A. 0. 1 2 3 1 2 3 I I , I 1 ,

28% 23s -

FIG. 5. Northern blot analysis of RNA from a hepatoma cell line (kzne I), a human fibroblast line AC (Zune 2), and a fibroblast line CG (kzne 3) with pAS1 cDNA (A) and 3’ region of pCGcl3 cDNA (B) as probe. The RNA filter was first hybridized to pAS1 probe then rehytxidized to El’ pCGcl3 probe after stripping off the first probe by washing the filter in ~50% formamide, 10 mM Tris-HCl, pH 75, at 65 ‘C for 1 h.

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A Splice Acceptor Site Mutation in Human Argininosuccinate Synthetase

from a human hepatoma cDNA library using the 3’ DNA fragment of pCGcl3 as a probe. The cDNA obtained is des- ignated as “PAS-L.” Sequence comparison of pAS1 and PAS- L, which were cDNA from 1.7- and 2.7-kb RNA, respectively, showed that the entire 3’ end sequence of pAS1 is within the sequence of PAS-L (data not shown). This result suggests that the 2.7-kb RNA is a transcript that bypasses the poly- adenylation signal employed by the 1.7-kb RNA. The PAS-L, the cDNA of 2.7-kb RNA, has its 3’ end extended about 800 nucleotides from the 3’ end of pAS1 clone. No open reading frame longer than 50 amino acids was found within this 800- nucleotide region. Within this region, the restriction enzyme mapping and DNA sequence analysis showed that the se- quence is colinear to that in genomic DNA (data not shown). The clone, PAS-L, is about 200 nucleotides short at 3’ end. Whether any RNA splicing involved in this region is un- known. DNA sequence analysis of PAS-L showed that there is a tract of poly(GT) at the 3’untranslated region of this 2.7-kb RNA (Fig. 6). This sequence is located at 23 nucleotides downstream from the poly(A) addition site of the 1.7-kb RNA (Fig. 6). The number of GT unit in this cDNA is 22. However, length variation was found when genomic clone from another individual was sequenced which showed 19 tandem repeats of the GT unit (data not shown).

In order to understand the defect in pCGcl3, both pCGcl3 and PAS-L DNAs were sequenced and compared. In pCGcl3, there is a deletion of 395 nucleotides beginning at the exon 16 and ending at an AG dinucleotide (Fig. 7). Apparently, this AG dinucleotide served as a splicing acceptor site. The result suggests that the production of the aberrant transcript of pCGcl3 was due to the activation of a downstream cryptic acceptor site. The deleted region includes both translation termination codon and the major polyadenylation signal. If the RNA is translated, the mutant protein will replace the C- terminal 15 amino acids of the wild type protein with a 55 amino acid peptide of different sequence.

DISCUSSION

This study demonstrates that a G- to C- substitution in splice acceptor site (AG to AC) of intron 15 of argininosuccin- ate synthetase is responsible for the deficiency of this enzyme in citrullinemia patient CG. The mutation leads to the pro- duction of aberrantly spliced RNA. By cDNA analysis, at least three abnormal splicing events can occur because of this mutation. This resulted in reading frame-shifting at the C- terminal end of argininosuccinate synthetase. Although the mutation only affects the last 15 amino acids of this 45- kilodalton protein, the resulting protein not only has lost its enzyme activity but also has no cross-reaction material being

A. A7CG

-7 ,

FIG. 6. A, DNA sequence in the vicinity of the poly(d(CA) .d(GT)) tract. The d(CA) dinucleotide was repeated 22 times. B, DNA se- quence at the 3’ end of the argininosuccinate synthetase gene. The poly(dGT) region is underlined. The polyadenylation signal for 1.7- kb RNA is &Led, and the poly(A) addition site is marked with an arrow.

Normal

CG cl3

L,Ai h&AA& Normal cci cl3

A7 GCi ATCG -

FIG. 7. A, splicing pattern of mutant RNA (CGcl3). The mutation G- to C- in the highly conserved AG sequence is marked. The UAG and AUAAAA are translation termination codon and polyadenylation signal, respectively. B, comparison of the normal cDNA (PAS-L) and mutant cDNA (pCGcl3) sequence in the vicinity of cryptic acceptor site. The dinucleotide AG that served as a cryptic acceptor site in RNA of CGc13 is ~o&cf.

A.

rnllNt3 l.,-kb

Y--X B.

-.,~A~ fUAAAA

++-&,-+A$ ,;,b- i-J+

=..zj *’ d’

FIG. 8. A, snhcing nattern of normal human argininosuccinate synthetase transcripts. The box represents exon; the ~/WI Line repre- sents intron. Because the sequence at the 3’ end of the 2.7-kb RNA is not available, it is not certain whether the end is colinear to that in the primary transcript and is represented by the box with a &Led Line. The UAG and AUAAAA are translation termination codon and polyadenylation signal, respectively. The drawing is not to scale. & splicing patterns of three aberrant argininosuccinate synthetase tran- scripts in citrullinemia CG. The mutation G-to C- in highly conserved AG sequence is marked. The thick Line representing intron remains unspliced.

detected (1). Apparently the C-terminal sequence of this protein is important for maintaining its stability.

By Sl nuclease mapping and cDNA analysis (pCGc5), the major argininosuccinate synthetase RNA in this cell line is the one with a seven-nucleotide deletion in exon 16. The deletion is the result of aberrant splicing that has joined exon 15 to a cryptic acceptor site, seven nucleotides downstream from the authentic splice site. If this RNA is translated, the mutant protein will replace the C-terminal 14 amino acids with a 39-amino acid peptide of different sequence.

By cDNA analysis, it was shown that in addition to this site, another downstream cryptic acceptor site on the last exon can also be activated. The resulting RNA joined exon 15 to a sequence that only presents in a 2.7-kb minor species of argininosuccinate synthetase RNA (pCGcl3). Sequence comparison of cDNAs and genomic DNA suggested that this 2.7-kb RNA is the one that bypasses the polyadenylation signal, ATAAAA (Ref. 16, Fig. 8). The inefficiency of this signal in polyadenylation may have resulted from its sequence variation from the consensus sequence, AATAAA (17, 18). Nevertheless, both 2.7- and 1.7-kb RNA encounter the same

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19720 A Splice Acceptor Site Mutation in Human Argininosuccinate Synthetase

translation termination site; therefore, both transcripts should encode identical proteins. Furthermore, the 2.7-kb RNA is present in every cell type examined and the ratio of these two species of RNA is about constant, suggesting that this bypassing phenomenon is not regulated. Interestingly, a tract of poly(dCA.dGT) was found at the 3’ end of the gene. This sequence is located immediately downstream from the last exon of the 1.7-kb RNA and this sequence is transcribed and present on the mature 2.7-kb RNA. The poly(dCA. GT) family has been reported to locate at the 5’ or 3’ end of several genes (19). However, in those instances, this repeat sequence is located several kilobases from the stably transcribed portion of the genes. In this study, length polymorphism of the repeat unit was found as has been reported (20). However, the length variation alone is not large enough to serve as a DNA poly- morphic marker (20). Poly(dCA . dGT) has a number of unique physico-chemical properties (21, 22) and has been suggested to have a regulatory function (19, 23). The role of this se- quence on argininosuccinate synthetase expression remains to be studied.

In this study, a third type of abnormal splicing event was also detected. DNA sequence analysis suggested that the intron 15 remained unspliced in this class of RNA. Although the entire intron in this cDNA has not been sequenced, the presence of both splice donor and acceptor sites of intron 15 in this cDNA suggests that no small splicing has occurred in this intron. The result of Sl nuclease analysis further showed that this RNA is a minor species that is present in only about 2% of mature argininosuccinate synthetase RNA.

This study demonstrated that a naturally occurring splice site mutation in the last intron of argininosuccinate synthe- tase gene can result in multiple splicing abnormalities (Fig. 8B). Similarly, the study of the defects in ,&globin genes present in several forms of @-thalassemia has provided ex- amples of natural splicing mutants (24). Mutations in /3- thalassemia that are known to affect the 3’ splice site include one which is a small deletion involving the 3’ acceptor site and the upstream polypyrimidine stretch of the first intron; another is a single base mutation in the splice acceptor (AG to GG) at the second intron of P-globin gene (25, 26). The former mutant was shown to abolish splicing of that intron, whereas the latter resulted in activation of a cryptic acceptor site in the intron (25,26). The nature of the mutation studied here resembles the second mutation described in P-thalasse- mia in that both involved mutation in the 3’ splice consensus sequence. However, the mutation in the fi-globin gene resulted in the activation of a cryptic site upstream from the correct splice site, whereas in the argininosuccinate synthetase mu- tation studied here, at least three abnormally spliced path- ways can be demonstrated (Fig. 8B). At the 5’ end of both cryptic AG sequences, a conserved branch point region (UUAAU) and a pyrimidine-rich stretch could be identified although the cryptic site closer to the authentic site is much more efficient than the other. It has been suggested that splicing and polyadenylation machinery communicate across the terminal exon to direct the spliceosome assembly (27). Since exon 16 is the last exon in this gene and there are two polyadenylation signals for argininosuccinate synthetase RNA, the two cryptic sites may respond to the spliceosome assembly with respect to individual polyadenylation recogni- tion signals. This may explain why there are two cryptic sites in use and also their relative efficiencies. In addition, the RNA analysis showed that there were only 2.3- and 1.7-kb

RNAs detected in citrullinemia CG (Fig. 5). If the upstream cryptic site was also in use in processing the larger species of RNA in citrullinemia CG, then the 2.7-kb RNA should also be detected. The fact that only 2.3-kb RNA was detected in citrullinemia CG suggests that most if not all of the larger species of RNA was spliced to the downstream cryptic site. The result supports the hypothesis that machinery of splicing and polyadenylation communicates to each other at the last exon.

The study of RNA splicing reaction in vitro has provided important information on its mechanisms (28). It has been demonstrated that formation of the 3’ splice complex may be an essential step in the formation of the stable 5’ nucleopro- tein complex and that constructed mutation at the 3’ splice AG sequence reduced 5’ cleavage and abolishes splicing (29). However when tested in uiuo, the same mutations elicit effi- cient splicing at a cryptic, rather than the correct, 3’ splice site. Our study of acceptor site mutation demonstrated that indeed the 3’ site mutation does sometimes abolish the splic- ing reaction in ho. The mutation described here should provide examples to allow further elucidation of the mecha- nism of RNA splicing.

Acknowledgments-We thank Drs. A. L. Beaudet and W. E. O’Brien for providing the citrullinemia fibroblast cell lines CG and AC and intron 14 probe.

1.

2.

3.

10.

11.

12.

13. 14. 15.

16.

17. 15. 19.

20.

21. 22. 23.

24.

25.

26.

27.

25. 29.

REFERENCES

Beaudet, A. L., O’Brien, W. E., Bock, H.-G. O., Freytag, S. O., and Su, T.- S. (1956) Adu. Hum. Genet. 15, 161-196

Su, T. S., Nussbaum, R. L., Airhart, S., Ledbetter, D. H., Mohandas, T., O’Brien, W. E., and Beaudet, A. L. (1954) Am. J. Hum. Genet. 36,954- 964

Shih, V. E. (1975) in The Metabolic Basis of Inherited Disease (Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., eds) 4th Ed., pp. 362- 356, McGraw-Hill Book Co., New York

Su, T.-S., Beaudet, A. L., and O’Brien, W. E. (1953) Nature 301,533~534 Blin, N., and Stafford, D. W. (1976) Nucleic Acids Res. 3,2303-2305 Gli2s,ii V., Crkvenlakov, R., and Byus, C. (1974) Bwchemlstry 13, 2633-

Aviv, H., and Leder, P. (1972) Proc. Natl. Acad. Sci. CT, S. A. 69, 1405- 1412

Thomas, P. S. (1980) Proc. N&l. Acad. Sci. U. S. A. 77,5201-5205 Maniatis, T., Fritsch, E. F., and Sambrook, J. (1952) in Molecular Cloning:

A Laboratory Mom&, pp. 109-112, 252-291, Cold Spring Harbor Labo- ratory, Cold Spring Harbor, NY

Huynh, T. V., Young, R. A., and Davis, R. W. (1955) in DNA Cloning: A Practical Aooroach (Glover, G. M., ed) Vol. 1, pp. 49-78, IRL Press, Oxford .

Su. T.-S., Bock, H.-G. O., O’Brien, W. E., and Beaudet, A. L. (1981) J. BioL &em. 256,11826-11531

Sawer. F.. Nicklen. S.. and Coulson. A. R. (1977) Proc. Natl. Acad. Sci. U. S-A.’ 74,541 63-5;16f

Maxam, A. M., and Gilbert. W. (1950) Methods Enzvmol. 65.499-560 Enders, G. H., Ganem, D., and Varmus, H. (1955) f%) 42. o47-3o8 , --- _-, --. I-- Melton, D. A., Krieg, P. A., Rebagliatj, M. R., M aniatis, T., Zinn, K., and

Green, M. R. (19( 34)Nudeic AcidsRes. 12, 7035-7056 Bock? H.-G. O., Su, -. -., - -~~.~~, , T.-S.. O’Brien. W. E.. and Beaudet. A. L. (1953) N&&c ~

AczdsRes. 11,6505-6512 Fitzgerald, M., and Shenk, T. (1951) Cell 24, 251-260 Proudfoot, N. J., and Brownlee, G. G. (1974) Nature 252,359-362 Braaten, D. C., Thomas, J. R., Little, R. D., Dickson, K. R., Goldberg, I.,

Schlessineer. D.. Ciccodicola. A.. and D’Urso. M. (1958) Nucleic Acids Res. 16,565-881

Kashi, Y., Tikochinsky, Y., Genislav, E., Iraqi, F., Nave, A., Beckmann, J. S.. Gruenbaum. Y.. and Soller, M. (1990) Nucleic Acids Res. 18, 1129- 1132

Haniford, D. B., and Pulleyblank, D. E. (1953) Natwe 302, 632-634 Htun, H., and Dahlberg, J. E. (1989) Science 243, 1571-1576 Pardue, M. L., Lowenhaupt, K., Rich, A., and Nordheim, A. (1987) EMBO

J. 6,1781-1789 Treisman, R., Orkin, S., and Maniatis, T. (1983) in Globin Gene Expression

and Hemato huis, A., eds P

o&c Differentiation (Stamatoyannopoulos, G., and Nien- pp. 99-121. Alan R. Liss, Inc., New York

Orkin, S. H., Sexton, J. P., Goff, S. C., and Kazazian, H. H., Jr. (1983) J. BioL Chem. 258,7249-7251

Antonarakis, S. E., Irkin, S. H., Cheng, T., Scott, A. F., Sexton, J. P., Trusko, S. P., Charache, S., and Kazazian, H. H., Jr. (1954) Proc. N&L Acad. Sci. U. S. A. 81,1154-1155

Rol:l;on, B. L., Cote, G. J., and Berget, S. M. (1990) Mol. Cell. Biol. 10,

Aebi, M., and Weissmann, C. (1957) Trends Genet. 3,102-107 Aebi, M., Horning, H., Padgett, R. A., Reiser, J., and Weissmann, C. (1956)

Cell47,555-565

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T S Su and L H Linabnormalities in the human argininosuccinate synthetase locus.

Analysis of a splice acceptor site mutation which produces multiple splicing

1990, 265:19716-19720.J. Biol. Chem. 

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