deletion analysis of a unique 3'splice site indicates that alternating

Deletion analysis of a unique 3' splice site indicates that alternating guanine and thymineresidues represent an efficient splicing signal

C.Simon Shelley* and Francisco E.Baralle

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OXI 3RE,UK

Received February 3, 1987; Revised and Accepted April 6, 1987

ABSTRACT

The 3' splice site of the second intron (I2) of the human apolipoprotein-AII gene,(GT)16GGGCAG, is unique in that, although fully functional, a stretch of alternatingguanine and thymine residues replaces the polypyrimidine tract usually associated with3' splice junctions. The transient expression of successive 5' deletion mutants hasdefined the minimum number of nucleotides at the 3' end of apo-AII 12 that arerequired to direct efficient splicing. Processing in two cell-types, representing apo-All producing and non-producing tissue was identical; in both, only by removing all theGT repeats did the 3' splice site of apo-AII 12 become completely non-functional.Similar deletion analyses of "classic" 3' splice sites, which conform to the consensussequence (Y)nNYAG, have indicated that a minimum of 14 nucleotides of thepolypyrimidine tract are required for detectable levels of processing to take place.Here we report that the six nucleotides (GT)2GG, which directly replace this tract in adeletion mutant of the 3' splice site of apo-AII 12 are sufficient to direct the splicingprocess efficiently and correctly.

INTRODUCTION

Analysis of the exon-intron boundaries has revealed that introns invariably beginwith the 5' nucleotides GT and end with the 3'nucleotides AG [1]. The identification ofover 130 5' and 3' splice sites has extended the original "GT---AG" rule so theconsensus sequences are now, 5' splice site: 5'-C/AAG:GTAC/GAGT-3 and 3' splicesite: (Y)nNYAG:G [2]. After the initial cleavage of the intron at the 5' end, theguanosine residue in the 5' GT attacks an adenosine residue close to the 3' end of theintron to form a 2'-5' phosphodiester bond, called the branch site [3], therebyproducing a so-called lariat structure [4-7]. The adenosine nucleotide involved in thebranch site has been identified for several introns and has been found to be located inmammalian genes 18 to 37 nucleotides upstream from the AG dinucleotide ending theintron [6,8-9]. Deletion analysis of a number of higher eukaryotic introns [10-14] hasdemonstrated that most of the intron is dispensable without deleterious effect on RNAsplicing. Indeed, the smallest naturally occurring intron reported to date is only 38

nucleotides long [15]. In addition, these studies have found that only the first six

nucleotides at the 5' end and between 20 and 24 nucleotides at the 3' end are required

C I RL Press Limited, Oxford, England.

Nucleic Acids ResearchVolume 15 Number 9 1987

3787

Nucleic Acids Research

for efficient and accurate splicing of two different globin introns [10-11]. In all the

mammalian introns examined so far, the region proximal to the 3' splice site is rich in

pyrimidines, a conservation that implies an important role some of these nucleotides in

the splicing process [9-11,16-17]. The adenosine branch nucleotide is located within

the general consensus sequence 5'-YTRAY-3' [41,18] just upstream of the pyrimidine-

rich stretch of nucleotides. Limited information is available about the nucleotide

composition of this tract that is required for efficient and correct splicing.

A number of naturally occurring mutations have been reported which

demonstrate the importance of the 3' splice site consensus sequence to gene expression

in vivo. For example, a point mutation changing the AG dinucleotide to GG in the

second intron of the 8-globin gene abolishes splicing at the normal site. This is

replaced by processing at a cryptic 3' splice site and results in a low level of

aberrantly spliced transcripts [19].In addition to defects within the normal 3' splice site, mutations can also occur

in regions of the gene not normally involved in the splicing process. These can cause

an altered phenotype by mutation towards an important consensus sequence. Such a

point mutation in the first intron of the 0-globin gene creates a 3' splice site so close

to the 3' end of the intron that it causes V' thalassemia by interfering with splicing at

the normal 3' splice site [20-23].Although differing considerably from the consensus sequence [2] the 3' splice site

(GT)16GGGCAG of the second intron (12) of the human apolipoprotein-AII (apo-AII)

gene directs accurate and efficient splicing of both hepatic and intestinal apo-AIIprimary transcripts [24-25]. It has previously been shown that the polypyrimidinetract of the "classic" intron 3' splice site sequence, (Y)nNYAG, is required for

cleavage at the 5' donor site and subsequent lariat structure formation during splicing[9,17]. The replacement of these polypyrimidines in apo-AII 12 suggests that this

function may be mimicked by the sequence: (GT)16GG. Consequently, in order to

assess the importance of this GT repeat to the splicing process, a series of expressionconstructs were generated in which it was successively deleted. This then allowed the

minimum number of nucleotides at the 3' end of apo-AII 12 that could act as a viable 3'

splice site to be defined and compared with that reported for "classic" 3' splice sites.

MATERIALS AND METHODS

General ProceduresPurification of DNA, ligation and labelling reactions, restriction enzyme

digestion, Bal3l digestion, gel electrophoresis and S1 nuclease analysis were performedaccording to established procedures as described by Maniatis et al. [31].Construction of Plasmids

See text and appropriate figure legends.

3788


Cell Growth

Hep-G2 [32-35] cells were grown at 37°C under 5% CO2 in MEM medium

supplemented with 10% FCS and 2 mM glutamine. DMEM medium supplemented with2 mM glutamine and 10% or 20% FCS was used to grow HeLa [36] cells. In continuous

culture, cells were harvested by trypsinization, seeded at a 1 : 5 dilution and fed with

fresh medium every day. All cell lines were grown in 80 cm2 tissue culture flasks

(Nunc) and 150 x 15 mm tissue culture dishes (Lux).

DNA TransfectionHep-G2 and HeLa cells were transfected with plasmid DNA by the calcium

phosphate co-precipitation method [37] with the following modification: 3 hrs before

transfection the cells were fed with fresh medium, 100 jLg of each plasmid was used to

transfect two 150 x 15 mm dishes of subconfluent cells. Transfection was for 4 hrs,

after which the medium was replaced.

Preparation of RNA

Total cellular RNA was prepared by lysis of cells in 5 M guanidinium isothio-

cyanate, 50 mM Tris-HCI, pH 7.6, 10 mM EDTA, 0.1 M $-mercaptoethanol [38]. RNA

was pelleted through a cushion of 5.7 M CsCl, 0.1 M EDTA in a Beckman SW50 rotor at

28 000 rpm and 200C for 24 hrs [39-40]. RNA pellets were resuspended in sterile

distilled water containing 10 mM ribonucleoside-vanadyl complex (Biolabs), ethanol

precipitated and stored as aqueous solutions at -200C.

RESULTS

In a hybrid intron, deletion of the (GT)16 repeat of apo-AII activates a nearby cryptic

3' splice site

The construction of the first series of (GT)16 deletion mutants is described in

Figure 1. In brief, thse involved the generation of a hybrid intron within the 3'

terminal exon of the al-globin gene present in pSVedaclW [27]. The 5' end of this

intron represented apo-AII 13 sequences, while the 3' end represented successive Bal3lgenerated 5' deletions of apo-AII 12. This series of constructs therefore differed only

in the number of nucleotides of the apo-AII 12 acceptor site they contained. The five

constructs produced, pSVed alW/AII/13-I2Awt, L 19, A 29, A 31 and 1 37, contained

respectively 47, 28, 18, 16 and 10 bp of apo-AII 12 immediately upstream of the 3'splice junction (Fig.1). These recombinants were transfected into HeLa cells and,after 48 hours, total RNA was extracted and analysed by Si nuclease protection.

Preliminary S1 nuclease analysis indicated that transcripts arising from the

different apo-AII 12 deletion constructs were processed to the same degree at the

donor site of apo-AII 13 (data not shown).To investigate the pattern of splicing at the 3' end of the deleted hybrid introns,

3789


(A) i/ SAM

ECO

| SVED-SwAV31 2

BST El

HINI

iii/ 13f 2 _ ; 20 bp

Pjl Dd.0EXON

EXON 3 EXON3

(B) *COCTAG 4C00_

IGT\rG_A 12 - 19

(C) 243 bp t 12 ACCEPTOR

Hin30 EXON 2) 290 Op(wt) CRYPTIC ACCEPTOR

778 boI.t) - PROBE

Figure 1. Construction of expression plasmids containing deletions of the 3' splice siteof apo-AII 12(A). Construction of pSVed alW/AII/I3-12 A wt-37(i) Schematic restriction map of the parent plasmid vector pSVed alW [271. Stripedand filled boxes represent human a-globin and apo-AII exons respectively. The (GT)16tract is denoted by an open box. A, deletion of nucleotides 1426 to 2490 of pBR322.E and 0, simian virus 40 enhancer and origin of replication sequences.(ii) A 291 bp RsaI/Hinfl apo-AII gene fragment isolated from pAIITSM.O [24] wasinserted into the BstEII site of pSVedaIW. This fragment contained the 76 bp at the 3'end of apo-AII E3 (filled box) and the 215 bp at the 5' end of apo-AII 13 (thick line).(iii) A 93 bp PvuII/DdeI apo-AII gene fragment isolated from pAIITS)4.0 was insertedinto the XbaI site of the construct generated above. A series of Bal3l generateddeletions of this fragment were also inserted into the XbaI site. These were producedby digesting pAIITS4.0 with PvuII, followed by a time course of Bal31 nucleasedigestions, inactivation and finally restriction with DdeI. The fragments inserted intothe XbaI site therefore contained varying lengths of the 3' end of apo-AII 12 (dashedline) and a fixed length (46 bp) of the 5' end of apo-AII 13 (filled box).(B). Linear representation of the hybrid region of the human al-globin/apo-AII gene

present in pSVed cxl W/13-12 Awt-37.See above for conventions used. Note that the intron generated within alE3 is ahybrid between the 5' end of apo-AII I3 and the 3' end of apo-AII 12. The apo-AII 12sequence present in each construct is shown as is the cryptic 3' splice site sequence5'-TCTCTAG-3'. The boxed thick line represents apo-AII 13 sequence which becomesexon sequence in pSVedalW/AII/13-12 Awt-37. The RsaI restriction site in alE3 ismarked. This site was used to produce the fragments employed in S1 nucleaseprotection analysis.(C). The kinase radiolabelled RsaI/HindIII fragments isolated from pSVedalW/AII/13-

12 Awt, 19, 29, 31 and 37 (PROBE).These were used in S1 nuclease protection analysis of the splicing pattern at the 3' end

3790


of the hybrid introns (Fig.2). The length of the fragment isolated from pSVedalW/AII/I3-12,A wt is presented. The labelling site is marked by an asterisk. The arrowheadat the 3' end of the probes indicates that they extend to the HindIlI site in alE2.Above the probe fragments, are indicated the regions which are protected from Sinuclease digestion by RNA hybridization. The RNA species involved in this protectionmay be spliced at the normal 3' splice site of apo-AII I2 (I2 ACCEPTOR) or at thecryptic site upstream (CRYPTIC ACCEPTOR). In addition, the probes can be partiallyprotected by RNA which contains the hybrid intron but not the intron (al-globin 12)immediately of upstream (Fig.2).

an Si nuclease probe was constructed from each mutant. In each case this was a

kinase-labelled RsaI/HindIll fragment which spanned the hybrid intron and also theintron immediately upstream (cal-globin 12) (See Fig.1). Consequently, when al-globin12 is spliced, intermediates arising from the inefficient splicing of the downstream

intron (apo-AII I3/12) could be detected. The extent to which these probes were

protected from nuclease digestion by hybridization to RNA extracted from theappropriately transfected cells is shown in Figure 2. This indicates that as the 3'splice site of apo-AII 12 is successively deleted, processing of the primary transcriptprogressively shifts away from this site to a previously non-functional cryptic 3' splicesite upstream (see Fig.1). There is little change in the overall net efficiency of splicingas the proportion of processed and unprocessed transcripts remains roughly constant.

These deletion experiments demonstrate that 28 nucleotides of the 3' end of apo-

All 12 are sufficient to direct efficient processing. This sequence contains eleven ofthe original sixteen GT repeats and probably lacks the usual lariat branch point of apo-AII 12 [24]. Removal of a further five GT repeats (L 29) causes a loss of 50% activityto the upstream cryptic acceptor site (Fig.5). However not until only two GT repeatsremain ( A37) does the apo-AII 12 3' splice site lose all activity to this cryptic site. Inthis final construct (A 37) the AG dinucleotide of the cryptic 3' splice site is onlyseparated from that of apo-AII 12 by eight nucleotides. There have been a number ofstudies both of naturally occurring and experimentally produced mutants whichindicate that such close proximity can in some cases lead to the interference by theupstream dinucleotide of splicing directed by the otherwise functional downstreamacceptor [20-23,28]. Consequently, it was possible that the deletion of the 3' splicesite of apo-AII 12 present in pSVed aclW/AII/13 -12 A 37 was non-functional, not becauseof intrinsic incompetence, but due to interference from the AG dinucleotide only eightnucleotides upstream.

Demonstration of interference, by a cryptic acceptor site, of processing directed by

deletions of the apo-AII 12 3' splice siteTo test the possibility of acceptor interference in the first series of experiments,

a second set of apo-AII 12 deletion constructs was generated (Fig.3) In principle these

3791


~ .it T ii 1

_ 43t ~~~~~~~~~~~~296

4-n- PROBE

w:^ UNSPLICED._.C,lSzz2r* 517

396

s o 0;=!~~~~~~~~hz 298

urni BguS ~ CRYPTIC

4-AU 12a

_ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~-I^r-2 20Figure 2. Si Nuclease analysis of splicing at the 3' end of the hybrid intronscontaining deletions of the 3' splice site of apo-AII 12.

Autoradiogram of the size fractionated products of Si nuclease analysis of RNAextracted from HeLa cells transfected with pSVed alW/Al/13-12 tkwt, 19, 29, 31 or 37.The probes used were individually generated from each of these constructs. In everycase this probe was an isolated kinase-radiolabelled RsaI/HindIII fragment (Fig.i).Each probe was hybridized with 10 >tg of total RNA extracted from untreated HeLacells (U) and separately hybridized with 10 9g of RNA extracted from appropriatelytransfected (T) HeLa cells. These hybrids were then digested with Si nuclease and theproducts size fractionated through a 5% denaturing polyacrylamide gel in parallel withsize markers. The resulting gel was autoradiographed for 36 hrs. Size markers areindicated by open arrows and their lengths given in nucleotides. The position of theintact probe (PROBE) is indicated by a filled arrow. Similarly indicated are thefragments of the probe which were protected from Si nuclease digestion by RNAhybridization. The RNA species involved in this protection either contain the hybridapo-AII 13/12 intron (UNSPLICED) or have had it spliced-out. This splicing can beeither at the normal 3' splice site of apo-AII I2 (All 12) or at the cryptic site upstream(CRYPTIC) (see Fig.1).

3792


differed from the original series only in that the AG dinucleotide of the apo-AII 12

acceptor was separated from that of the cryptic site by the insertion of a 33nucleotide spacer fragment. This fragment originated from a fibronectin cDNA clone

(pFH23) [291 and contained no AG dinucleotides. The six constructs produced,

pSVedlW/FN/AII/I3-I2Lwt, A7, L\17, I25, A37 and A41, contained respectively

47, 40, 30, 22, 10 and 6 bp of apo-AII 12 immediately upstream from the 3' splice site

(see Fig.3). Each of these recombinants was transfected separately both into apo-AII

non-producing (HeLa) cells and into apo-AII producing (Hep-G2) cells. After 48 hours,

total RNA was extracted from these cells and analysed by Si nuclease protection.

Accurate RNA processing at the 5' splice site of the hybrid introns was assumed

given the results from the first series of deletion constructs (data not shown).Processing at the 3' end of these introns was assessed by Si nuclease probes specific

for each recombinant. In each case this was a kinase-labelled Ball/HindIII fragmentanalagous to the RsaI/HindIII fragments employed to analyse the transcripts produced

from the original set of deletion mutants (see Figs. 1 and 2). The SI nuclease

protection procedure was performed on RNA extracted from transfected and non-

transfected cells. The results of transient expression in HeLa cells are presented in

Figure 4. These are identical to the results obtained by transient expression in HepG2

cells (data not shown) and indicate that as nucleotides are successively removed from

the 5' end of the 3' splice site of apo-AII I2, it progressively loses activity to the same

upstream cryptic acceptor sequence described in the first series of deletion

experiments. However, in this second set of experiments, fewer GT repeats of the

apo-AII 12 acceptor are required to direct efficient processing of the primary

transcript (Fig.5). A 3' splice site containing eight GT repeats totally outcompetes the

cryptic sequence while one containing two repeats relinquishes only 25% of its

activity. This is in contrast to the original deletion series where an acceptor with two

GT repeats lost all activity to the cryptic site. Rather, when these two 3' splice sites

are more widely separated, only by removing all of the GT repeats can processing at

the apo-AII 12 3' splice junction be completely abolished. In this final construct (L&41)the AG dinucleotides involved are some 37 nucleotides apart, well beyond 17

nucleotides, the greatest distance at which one acceptor has been found to interfere

with processing at another [221 In the first set of constructs this crucial separationwas not maintained probaly resulting in interference. This would then explain why in

the original series of experiments apparently more the apo-AII 12 acceptor was

required for efficient splicing that was indicated by the second series (Fig.5).

3793


(A) i/ H"DECOIU,

PSTI

Hi

Ul

W,

IV/

(B)

(C)*h ) EXON 2)

I-

Figure 3. Construction of expression plasmids containing deletions of the 3' splice siteof apo-AII 12 which eliminate the possibility of interference by cryptic 3' splice sites.(A). Construction of pSVed a1W/FN/AII/13-12 A wt-41.Ci) Schematic restriction map of the parent plasmid vector pSVed alW [27]. SeeFig.1 for conventions used.(ii) A 291 bp RsaI/Hinfl apo-AII gene fragment isolated from pAIITSA4.O [24] insertedinto the BstEii site of pSVed a1W. See Fig.1 for conventions used.(iii) A 61 bp Sau3A1 fibronectin cDNA fragment isolated from pFH23 [29] insertedinto the XbaI site of the construct generated above.(iv) A 93 bp PvuII/DdeI apo-AII gene fragment isolated from pAIITSM4.O inserted intothe SacI site of the construct generated immediately above. A series of Bal31generated deletions of this fragment were also inserted into the SacI site (see Fig.1,and also for conventions used).(B). Linear representation of the hybrid region of the human al-globin/fibronectin/apo-AII genes present in pSVedacW/FN/I3-12Awt-41. See Fig.1 for conventions used.Note that the intron generated within alE3 is a hybrid between the 5' end of apo-AII13, fibronectin cDNA and the 3' end of apo-AII I2. The apo-AII 12 sequence in eachconstruct is shown, as is the cryptic 3' splice site sequence, 5'-TCTCTAG-3'. Notethat these two sequences are widely separated compared to their relative positioningin the constructs presented in Figure 1. The thin line represents fibronectin exonsequence which becomes intron sequence in pSVedalW/FN/AII/13-12 A wt-41. The BalIrestriction site in ot1E3 is marked. This site was used to produce the fragmentsemployed in S1 nuclease protection analysis.(C). The kinase-radiolabelled Ball/HindIII fragments isolated from pSVed alW/FN/

3794


AII/13-12 Awt, A 7, A17, A25, A37 and A41 (PROBE). These were used in Si nucleaseprotection analysis of the pattem of splicing at the 3' end of the hybrid introns (seeFig.4). The length of the fragment isolated from pSVedalW/FN/AII/13-I2 t&wt ispresented. The labelling site is marked by an asterisk. The arrowhead at the 3' end ofthe probes indicates that they extend to the HindIII site in alE2. Above the probefragments, are indicated those regions which are protected from Si nuclease digestionby RNA hybridization. The RNA species involved in this protection are either splicedat the normal 3' splice site of apo-AII I2 (12 ACCEPTOR) or at a cryptic site upstream(CRYPTIC ACCEPTOR). In addition, the probes can be partially protected by RNAwhich contains the hybrid intron, but not the intron immediately upstream (see Fig.4).

DISCUSSIONThe construction of successive 5' deletions has defined the minimum number of

nucleotides at the 3' end of apo-AlI 12 that are required to direct efficient splicing intransient expression systems. It is unlikely that this definition incorporates theeffects of interference by upstream 3' splice sites. Processing in two cell types,representing apo-AII producing (Hep-G2) and non-producing (HeLa) tissues, wasidentical; in both, 22 nucleotides of the apo-AII 12 acceptor sequence were sufficientto direct the efficient removal of a hybrid intron from primary transcripts. Thissequence contained eight of the sixteen GT repeats normally present at the 3' end ofapo-AII 12. Removal of a further six GT repeats caused this acceptor to lose 25% ofits activity to a previously unused cryptic 3' splice site upstream. However, only byremoving all the GT repeats did the 3' splice site of apo-AII 12 become completely non-functional. Similar deletion analysis has been performed on "classic" 3' splice siteswhich conform to the consensus sequence (Y)nNYAG [2]. These have indicated that aminimum of 14 nucleotides of the polypyrimidine tract are required for any detectablelevel of processing to take place [10,11]. Here we report experiments which indicatethat the six nucleotides (GT)2GG which directly replace this tract in a deletion of the3' splice site of apo-AII 12 are sufficient to direct a relatively high level of splicing.Therefore the apo-AII 12 3' splice site (GT)2GGGCAG represents a splicing signalwhich is as effective as its "classic" counterparts [251. This finding is somewhat atodds with the study of Van Santen and Spritz [11] where a similar 3' splice site,(GT)11GCGCGAG, was artificially generated which was non-functional. In this studythe GT repeat was regarded as a neutral spacer sequence located upstream ofsuccessive 5' acceptor site. The discrepancy between the activity of the(GT)11GCGCGAG acceptor and the related apo-AII 12 splice site may be explained bythe recent report by Reed and Maniatis [26] suggesting the sequences immediatelyflanking introns also play an important role in the splicing process. Under the correctcircumstances, therefore, it appears that during splicing a (GT)2GG sequence canmimic the function usually performed by a tract of polypyrimidines. The looselydefined pyrimidine tract may act by means of secondary structure as a recognition

3795


U T U T U T U T U T U T

750 PROBE

5- UNSPLICED517 C~

298 t

_ , +-- CRYPTIC

220at. 4

m- Ci U"-----4-AU 12

154̂

Figure 4. Si nuclease analysis of the pattern of splicing at the 3' end of the hybridintrons transcribed from pSVedalW/FN/AII/I3-I2 Awt-41 transfected into HeLa cells.

Autoradiogram of the size fractionated products of Si nuclease analysis of RNAextracted from HeLa cells transfected with pSVeda1W/FN/AII/l3-I2Awt, A7, A17,A25, A37 or A 41. The probes used were individually generated from each of theseconstructs. In every case this probe was an isolated kinase-labelled BalI/HindllIfragment (Fig.3). Each probe was hybridized with 10 ILg of total RNA extracted fromuntreated HeLa cells (U) and separately to 10 pLg of total RNA extracted fromappropriately transfected HeLa cells (T). These hybrids were then digested with SInuclease and the products size fractionated through a 5% denaturing polyacrylamidegel in parallel with size markers. The resulting gel was autoradiographed for 36 hrs.Size markers are indicated by open arrows and their lengths given in nucleotides. Theposition of the intact probe (PROBE) is indicated by a filled arrow. Similarly indicatedare the fragments of the probe which were protected from S1 nuclease digestion byRNA hybridization. The RNA species involved in this protection either contain theunspliced hybrid intron apo-AII 13/12/FN (UNSPLICED) or have had it removed. Thissplicing can be either at the normal 3' splice site of apo-AII 12 (All 12) or at a crypticsite upstream (CRYPTIC) (see Fig.3). This cryptic site is the same as that utilized inthe transcripts produced from pSVed a1W/AII/13-12 A 31 and 37 (Figs. 1 and 2).

3796


100 I 0 Pk

90

80

70

60

50

14040

30

20

20

0

16 15 14 13 12 11 10 9 8 7 6 S 4 3 2 1 0

N GT FPETS

Fiqure 5. Relative efficiency of the cryptic 3' splice site and the deletions of theapo-AII 12 3' splice junction.

The graph shows the results of quantitative densitometry of the autoradiographspresented in Figures 2 and 4. It indicates the usage of the apo-AII 12 3' splice site inthe transcripts produced from pSVedalW/AII/I3-I2 Awt-37 (dashed line) andpSVedcl W/FN/AII/I3-I2,Awt-41 (solid line). The graph correlates the number of apo-All 12 acceptor "GT repeats" with the percentage of transcripts spliced at that sitecompared to the cryptic acceptor. Together these two sites account for all thesplicing that occurs at the 3' end of the hybrid introns presented in Figures 1 and 3.

signal for a splicing factor and/or a spacer sequence, separating the AG dinucleotidefrom the lariat branch site. Therefore a more strictly defined secondary structureconferred by the GT repeat could serve the same purpose. There is somecircumstantial evidence to suggest that the GT repeat has such a defined secondary

structure. When single-stranded DNA containing the (GT)16 repeat was sequenced bythe chemical degradation procedure the ladder of resulting DNA fragments becameprogressively and rapidly fainter in the region of the repeat in the 3' to 5' direction(data not shown). This phenomenon, which has been observed in GT repeat sequencesdemonstrated to represent Z-DNA [30], could reflect secondary structure also presentin the single-stranded apo-AII primary RNA transcript.

In vitro splicing experiments have demonstrated that when the pyrimidinesnormally associated with 3' splice sequences are delted, both cleavage at the 5' donorsite and lariat formation are prevented [9,17]. Whether the "GT repeat acceptor" ofthe second intron of the human apo-AII 12 gene is involved in the same splicingmechanism as the classic "polyrimidine acceptor" remains to be determined.

3797


AcknowledgementsWe gratefully thank Mr K.C. Mabbatt, Mr P. Thornton-Evison, Mr S.A.

Buckingham and Miss C. Lee for art work and photography. Our thanks are also due to

Dr N.J. Proudfoot for providing the plasmid pSVedalW. This work was supported by

grants to F.E.B. from the Medical Research Council of Great Britain (grant no.

G8309498CB) and the British Heart Foundation (grant no. 83/30). C.S.S. held an MRC

studentship.

*Present address: Department of Immunology, The Children's Hospital, Harvard Medical School, 300Longwood Avenue, Boston, MA 02115, USA

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