Volume 15 Number 9 1987
Nucleic Acids Research
Deletion analysis of a unique 3' spike site indicates that alternating guanine and thymine
residues 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 0X1 3RE,
UK
Received February 3, 1987; Revised and Accepted April 6, 1987
ABSTRACT
The 31 splice site of the second intron (12) of the human apollpoprotein-AII gene,
(GT)igGGGCAG, is unique in that, although fully functional, a stretch of alternating
guanine and thymine residues replaces the polypyrtmidine tract usually associated with
3' splice junctions. The transient expression of successive 51 deletion mutants has
defined the minimum number of nucleotides at the 3' end of apo-AII 12 that are
required to direct efficient splicing. Processing in two cell-types, representing apoAII producing and non-producing tissue was identical; in both, only by removing all the
GT repeats did the 3' splice site of apo-AII 12 become completely non-functional.
Similar deletion analyses of "classic" 31 splice sites, which conform to the consensus
sequence (Y)nNYAG, have Indicated that a minimum of 14 nucleotides of the
polypyrimidine tract are required for detectable levels of processing to take place.
Here we report that the six nucleotides (GT^GG, which directly replace this tract in a
deletion mutant of the 3' splice site of apo-AII 12 are sufficient to direct the splicing
process efficiently and correctly.
INTRODUCTION
Analysis of the exon-intron boundaries has revealed that introns Invariably begin
with the 5' nucleotides GT and end with the 3'nucleotldes AG [1]. The identification of
over 130 5' and 3' splice sites has extended the original "GT
AG" rule so the
consensus sequences are now, 5' splice site: 5 ' - C / ^ A G : G T ^ / Q A G T - 3 ' and 3' splice
site: (Y)nNYAG:G [2]. After the initial cleavage of the Intron at the 5' end, the
guanosine residue in the 51 GT attacks an adenoslne residue close to the 3' end of the
intron to form a 2'-5' phosphodlester bond, called the branch site [3], thereby
producing a so-called lariat structure [4-7]. The adenoslne nucleotide Involved In the
branch site has been identified for several introns and has been found to be located In
mammalian genes 18 to 37 nucleotides upstream from the AG dlnucleotide ending the
intron [6,8-9], Deletion analysis of a number of higher eukaryotic introns [10-14] has
demonstrated that most of the intron is dispensable without deleterious effect on RNA
splicing. Indeed, the smallest naturally occurring Intron reported to date is only 38
nucleotides long [151 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
© IRL Press Limited, Oxford, England.
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for efficient and accurate splicing of two different globin introns [10-111 In all the
mammalian introns examined so far, the region proximal to the 31 splice site is rich in
pyrimldines, a conservation that implies an important role some of these nucleotides in
the splicing process [9-11,16-171 The adenosine branch nucleotide is located within
the general consensus sequence 5'-YTRAY-3' [41,18] just upstream of the pyrimldinerich 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
jn vivo. For example, a point mutation changing the AG dinucleotide to GG in the
second intron of the B-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 (5-globin gene creates a 31 splice site so close
to the y end of the intron that it causes B+ thalassemia by interfering with splicing at
the normal 31 splice site [20-23].
Although differing considerably from the consensus sequence [2] the 3' splice site
(GT)igGGGCAG of the second intron (12) of the human apollpoprotein-AII (apo-AII)
gene directs accurate and efficient splicing of both hepatic and intestinal apo-AII
primary transcripts [24-251 It has previously been shown that the polypyrimidine
tract 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
[9,171 The replacement of these polypyrimidines in apo-AII 12 suggests that this
function may be mimicked by the sequence: (GT)igGG. Consequently, in order to
assess the importance of this GT repeat to the splicing process, a series of expression
constructs 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 Procedures
Purification of DNA, llgation and labelling reactions, restriction enzyme
digestion, Bal31 digestion, gel electrophoresis and SI nuclease analysis were performed
according to established procedures as described by Maniatis et al. [311
Construction of Plasmids
See text and appropriate figure legends.
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Cell Growth
Hep-G2 [32-35] cells were grown at 37°C under 5% CO2 in MEM medium
supplemented with 10% F"CS and 2 mM glutamine. DMEM medium supplemented with
2 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 cm^ tissue culture flasks
(Nunc) and 150 x 15 mm tissue culture dishes (Lux).
DNA Transfection
Hep-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 ng 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 isothiocyanate, 50 mM Tris-HCl, pH 7.6, 10 mM EDTA, 0.1 M B-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 20°C 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 -20°C.
RESULTS
In a hybrid intron, deletion of the (GT)i/; repeat of apo-AH activates a nearby cryptic
3' splice site
The construction of the first series of (GT)^g 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 pSVedalW [27]. The 5' end of this
intron represented apo-AH 13 sequences, while the 3' end represented successive Bal31
generated 5' deletions of apo-AH 12. This series of constructs therefore differed only
in the number of nucleotides of the apo-AH 12 acceptor site they contained. The five
constructs produced, pSVedalW/AU/I3-I2Awt, & 19, A 29, &31 and A 37, contained
respectively 47, 28, 18, 16 and 10 bp of apo-AII 12 immediately upstream of the 31
splice junction (Fig.l). These recomblnants were transfected into HeLa cells and,
after 48 hours, total RNA was extracted and analysed by SI nuclease protection.
Preliminary SI 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-AH D (data not shown).
To investigate the pattern of splicing at the 3' end of the deleted hybrid introns,
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(C)
Figure 1. Construction of expression plasmids containing deletions of the 3' splice site
of apo-AII 12
(A). Construction of pSVedalW/AII/I3-I2 Awt-37
(I)
Schematic restriction map of the parent plasmid vector pSVedalW [27], Striped
and filled boxes represent human ct-globin and apo-AII exona respectively. The (GT)is
tract is denoted by an open box. A , deletion of nucleotides 1426 to 2490 of pBR322.
E and O, simian virus 40 enhancer and origin of replication sequences.
(ii) A 291 bp Rsal/Hinfl apo-AII gene fragment Isolated from pAIITSVi.O [24] was
Inserted into the BstEIl site of pSVedolW. This fragment contained the 76 bp at the 3"
end of apo-AII E3 (filled box) and the 215 bp at the 51 end of apo-AII 13 (thick line).
(ill) A 93 bp PvuII/Ddel apo-AII gene fragment isolated from pAHTSJA.O was inserted
into the Xbal sits of the construct generated above. A series of Bal31 generated
deletions of this fragment were also inserted into the Xbal site. These were produced
by digesting pAIITSM.O with PvuII, followed by a time course of BaI31 nuclease
digestions, inactivation and finally restriction with Ddel. The fragments inserted into
the Xbal site therefore contained varying lengths of the 3' end of apo-AII 12 (dashed
line) 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 pSVedotlW/B-I2 Awt-37.
See above for conventions used. Note that the Intron generated within O.1E3 is a
hybrid between the 51 end of apo-AII 13 and the 3' end of apo-AII 12. The apo-AII 12
sequence present in each construct is shown as is the cryptic 3' splice site sequence
5'-TCTCTAG-3'. The boxed thick line represents apo-AII D sequence which becomes
exon sequence in pSVedolW/AII/I3-I2Awt-37. The Rsal restriction site in alE3 is
marked. This site was used to produce the fragments employed in SI nuclease
protection analysis.
(C). The kinase radiolabelled Rsal/Hlndin fragments isolated from pSVedalW/AII/D12 Awt, 19, 29, 31 and 37 (PRO8E).
These were used in SI nuclease protection analysis of the splicing pattern at the 3' end
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of the hybrid introns (Fig.2). The length of the fragment isolated from pSVedalW/
AII/I3-I2A wt is presented. The labelling site Is marked by an asterisk. The arrowhead
at the 3' end of the probes indicates that they extend to the Hindlll site in alE2.
Above the probe fragments, are indicated the regions which are protected from SI
nuclease digestion by RNA hybridization. The RNA species involved in this protection
may be spliced at the normal 3' splice site of apo-AII 12 (12 ACCEPTOR) or at the
cryptic site upstream (CRYPTIC ACCEPTOR). In addition, the probes can be partially
protected 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 Rsal/Hindin fragment which spanned the hybrid intron and also the
intron immediately upstream (al-globin 12) (See Fig.l). Consequently, when al-globin
12 is spliced, intermediates arising from the inefficient splicing of the downstream
intron (apo-AII D/I2) could be detected.
The extent to which these probes were
protected from nuclease digestion by hybridization to RNA extracted from the
appropriately 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 transcript
progressively shifts away from this site to a previously non-functional cryptic 3' splice
site upstream (see Fig.l). There is little change in the overall net efficiency of splicing
as the proportion of processed and unprocessed transcripts remains roughly constant.
These deletion experiments demonstrate that 28 nucleotldes of the 3' end of apoAII 12 are sufficient to direct efficient processing. This sequence contains eleven of
the original sixteen GT repeats and probably lacks the usual lariat branch point of apoAII 12 [24]. Removal of a further five GT repeats (£29) causes a loss of 50% activity
to the upstream cryptic acceptor site (Fig.5). However not until only two GT repeats
remain ( A37) does the apo-AII 12 3' splice site lose all activity to this cryptic site. In
this final construct (A 37) the AG dinucleotide of the cryptic 3' splice site Is only
separated from that of apo-AII 12 by eight nucleotldes. There have been a number of
studies both of naturally occurring and experimentally produced mutants which
indicate that such close proximity can in some cases lead to the interference by the
upstream dinucleotide of splicing directed by the otherwise functional downstream
acceptor [20-23,28]. Consequently, it was possible that the deletion of the 3' splice
site of apo-AII 12 present in pSVedalW/AII/I3-I2 A37 was non-functional, not because
of intrinsic incompetence, but due to interference from the AG dinucleotide only eight
nucleotides upstream.
Demonstration of interference, by a cryptic acceptor site, of processing directed by
deletions of the apo-AII 12 3' splice site
To 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
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AWt
U
T
A19
U
A 29
T
U
T
A31
U
T
A37
lT~T
<3=1296
PROBE
UNSPLICED
517
<=396
298
CRYPTIC
Figure 2.
SI Nuclease analysis of splicing at the 3' end of the hybrid Introns
containing deletions of the 3' splice site of apo-AII 12.
Autoradiogram of the size fractionated products of SI nuclease analysis of RNA
extracted from HeLa cells transfected with pSVedalW/AII/B-I2 Awt, 19, 29, 31 or 37.
The probes used were individually generated from each of these constructs. In every
case this probe was an isolated kinase-radiolabelled Rsal/HindDI fragment (Fig.l).
Each probe was hybridized with 10 u.g of total RNA extracted from untreated HeLa
cells (U) and separately hybridized with 10 (ig of RNA extracted from appropriately
transfected (T) HeLa cells. These hybrids were then digested with SI nuclease and the
products size fractionated through a 5% denaturing polyacrylamide gel in parallel with
size markers. The resulting gel was autoradiographed for 36 hra. Size markers are
indicated by open arrows arid their lengths given in nucleotldes. The position of the
intact probe (PROSE) is indicated by a filled arrow. Similarly indicated are the
fragments of the probe which were protected from SI nuclease digestion by RNA
hybridization. The RNA species involved In this protection either contain the hybrid
apo-AII 13/12 intron (UNSPLICED) or have had it spliced-out. This splicing can be
either at the normal 3' splice site of apo-AII 12 (ATI 12) or at the cryptic site upstream
(CRYPTIC) (see Fig.l).
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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 33
nucleotide spacer fragment. This fragment originated from a fibronectin cDNA clone
(pFH23) [29] and contained no AG dinucleotides. The six constructs produced,
pSVedalW/FN/AII/I3-I2Awt, A7, A17, C25, 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/HindlH fragment
analagous to the Rsal/Hindlll 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 nontransfected 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 12, 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 (A41)
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 [22]. In the first set of constructs this crucial separation
was 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).
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(C)
Figure 3. Construction of expression plasmida containing deletions of the 3' splice site
of apo-AII 12 which eliminate the possibility of interference by cryptic 3' splice sites.
(A). Construction of pSVedalW/FN/AII/D-I2 Awt-41.
(i)
Schematic restriction map of the parent plasmid vector pSVedalW [27]. See
Fig.l for conventions used.
(ii) A 291 bp R88l/Hlnfl apo-AII gene fragment isolated from pAIITSM.O [24] inserted
Into the B8tEii site of pSVedolW. See Fig.l for conventions used.
(iii) A 61 bp Sau3Al fibronectin cDNA fragment Isolated from pFH23 [29] inserted
into the Xbal site of the construct generated above.
(iv) A 93 bp PvuII/Ddel apo-AII gene fragment isolated from pAIITSJA.O inserted into
the Sad site of the construct generated immediately above. A series of Bal31
generated deletions of this fragment were also inserted into the Sad site (see Fig.l,
and also for conventions used).
(B). Linear representation of the hybrid region of the human al-globin/flbronectin/
apo-AII genes present in pSVedalW/FN/I3-I2 Awt-41. See Fig.l for conventions used.
Note that the intron generated within alE3 is a hybrid between the 51 end of apo-AII
13, fibronectin cDNA and the 3' end of apo-AII 12. The apo-AII 12 sequence in each
construct is shown, as is the cryptic 31 splice site sequence, 5'-TCTCTAG-3'. Note
that these two sequences are widely separated compared to their relative positioning
in the constructs presented in Figure 1. The thin line represents fibronectin exon
sequence which becomes intron sequence in pSVedalW/FN/AII/B-I2 Awt-41. The Ball
restriction site In alE3 is marked. This site was used to produce the fragments
employed in SI nuclease protection analysis.
(C). The kinase-radiolabeUed Ball/Hlndm fragments isolated from pSVedalW/FN/
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An/I3-I2Awt, A 7, A17, A25, A37 and A41 (PROBE). These were used in SI nuclease
protection analysis of the pattern of splicing at the 31 end of the hybrid introns (see
Fig.4). The length of the fragment isolated from pSVedalW/FN/AII/D-I2 Awt is
presented. The labelling site is marked by an asterisk. The arrowhead at the 3' end of
the probes indicates that they extend to the HindlH site in alE2. Above the probe
fragments, are indicated those regions which are protected from SI nuclease digestion
by RNA hybridization. The RNA species involved in this protection are either spliced
at the normal 3' splice site of apo-AII 12 (12 ACCEPTOR) or at a cryptic site upstream
(CRYPTIC ACCEPTOR). In addition, the probes can be partially protected by RNA
which contains the hybrid lntron, but not the intron immediately upstream (see Fig.4).
DISCUSSION
The construction of successive 5' deletions has defined the minimum number of
nucleotides at the 3' end of apo-AII 12 that are required to direct efficient splicing in
transient expression systems. It is unlikely that this definition incorporates the
effects of interference by upstream 3' splice sites. Processing in two cell types,
representing apo-AII producing (Hep-G2) and non-producing (HeLa) tissues, was
identical; in both, 22 nucleotides of the apo-AII 12 acceptor sequence were sufficient
to direct the efficient removal of a hybrid intron from primary transcripts. This
sequence contained eight of the sixteen GT repeats normally present at the 3' end of
apo-AII 12. Removal of a further six GT repeats caused this acceptor to lose 25% of
its activity to a previously unused cryptic 3' splice site upstream. However, only by
removing all the GT repeats did the 31 splice site of apo-AII 12 become completely nonfunctional. Similar deletion analysis has been performed on "classic" 3' splice sites
which conform to the consensus sequence (Y)nNYAG [2]. These have indicated that a
minimum of 14 nucleotides of the polypyrimidine tract are required for any detectable
level of processing to take place [10,11]. Here we report experiments which indicate
that the six nucleotides (GT^GG which directly replace this tract in a deletion of the
y 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^GGGCAG represents a splicing signal
which is as effective as its "classic" counterparts [251 This finding is somewhat at
odds with the study of Van Santen and Spritz [11] where a similar 3' splice site,
(GT)2iGCGCGAG, was artificially generated which was non-functional. In this study
the GT repeat was regarded as a neutral spacer sequence located upstream of
successive 5' acceptor site.
The discrepancy between the activity of the
(GT)nGCGCGAG acceptor and the related apo-AII 12 splice site may be explained by
the recent report by Reed and Maniatis [26] suggesting the sequences immediately
flanking introns also play an important role in the splicing process. Under the correct
circumstances, therefore, it appears that during splicing a (GT^GG sequence can
mimic the function usually performed by a tract of polypyrimidines. The loosely
defined pyrimidine tract may act by means of secondary structure as a recognition
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, 7
u
T
U
A17
T
U
A25
T
U
A37
T
U
A41
T
U
T
PROBE
UNSPLICED
517.
CRYPTIC
AH 12
154<
I
Figure 4. SI nuclease analysis of the pattern of splicing at the 3' end of the hybrid
introns transcribed from pSVedalW/FN/AII/I3-I2 Awt-41 transfected into HeLa cells.
Autoradiogram of the size fractionated products of SI nuclease analysis of RNA
extracted from HeLa cells transfected with pSVedalW/FN/AII/D-I2A wt, A 7, A17,
A25, A37 or A 41. The probes used were individually generated from each of these
constructs. In every case this probe was an isolated klnase-labelled Ball/Hindlll
fragment (Fig.3). Each prob8 was hybridized with 10 ng of total RNA extracted from
untreated HeLa cells (U) and separately to 10 ng of total RNA extracted from
appropriately transfected HeLa cells (T). These hybrids were then digested with SI
nuclease and the products size fractionated through a 5% denaturing polyacrylamide
gel 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. The
position of the intact probe (PROBE) is indicated by a filled arrow. Similarly indicated
are the fragments of the probe which were protected from SI nuclease digestion by
RNA hybridization. The RNA species involved in this protection either contain the
unspliced hybrid intron apo-AH I3/I2/FN (UNSPLICED) or have had it removed. This
splicing can be either at the normal 3' splice site of apo-AII 12 (All 12) or at a cryptic
site upstream (CRYPTIC) (see Fig.3). This cryptic site Is the same as that utilized in
the transcripts produced from pSVedotlW/AII/B-I2 4,31 and 37 (Figs. 1 and 2).
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Nucleic Acids Research
*6
O
M
MO OT
flHTATJ
Figure 5. Relative efficiency of the cryptic 3' splice site and the deletions of the
apo-AII 12 3' splice junction.
The graph shows the results of quantitative densitometry of the autoradiographs
presented in Figures 2 and 4. It indicates the usage of the apo-AII 12 3' 3plice site in
the transcripts produced from pSVedalW/AII/D-I2 Awt-37 (dashed line) and
pSVedalW/FN/AII/D-I2 Awt-41 (solid line). The graph correlates the number of apoAII 12 acceptor "GT repeats" with the percentage of transcripts spliced at that site
compared to the cryptic acceptor. Together these two sites account for all the
splicing 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 dinucleotlde
from the lariat branch site. Therefore a more strictly defined secondary structure
conferred by the GT repeat could serve the same purpose.
There is some
circumstantial evidence to suggest that the GT repeat has such a defined secondary
structure. When single-stranded DNA containing the (GT)ig repeat was sequenced by
the chemical degradation procedure the ladder of resulting DNA fragments became
progressively and rapidly fainter in the region of the repeat in the 3' to 51 direction
(data not shown). This phenomenon, which has been observed in GT repeat sequences
demonstrated to represent Z-DNA [30], could reflect secondary structure also present
in the single-stranded apo-AII primary RNA transcript.
In vitro splicing experiments have demonstrated that when the pyrimldines
normally associated with 3' splice sequences are delted, both cleavage at the 5' donor
site and lariat formation are prevented [9,17]. Whether the "GT repeat acceptor" of
the second intron of the human apo-AII 12 gene is involved in the same splicing
mechanism as the classic "polyrimldine acceptor" remains to be determined.
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Acknowledgements
We gratefully thank Mr K.C. Mabbatt, Mr P. Thornton-Evlson, 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, 300
Longwood Avenue, Boston, MA 02115, USA
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