From the
Expression of the muscle-specific 2a isoform of the
sarco/endoplasmic reticulum Ca
The Ca
Tissue-dependent alternative processing of the SERCA2 transcripts
gives rise to four different mRNAs (classes 1-4), which encode
for the two different SERCA2 protein isoforms (Fig. 1 A).
The class 1 mRNA is generated in the heart, slow-twitch skeletal
muscle, and smooth muscle by a splicing process, removing an optional
intron at the 3`-end of the SERCA2 messenger. The predominant
non-muscle class 2 mRNA is formed by polyadenylation at a site that
lies in between the muscle-specific 5`-donor and 3`-acceptor splice
sites. The class 3 mRNA is found in all tissues and contains the
muscle-specific intron while it is polyadenylated at a downstream site,
which is also used in the class 1 messenger. Finally, the
brain-specific class 4 mRNA is formed by a splicing event using a
second 5`-donor splice site downstream of the muscle donor site and the
same 3`-acceptor site as in class 1 mRNA. Class 1 encodes the
muscle-specific SERCA2a isoform, while classes 2-4 all encode the
non-muscle SERCA2b isoform.
In this study, we further tested
this hypothesis and also characterized some of the spatial and sequence
elements of the SERCA2 gene that are required for tissue-specific
transcript processing. We focused on the donor sites (the
muscle-specific upstream 5`D1 and the neuronal specific downstream
5`D2) and on the polyadenylation site located in the alternatively
spliced intron (pA
Mutations in the construct were introduced using the Altered Sites
mutagenesis system (Promega). An XbaI- XbaI or an
XbaI- AflII restriction fragment from pCM
Different
intron deletions, shortening the distance between the muscle-specific
5`-splice donor and 3`-acceptor site, were made. Del1 was created by
removing the AflII- EcoNI restriction fragment (1924
nt) from exon 23-24, blunt-ending the vector by Klenow fill-in,
and recircularizing it. For Del2, the BstEII- BstEII
restriction fragment (1047 nt) was removed from exon 24, and the vector
was recircularized. For making further intron deletions, a new unique
restriction site was created by site-directed mutagenesis near the
5`-end of the intron (position 1842). At this position, a T was changed
into a G, resulting in an Asp718 recognition site
(GGTACC). This mutation was named Mut6. Del3 was created
starting from Mut6 by removing an Asp718- BstEII
restriction fragment (3104 nt) and blunt-ending and recircularizing the
vector. In this way, the largest part of the intron was removed, only
leaving 284 nucleotides. Del4, Del5, and Del6 were constructed by
removing an Asp718- AflII fragment (1004 nt), an
Asp718- XhoI fragment (2438 nt), and an
Asp718- EcoNI fragment (2928 nt), respectively, from
Mut6, blunt-ending, and religating the plasmid.
The second donor
site 5`D2 was mutated by first subcloning the
AflII- XhoI fragment into pALTER. This donor site
(AC/GTGAGT) was converted into a full-consensus donor site
(AG/GTGAGT), which resulted in Mut7 after ligating the
AflII- XhoI fragment back into the construct. A
further modification of Mut7 was made by first exchanging the
XbaI- XbaI fragment from Mut7 with the corresponding
fragment from Mut6. This fragment contains an Asp718 site,
which could then be used to remove an Asp718- SwaI
fragment (824 nt) upstream of the consensus 5`D2.
In this study, the structural and spatial requirements
imposed on the processing sites for obtaining a muscle-specific SERCA2
gene transcript were assessed by means of a mutational analysis of a
minigene containing the SERCA2 3`-end. It was concluded from a previous
study that the activation of splicing is the mechanism that is
responsible for obtaining the class 1 transcript encoding the
muscle-specific SERCA2a isoform
(15) . In this study, we looked
more specifically at the influence of the relative strength of splice
and polyadenylation sites, which are involved in generating the
different transcripts. This was done to further delineate the sequence
requirements for obtaining a tissue-specific processing of the
transcript. This knowledge is a prerequisite for unraveling the exact
mechanisms regulating the differential transcript processing. We
conclude from our data that regulated expression of the SERCA2 gene as
observed during differentiation of BC
A second factor contributing to the
inefficiency of splicing in non-differentiated cells is the long
terminal intron in the SERCA2 transcript. An inverse relation was
observed between the length of the intron and the efficiency of
splicing in fibroblasts as well as in non-differentiated
BC
A third
potential site, which could be responsible for inefficient splicing in
non-muscle cells, could be the branch point and 3`-acceptor site.
However, the sequence of the branch point/3`-acceptor region of the
SERCA2 gene fits reasonably well with the mammalian consensus sequence
(SERCA2, GTG AT(Py)
Replacing the upstream polyadenylation site by a
well characterized strong synthetic polyadenylation site
(26) interfered with the normal SERCA2 transcript processing and
nearly completely abolished the differentiation-dependent up-regulation
of splicing. This experiment showed that a weak upstream
polyadenylation site is a necessary condition for obtaining regulated
transcript processing. The second donor site 5`D2 lies downstream of
the muscle-specific 5`D1 and is only used in neuronal cells. Optimizing
the 5`D2 donor site drastically altered the splicing pattern during
myogenic differentiation, as class 1 mRNAs were no longer formed.
However, the neuron-specific class 4 transcript could be detected both
in undifferentiated and differentiated muscle cells, indicating that
the consensus 5`D2 donor site completely outcompeted the more distal
5`D1 site. Therefore, the appearance of class 1 mRNAs during myogenic
differentiation is only possible when both the upstream poly(A) site
and the second donor splice site are weak processing sites.
We are grateful to Dr. N. Proudfoot for providing the
pUC-SPA vector. We acknowledge the skillful assistance of A.
Florizoone, M. Crabbé, Y. Parijs, and L. Bauwens.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
ATPase (SERCA2)
requires activation of an otherwise inefficient splicing process at the
3`-end of the primary gene transcript. The sequence and topology
requirements for this regulated splicing event were studied in the
BC
H1 myogenic cell line using a minigene containing the
3`-end of the SERCA2 gene. In undifferentiated BC
H1 cells,
the splice process is made inefficient by the presence of a weak
muscle-type 5`-donor site (5`D1) and a long terminal intron. Both
optimizing the 5`D1 and decreasing the length of the muscle-specific
intron, induced muscle-type splicing in undifferentiated myogenic
cells. Moreover, the induction of muscle-type transcripts was only
observed when two competing processing sites, the polyadenylation site
(pA
) used in non-muscle cells and the second neuronal
5`-donor site (5`D2), were weak. Indeed, making 5`D2 consensus induced
neuronal-type splicing in undifferentiated myocytes and prevented the
appearance of muscle-type transcripts. Similarly, replacing the
polyadenylation site (pA
) with a strong site almost
completely inhibited muscle-type splicing after myogenic
differentiation. We conclude that weak processing sites and a long
terminal intron are required for tissue-dependent mRNA processing of
the SERCA2 transcript.
-transport ATPases of the sarcoplasmic
or endoplasmic reticulum mediate the uptake of Ca
into intracellular stores, such as the sarcoplasmic or
endoplasmic reticulum. They are encoded by three different
SERCA
(
)
genes, and additional alternative
processing of the primary gene transcripts leads to five different
protein isoforms. These are expressed in a tissue-specific and
developmentally regulated way
(1) . SERCA1 is only expressed in
fast-twitch skeletal muscle, and alternative splicing generates an
adult (SERCA1a) and a neonatal (SERCA1b) isoform
(2, 3) . SERCA2 is expressed in all other tissues.
Tissue-specific differential processing of the 3`-end of the SERCA2
mRNA gives rise to two distinct protein isoforms, which differ in their
C-terminal part
(4, 5, 6, 7) . In
SERCA2b, a tail of 49 amino acids replaces the last 4 amino acids of
SERCA2a. This divergence in the C-terminal part of the protein is
responsible for a functional difference between SERCA2a and SERCA2b
(8, 9) . The muscle-specific SERCA2a has a lower
Ca
affinity and a higher turnover rate for
Ca
transport and for ATP hydrolysis compared with the
SERCA2b isoform. It has recently been demonstrated that the last 12
residues in SERCA2b are of critical importance for eliciting the
functional difference between the SERCA2 isoforms
(10) . These
isoforms have a tissue-specific expression pattern: the SERCA2b isoform
is present in non-muscle tissues and in smooth muscle while SERCA2a is
typical of heart, slow-twitch skeletal muscle and is also present in
smooth muscle. Recent in situ hybridization studies have
detected SERCA2a mRNAs very early during embryological development in
the cardiogenic plate of the presomite rat embryo (day 10 post-cotum)
(11, 12) . This illustrates the early occurrence of
SERCA2 isoform diversity during cardiac development, even before the
first cardiac contractions are present. The expression of the SERCA3
gene was recently shown to be particularly high in platelets, lymphoid
cells, and in some endothelial cells
(13, 14) .
Figure 1:
Processing of
pCMSERCA2 transcripts during differentiation of transfected
BC
H1 cells. A, schematic representation of the
pCM
SERCA2 construct. The 3`-end of the SERCA2 gene consisting of
exons 21-25 followed by the downstream flanking region
( DFR) was cloned after a cytomegalovirus-immediate early
promotor ( CMV-IE) and after a rabbit
-globin genomic
sequence ( grayshading). Coding and non-coding
sequences are represented by wide and narrowboxes, respectively. Partial exon sequences are between
parentheses. The processing signals present in SERCA2 are
indicated as follows: 5`D1 and 5`D2, 5`-donor splice sites 1 and 2,
respectively; 3`A, 3`-acceptor splice site; pA
and
pA
, upstream ( u) and downstream ( d)
polyadenylation signal. The numbers refer to the alternative
processing patterns of the 3`-end of SERCA2 giving rise to different
mRNA classes. The relative positions of the sense and antisense PCR
primers used to amplify the class 1 and class 2/3 RNAs are depicted
beneath the sequence. B, processing of pCM
SERCA2
transcripts during differentiation. BC
H1 cells were stably
transfected with pCM
SERCA2, and total RNA extracts were prepared
at 0, 2, 4, 7, and 10 days after the onset of differentiation.
Oligo(dT)-primed cDNA was amplified by PCR using the U1 sense primer
and the C2 and P2 antisense primers (25 cycles). In the control lane
( C), the cDNA samples were replaced by water. The samples were
run on a 6% polyacrylamide gel for about 120 min at 12 V/cm. Molecular
mass marker III (Boehringer Mannheim) was used as standard.
Representations of the amplified fragments and the primers are shown at
the left of the gel.
In a previous study
(15) , we
used a minigene containing the 3`-end of the SERCA2 gene to study the
tissue-dependent mRNA processing. In transfected 10T1/2 and
BCH1 cells, the minigene transcripts were shown to be
processed similarly to the endogenous SERCA2 gene transcripts. In
undifferentiated BC
H1 myoblasts, SERCA2 minigene
transcripts were not spliced, while muscle-type splicing was induced
when the cells were allowed to differentiate into myocytes. In 10T1/2
fibroblasts, we could not observe muscle-type splicing, even when the
class 2-specific polyadenylation site was deleted. However, muscle-type
splicing in 10T1/2 fibroblasts was seen when the muscle-specific donor
splice site was optimized and/or when the length of the muscle-specific
intron was shortened. These experiments led us to propose that
cis-acting elements present in the primary SERCA2 transcript render
muscle-type splicing inefficient in non-muscle cells and that the
splicing process becomes activated during myogenic differentiation by
the expression of splice factors.
). The strength of these sites was
manipulated to assess their contribution to differentiation-dependent
transcript processing. We also further explored the inverse
relationship between intron length and efficiency of splicing. Our
results indicate that regulated splicing, i.e. induction of
splicing during myogenic differentiation, can only occur when all three
sites (5`D1, 5`D2, and pA
) are weak processing sites and
when the muscle-specific intron exceeds a critical length.
Cell Culture
BCH1 mouse
myoblasts (ATCC; CRL1443) and C3H/10T1/2 cells (ATCC; CCL226) were
cultured as previously described
(16) . For differentiation
studies, the cells were seeded at a density of 5000 cells/cm
in 175-cm
flasks in Dulbecco's modified
Eagle's medium supplemented with 20% (v/v) fetal calf serum, 3.5
mML-glutamine, 0.9% (v/v) non-essential amino acids,
85 µg of streptomycin/ml, and 85 units of penicillin/ml (Life
Technologies, Inc.). 3 days later, differentiation was induced by
switching the BC
H1 cells to differentiation medium
containing 0.5% (v/v) serum. The cultures were supplied with fresh
differentiation medium 2, 4, and 7 days after the onset of
differentiation. 10T1/2 fibroblasts were cultured in the same way as
the BC
H1 cells except that 10% fetal calf serum was used in
the growth medium.
pCM
The
construction of the minigene containing the 3`-end of the SERCA2 gene
has been previously described
(15) . The pCMSERCA2 Modifications
SERCA2
construct contains the 3`-end of the pig SERCA2 gene inserted behind a
cytomegalovirus immediate early promotor and a
SfaNI- EcoRI fragment of the rabbit
-globin gene.
All recombinant DNA manipulations were carried out according to
standard protocols
(17) . The sequence numbers refer to the
sequence published by Eggermont et al.(18) .
SERCA2
were subcloned into the pALTER vector. In Mut1, the splice donor site
5`D1 (TG/GTAAAG) (where the diagonal line indicates the end of the exon
and the beginning of the intron) was changed into a near consensus
sequence (TG/GTAAGT). To create Mut4, a new AflII-site
was introduced in the XbaI- AflII fragment at position
2653 by changing the T at this position into a C, thus obtaining
C/TTAAG. By transferring the XbaI- AflII fragment from
pALTER back to pCM
SERCA2, the AflII- AflII
fragment (193 nucleotides) containing the upstream polyadenylation site
(pA
) was deleted from the construct. A strong
polyadenylation signal (SPA) was introduced by ligating a blunt-ended
Asp718- XbaI fragment excised from a pUC-SPA vector
(kindly provided by N. J. Proudfoot, Oxford, United Kingdom) into the
blunt-ended AflII-site of Mut4. This resulted in
Mut4,SPA
. A combination of Mut4,SPA
with Mut1
was obtained by removing the XbaI- XbaI fragment from
Mut4 and exchanging it with the XbaI- XbaI fragment
from Mut1, containing the optimized donor site. This resulted in the
mutated construct pCM
SERCA2(Mut4,SPA
,Mut1). From this
construct, an AflII- EcoNI fragment (1924 nt) from
exon 23-24 was removed, and the vector was blunt-ended and
recircularized, resulting in the construct
pCM
SERCA2(Mut4,SPA
,Mut1,Del1). The SPA site was also
inserted just before the downstream polyadenylation site
pA
. Therefore, an Asp718 unique restriction site
was introduced in the minigene at position 6305 just upstream of the
pA
(Mut12). In this site, the blunt-ended
Asp718- XbaI frag-ment excised from the pUC-SPA vector
was ligated. This resulted in Mut12,SPA
.
Analysis of in Vivo Splicing
For analysis
of in vivo splicing, all plasmids were stably transfected into
BCH1 cells or 10T1/2 cells using the calcium-phosphate
coprecipitation method as described before
(15) . Total RNA was
isolated from transfected or untransfected BC
H1 or 10T1/2
cells according to the Chirgwin procedure
(19) .
Oligo(dT)-primed first-strand cDNA was synthesized typically in a
20-µl reaction volume containing 1 µg of total RNA, 200 units
of Moloney murine leukemia virus-reverse transcriptase (Life
Technologies, Inc.), 1
Moloney murine leukemia virus-reverse
transcriptase buffer (Life Technologies, Inc.), 1 mM dATP,
dCTP, dGTP, and dTTP (Boehringer Mannheim), 20 units of RNase inhibitor
(rRNasin, Promega), 200 ng of oligo(dT)
(Boehringer
Mannheim, Mannheim, Germany), and incubated at 37 °C for 1 h.
Portions of the first-strand cDNA mix (25%) were then PCR amplified
using Hot Tub
DNA polymerase (Amersham International,
Amersham, UK). The PCR reaction mixture (50 µl) contained 1
Tub buffer (low Mg
, Amersham International), 0.2
mM of each dNTP, 25 pmol of each forward and reverse
primer(s), and 2.5 units of Hot Tub
DNA polymerase. Each
cycle consisted of 1 min denaturation at 95 °C, 1 min annealing at
55 °C, and 1 min extension at 72 °C. The labeled amplification
products (20 µl of the final PCR mixture) were separated on a 6%
polyacrylamide gel. To ensure that no contaminating genomic DNA was
present in the cDNA samples, we performed control PCR amplifications
starting from RNA samples. These controls were always negative. The
SERCA2 primer sequences were derived from the pig genomic SERCA2
sequence
(18) and correspond to the following sequences: C2,
5`-AGACCAGAACATATCACT-3` (nt 2267-2284, exon 22); P2,
5`-GAAACATGGATACTTGGC-3` (nt 5858-5875, exon 25); U4,
5`-GGATCCTGTTCTGCTGCA-3` (nt 3241-3258, exon 23); and S2,
5`-GCACTAACCACCACCATC-3` (nt 3477-3494, exon 23). The U1 primer
(5`-GAGGTTCTTCGAGTCCTT-3`) corresponds to nt 299-316 (exon 2) of
the rabbit
-globin sequence as described by Van Ooyen et al.(20) . When two different antisense primers were used
simultaneously in combination with the same sense primer, it was
verified that the result was the same as when the antisense primers
were used separately.
pCM
BCSERCA2 Processing during Myogenic
Differentiation of BC
H1
Cells
H1 is a myogenic cell line, which is well
characterized with respect to muscle differentiation
(21, 22) . We have previously demonstrated that
muscle-type class 1 mRNAs derived from the endogenous SERCA2 gene are
induced during myogenic differentiation of BC
H1 cells
(16, 23) . Furthermore, when BC
H1 cells were
stably transfected with the pCM
SERCA2 minigene, class 1 mRNAs
derived from the minigene were also observed during differentiation
(Fig. 1 B and Van Den Bosch et al.
(15) ). Fig. 1shows a reverse transcriptase-PCR
experiment performed on BC
H1 cells that were stably
transfected with the pCM
SERCA2 construct. The relative position of
the different primers used for PCR amplification is shown in
Fig. 1A. For the amplification of muscle-specific class
1 messenger, the U1 sense primer located in exon 2 of
-globin and
the P2 antisense primer located in exon 25 were used. This gives a
617-nt-long PCR-product specific for the class 1 mRNAs derived from the
transfected minigene. For the amplification of class 2/3 messengers,
the same sense primer (U1) was used in combination with the C2
antisense primer located in exon 22. In this way, a 414-nt-long
fragment specific for class 2/3 mRNA was amplified. Fig. 1 B shows that in undifferentiated cells, only class 2/3 mRNAs could
be detected and that starting from day 4, the class 1 mRNA gradually
appeared during myogenic differentiation.
The Influence of the 5`D1 Donor Site Strength and of
Intron Length on Splice Regulation
We had previously
observed that transfection of 10T1/2 fibroblasts with a SERCA2 minigene
containing either an optimized 5`D1 site (Mut1) or a shortened intron
(Del1) resulted in the formation of muscle-type class 1 mRNAs
(15) . The effect of the intron deletion on splice activation
was shown to be a distance effect as the restoration of intron length
with a non-related DNA fragment again prevented muscle-type splicing in
10T1/2 fibroblasts. We therefore wanted to investigate whether these
modifications would interfere with the differentiation-dependent
activation of splicing in BCH1 cells. SERCA2 minigenes with
an optimized 5`D1 (Mut1) or a shortened intron (Del1) or a combination
of both modifications (Fig. 2 A) were stably transfected
in BC
H1 cells. Minigene mRNAs before and after
differentiation were analyzed via reverse transcriptase-PCR.
Fig. 2B shows that in undifferentiated cells transfected
with either the optimized 5`D1 or the shortened intron, besides the
class 2/3 mRNAs, low levels of class 1 mRNAs could also be detected.
Importantly, class 1 mRNAs for these two mutant constructs increased
during differentiation as illustrated in Fig. 2 B. In
contrast, a combination of both mutations resulted in the nearly
exclusive formation of class 1 mRNAs in undifferentiated cells with no
marked changes in mRNA profile during differentiation. From these data,
we concluded that each mutation on its own slightly increased the
likelihood of splicing in non-differentiated BC
H1 cells and
that they did not interfere with the differentiation-dependent
up-regulation of splicing. However, combining both modifications nearly
maximally activated splicing in non-differentiated cells, suggesting an
additive effect of these modifications on the splice process. Moreover,
the combination of both modifications apparently provoked a maximal
splice efficiency in undifferentiated cells as differentiation did not
produce a further increase in the class1 mRNA level.
Figure 2:
Effect of optimizing the 5`D1 donor splice
site of a deletion and of the combination of both modifications on the
muscle-specific splicing of pCMSERCA2 transcripts during
differentiation of BC
H1 cells. A, schematic
representation of the modifications made to the SERCA2 part of
pCM
SERCA2. In Mut1, the muscle-specific-donor splice site was
optimized, and in Del1 an AflII ( A)- EcoNI
( EN) fragment of 1924 nt was removed. B, effect of
the modifications on the processing of pCM
SERCA2 transcripts.
BC
H1 cells were stably transfected with the modified
pCM
SERCA2 constructs as indicated at the top of the gel.
Total RNA extracts were prepared at the moment that the cells were
switched to low serum medium (undifferentiated, U) and after
being cultured for 10 days in medium with low serum (differentiated,
D). Class 1 and class 2/3 transcripts were amplified and
separated as described in Fig. 1.
Effect of 5`D2 Donor Site Strength on Splicing
Efficiency and Splice Site Selection
We further examined
whether regulated splicing was also affected by changing the 5`D2 site.
This donor splice site is located downstream of 5`D1
(Fig. 3 A) and is only used in neuronal tissues and
neuronal cell lines
(18, 24) . Both 5`D1 and 5`D2 use
the same 3`-acceptor site. At first sight, the 5`D2 site could be
considered as a near consensus binding site as it deviates in only one
position (-1) from the mammalian consensus sequence (5`D2,
AC/GTGAGT; consensus, AG/GTRAGT). However, a statistical analysis of
mammalian donor sites
(25) showed that a G at this position was
strongly favored (78%), whereas a C in this position was only present
in 3% of the analyzed sites. This prompted us to investigate whether
the C at position -1 affected the processing strength of 5`D2.
When 5`D2 was mutated to a consensus donor splice site (Mut7, AC/GTGAGT
to AG/GTGATG), class 4 mRNAs were formed both in
undifferentiated and differentiated cells (figure 3 B,
lanes1 and 2). Moreover, no class 1 mRNAs
could be detected after differentiation of BCH1 cells
(Fig. 3 C, lanes1 and 2).
This indicates that 5`D1 and 5`D2 compete for the same 3`-acceptor site
but that in the context of the wild type gene, 5`D2 is too weak to
compete effectively with 5`D1 during differentiation. We then
investigated whether the competition between 5`D1 and 5`D2 could be
influenced by shortening the distance between both sites. We therefore
made a construct in which the distance between 5`D1 and the consensus
5`D2 was shortened by deleting an Asp718- SwaI
fragment of 827 nt (Mut7, Del7), thereby reducing the distance between
5`D1 and 5`D2 from 1473 to 646 nt. Undifferentiated cells transfected
with this construct contained both class 1 and class 4 mRNAs
(Fig. 3, B and C, lanes3 and
4). During differentiation, class 1 mRNAs were up-regulated,
whereas the level of class 4 mRNAs did not change markedly, indicating
that in this shortened construct, 5`D1 competes more efficiently with
the consensus 5`D2.
Figure 3:
Effect of optimizing the 5`D2 donor splice
site and of a deletion on the muscle- and neuronal-specific splicing of
pCMSERCA2 transcripts during BC
H1 differentiation.
A, schematic representation of the modifications made to the
SERCA2 part of pCM
SERCA2. In Mut7, the neuronal-specific donor
splice site was optimized, and in Del7 an Asp718
( As)- SwaI ( S) fragment of 827 nt was
removed. The relative position of the sense and antisense PCR primers
used to amplify the class 3 and class 4 RNAs are depicted above the sequence. B, effect of the modifications on the
neuronal-specific processing of pCM
SERCA2 transcripts.
BC
H1 cells were stably transfected with the modified
pCM
SERCA2 constructs indicated at the top of the gel.
Total RNA extracts were prepared from undifferentiated ( U) and
differentiated ( D) BC
H1 cells. The
oligo(dT)-primed cDNA was subjected to a PCR using the U4 sense primer
and the P2 antisense primer to amplify class 3 and class 4 (25 cycles).
From this PCR mixture, 1 µl was used for a second PCR (10 cycles)
with S2 as sense primer and P2 as antisense primer. Lane5 shows the amplification products starting from pig cerebellum RNA.
Representations of the amplified fragments and the primers are shown at
the left of the gel. C, effect of the modifications
on the muscle-specific processing of pCM
SERCA2 transcripts. Total
RNA extracts were prepared from undifferentiated ( U) and
differentiated ( D) BC
H1 cells, and class 1 and
class 2/3 transcripts were detected as described in Fig. 1. In the
control lane ( C), the cDNA samples were replaced by water. By
deleting the Asp718- SwaI fragment in Mut7,Del7, the
complementary sequence to primer C2 was removed so that no class 2/3
mRNAs could be detected using this primer.
The Influence of the Strength of the Polyadenylation
Site on Splice Regulation
We previously demonstrated that
deletion of the upstream polyadenylation site (pA) did not
result in the use of the upstream donor splice site in 10T1/2
fibroblasts
(15) . This argues against a simple competition
model between splicing and polyadenylation. We further investigated the
effect of polyadenylation site strength on transcript processing in
BC
H1 cells by replacing the upstream polyadenylation site
with a strong SPA, as described by Levitt et al.(26) (Fig. 4 A). As demonstrated in
Fig. 4B, deleting the upstream polyadenylation site
(Mut4) did not interfere with the differentiation-dependent activation
of splicing ( lanes1 and 2). Class 2/3 mRNAs
were the only detectable mRNAs in non-differentiated cells, and the
class 1 mRNA was induced after differentiation. However, introducing
the SPA at the pA
locus (SPA
) nearly completely
abolished the differentiation-dependent up-regulation of splicing
( lanes3 and 4). Polyadenylation at the
upstream site became the predominant process, both in undifferentiated
and differentiated cells. Therefore, increasing the efficiency of
polyadenylation at the pA
blocked the
differentiation-dependent formation of class 1 mRNAs. We then examined
the processing pattern for a construct in which both the efficiency of
splicing (optimized 5`D1 and shortened intron) and the efficiency of
polyadenylation at the pA
(SPA
) were augmented.
Fig. 4B ( lane5) shows that in
undifferentiated cells, the class 1 mRNA was the most abundant
messenger, indicating that in this case splicing nearly completely
outcompeted polyadenylation. Consequently, no further increase in class
1 mRNA was detected during differentiation (Fig. 4 B,
lane6). To investigate the influence of the strength
of the downstream polyadenylation site on splice regulation, we also
inserted a strong polyadenylation site at the end of exon 25
immediately upstream of the pA
(Fig. 4 A). As
shown in Fig. 4 C, the presence of a strong downstream
polyadenylation site did not significantly alter the
differentiation-dependent splice pattern. This indicated that the
strength of the downstream pA
is not important for splice
regulation.
Figure 4:
Effect of the introduction of a strong
synthetic polyadenylation site on the muscle-specific splicing of
pCMSERCA2 transcripts during BC
H1 differentiation.
A, schematic representation of the modifications made to the
SERCA2 part of pCM
SERCA2. In Mut1, the muscle-specific donor
splice site was optimized, and in Del1 an AflII
( A)- EcoNI ( EN) fragment of 1924 nt was
removed. In Mut4 an extra AflII site was created upstream of
the pA
site, and a fragment of 193 nt containing the
pA
site was lost during the recloning of the mutated
XbaI- AflII fragment into the pCM
SERCA2 vector.
The synthetic polyadenylation site was cloned in the AflII
site giving rise to SPA
. In Mut12, an Asp718 site
( As) was created just in front of the pA
site, and
SPA
was created by cloning the synthetic polyadenylation
site in this Asp718 site. PanelB shows the
effect of replacing the upstream polyadenylation site by the synthetic
polyadenylation site, while in panelC, the results
are given as the synthetic polyadenylation site is positioned just in
front of the downstream polyadenylation site. In both cases, the
BC
H1 cells were stably transfected with the modified
pCM
SERCA2 constructs indicated at the top of the gel.
Total RNA extracts were prepared from undifferentiated ( U) and
differentiated ( D) BC
H1 cells, and class 1 and
class 2/3 transcripts were detected as described in Fig. 1.
Representations of the amplified fragments and the primers are shown at
the left of the gel. Molecular mass marker III (Boehringer
Mannheim) was used as standard.
The Effect of Intron Length on Transcript
Processing
Finally, we analyzed in greater detail how
splicing is affected by changing the length of the terminal intron.
Previous experiments had shown that deleting a large part of the
terminal intron (Del1) resulted in the appearance of class 1 mRNAs in
undifferentiated BCH1 (Fig. 2) and in 10T1/2
fibroblasts
(15) . This effect is not sequence dependent but
distance dependent, as substituting the Del1 with a non-related
sequence restored the normal transcript processing
(15) . To
further explore the effect of intron length on splicing efficiency and
splice regulation, deletions of different length were created within
the terminal intron at different positions (Fig. 5 A).
These constructs were then transfected into 10T1/2 cells. The amount of
spliced SERCA2 transcript was found to be inversely related to the
length of the remaining intron. Fig. 5 B shows that
deletions of less than 1100 nt (Del2, Del4) had only a weak effect on
splice activation, whereas longer deletions of over 1900 nt (Del1,
Del5) resulted in a strong class 1 mRNA band. Interestingly, shortening
the intron with 514 nt from 1464 nt (Del1) to 950 nt (Del5) only had a
small effect on the class 1 mRNA level, whereas an additional deletion
of 490 nt from 950 nt (Del5) to 460 nt (Del6) strongly increased the
class 1 mRNA level in fibroblasts. The marked increase in class 1 mRNA
in Del6 as compared with Del5 could not be attributed to the removal of
a specific sequence element, as the same XhoI- EcoNI
fragment was also removed in Del2 without any effect on class 1 mRNA
levels. This suggested that once the intron was shortened below a
critical length, splicing became very efficient in non-muscle cells. To
further study the differentiation-dependent regulation of SERCA2
transcript processing, these deletions were transfected into
BC
H1 myogenic cells. This resulted in the detection of
class 1 messenger in undifferentiated cells in all cases. For the short
deletions (Del4, Del2, Del1, and Del5), we could still detect a further
up-regulation of about 65% of class 1 messenger during differentiation,
indicating that they did not interfere with the
differentiation-dependent up-regulation of splicing. In the long
deletions (Del3, Del6), no further significant up-regulation could be
detected during differentiation. It appeared that in these conditions,
a maximal stimulation of splicing already occurred in undifferentiated
conditions. This is illustrated in Fig. 5 B where only
the results for the shortest and the longest deletion are shown.
Figure 5:
Effect of deletions in the muscle-specific
intron on muscle-specific splicing in 10T1/2 cells and during
differentiation of BCH1 cells. Panel A, schematic
representation of the deletions made in the muscle-specific intron of
pCM
SERCA2. Del1 and Del2 were created using the restriction sites
present in the construct, while for the other deletions an
Asp718 site ( As) was created downstream of the 5`D1
donor splice site. A, AflII; B,
BstEII; EN, EcoNI; X,
XhoI. In panelB, the effect of the
different deletions on the muscle-specific splicing of pCM
SERCA2
transcripts in 10T1/2 cells is shown, while panelC illustrates the effect of the shortest (Del4) and the longest
(Del3) deletion on the processing of pCM
SERCA2 transcripts in
undifferentiated ( U) and differentiated ( D)
BC
H1 cells. 10T1/2 cells and BC
H1 cells were
stably transfected with the deletions indicated at the top of
the gel. Total RNA extracts were prepared, and class 1 transcripts were
amplified by PCR using the U1 sense primer and the P2 antisense primers
(10T1/2, 30 cycles; BC
H1, 25 cycles). Representations of
the amplified fragments and the primers are shown at the left of the gel. In the control lane ( C), the cDNA samples
were replaced by water.
H1 cells ( i.e. only class 2/3 mRNAs in non-differentiated cells and the induction
of class 1 mRNAs during differentiation) depends on three conditions:
(i) removal of the terminal intron is an inefficient process so that
splicing is a very improbable event in non-differentiated cells; (ii)
the processing sites (pA
and 5`D2) competing with the use
of the muscle-specific splice sites are weak and do not interfere with
muscle-type processing during differentiation; (iii) the efficiency of
the muscle-type splice process is increased during differentiation.
Terminal Intron Removal as an Inefficient
Process
To obtain regulated SERCA2 transcript processing,
splicing of the terminal intron has to be an inefficient process in
non-differentiated cells. We have identified two factors that
contribute to the low probability of splicing in non-differentiated
cells. First, the 5`D1 splice donor site is a weak processing site. The
5`D1 site deviates from the consensus U1snRNP binding site in three
positions (-2, +5, +6), suggesting that it is a weak
site. When the 5`D1 site was optimized, splicing was induced in
undifferentiated cells, implying that the wild type 5`D1 is indeed a
weak site. Splicing was further up-regulated during differentiation,
which indicates that changing the 5`D1 donor site to an optimal donor
site increased the probability of splicing in non-differentiated cells
but did not induce a maximal level of splicing in these conditions. The
observation that optimizing the 5`D1 donor was on its own not
sufficient for maximal splice up-regulation in non-differentiated cells
suggested that other factors were involved in lowering the efficiency
of terminal intron splicing.
H1 cells. The effect of the deletions cannot be ascribed
to the removal of a specific regulatory sequence, as deleting stretches
of approximately equal length but in different zones had nearly
identical effects on splice activation. In addition, the normal splice
pattern was restored by substituting the intron deletion (Del1) with a
non-related sequence derived from
-phage DNA
(15) . We also
observed that reducing the intron from its original length of 3389 to
950 nt had only a small effect on splice activation, whereas a further
reduction to 460 nt greatly increased splice efficiency. This suggests
that there is a critical intron length between 460 and 950 nt, above
which splicing becomes inefficient. Interestingly, this critical intron
length correlates with the average terminal intron length (681
nucleotides) as observed in vertebrates
(27) . We therefore
conclude that the relatively long terminal intron in the SERCA2 gene
contributes to the inefficiency of splicing as observed in
undifferentiated cells. An effect of intron length on splice efficiency
has only been previously described for the splicing of the mouse µ
heavy chain mRNA
(28, 29) . In this model, it was shown
that shortening the distance between exon C4 and exon M strongly
promoted the splicing pathway relative to a competing polyadenylation
process at a site located in the intervening sequence.
CAG versus consensus,
CT(G/C) AC(Py)
CAG)
(30, 31) . Moreover, the 3`-acceptor site was readily
used in non-muscle cells when either the 5`D1 was optimized or when the
intron was shortened. In view of this, we do not think that a block at
the level of the branch point/3`-acceptor significantly contributes to
the inefficiency of SERCA2 splicing.
The Polyadenylation Site (pA
When
splicing becomes activated during muscle differentiation, it has to
compete with two other processes. First, the messenger can also be
polyadenylated at the upstream polyadenylation site in the terminal
intron; second, a neuron-specific donor site (5`D2) downstream of the
muscle-specific donor site (5`D1) can be used. To explain why these
sites are poor competitors for the muscle-type splicing in
differentiated BC )
and the Second Donor Site (5`D2) Are Poor Competitors
H1 cells, we changed the strength of these
processing sites.
Increase in Splice Efficiency during Myogenic
Differentiation
Splicing of the SERCA2 terminal intron is
an improbable event in non-differentiated cells. Our data indicate that
the limitations imposed by the weak donor site and the long terminal
intron are overcome during myogenic differentiation. This is most
likely due to trans-acting factors induced during differentiation that
increase the likelihood of splicing such that it becomes the main
processing event for the SERCA2 gene transcript. The selection and
usage of the 5`-splice site is a complex process in which both snRNPs
(U1snRNP, U5snRNP, and U6snRNP) and protein splice factors (like
ASF/SF2 and SC35) are involved (for a recent review, see Horowitz and
Krainer
(32) ). A critical step for 5`-splice site selection and
usage seems to be the base pairing between the donor splice site and
the 5`-end of the U1snRNA. Consequently, the usage of the 5`D1 donor
site can be enhanced by factors that facilitate U1snRNP binding to the
donor splice site. Eperon et al.(33) have
demonstrated that SR proteins can stabilize binding of U1snRNP to
5`-splice sites. This stabilizing function may become especially
important when the donor site diverges from the consensus binding
sequence, as is the case for 5`D1. Trans-acting factors expressed
during differentiation must also overcome the inhibitory effect of the
long terminal intron. It has been demonstrated that splice factors of
the SR family interact at the 5`-donor site via the U1-70K protein and
also with the 3`-acceptor region via the U2AF protein,
suggesting that SR splice factors may play a role in approximating
donor and acceptor sites during spliceosome formation
(33, 34, 35, 36) . However, whether SR
proteins are involved in the activation of SERCA2 splicing either by
up-regulating the use of the 5`D1 or by bridging the long distance
between 5`D1 and the 3`-acceptor splice site remains an open question.
-ATPase; 5`D1, 5`-donor
splice site 1; 5`D2, 5`-donor splice site 2; pA
, upstream
polyadenylation site; pA
, downstream polyadenylation site;
SPA, strong polyadenylation signal; nt, nucleotide(s); PCR, polymerase
chain reaction.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.