©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sequence and Spatial Requirements for Regulated Muscle-specific Processing of the Sarco/Endoplasmic Reticulum Ca-ATPase 2 Gene Transcript (*)

Luc Mertens(§)(¶) , Ludo Van Den Bosch(§)(**) , Hilde Verboomen , Frank Wuytack , Humbert De Smedt , Jan Eggermont (§§)

From the (1) Laboratory of Physiology, University of Leuven (KULeuven), Campus Gasthuisberg, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca 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 BCH1 myogenic cell line using a minigene containing the 3`-end of the SERCA2 gene. In undifferentiated BCH1 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.


INTRODUCTION

The Ca-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) .

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.


Figure 1: Processing of pCMSERCA2 transcripts during differentiation of transfected BCH1 cells. A, schematic representation of the pCMSERCA2 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 pCMSERCA2 transcripts during differentiation. BCH1 cells were stably transfected with pCMSERCA2, 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 BCH1 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.

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). 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.


MATERIALS AND METHODS

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 BCH1 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 BCH1 cells except that 10% fetal calf serum was used in the growth medium.

pCMSERCA2 Modifications

The construction of the minigene containing the 3`-end of the SERCA2 gene has been previously described (15) . The pCMSERCA2 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) .

Mutations in the construct were introduced using the Altered Sites mutagenesis system (Promega). An XbaI- XbaI or an XbaI- AflII restriction fragment from pCMSERCA2 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 pCMSERCA2, 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 pCMSERCA2(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 pCMSERCA2(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.

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.

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 BCH1 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.


RESULTS

pCMSERCA2 Processing during Myogenic Differentiation of BCH1 Cells

BCH1 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 BCH1 cells (16, 23) . Furthermore, when BCH1 cells were stably transfected with the pCMSERCA2 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 BCH1 cells that were stably transfected with the pCMSERCA2 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 BCH1 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 BCH1 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 BCH1 cells. A, schematic representation of the modifications made to the SERCA2 part of pCMSERCA2. 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 pCMSERCA2 transcripts. BCH1 cells were stably transfected with the modified pCMSERCA2 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 BCH1 differentiation. A, schematic representation of the modifications made to the SERCA2 part of pCMSERCA2. 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 pCMSERCA2 transcripts. BCH1 cells were stably transfected with the modified pCMSERCA2 constructs indicated at the top of the gel. Total RNA extracts were prepared from undifferentiated ( U) and differentiated ( D) BCH1 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 pCMSERCA2 transcripts. Total RNA extracts were prepared from undifferentiated ( U) and differentiated ( D) BCH1 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 BCH1 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 BCH1 differentiation. A, schematic representation of the modifications made to the SERCA2 part of pCMSERCA2. 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 pCMSERCA2 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 BCH1 cells were stably transfected with the modified pCMSERCA2 constructs indicated at the top of the gel. Total RNA extracts were prepared from undifferentiated ( U) and differentiated ( D) BCH1 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 BCH1 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 pCMSERCA2. 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 pCMSERCA2 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 pCMSERCA2 transcripts in undifferentiated ( U) and differentiated ( D) BCH1 cells. 10T1/2 cells and BCH1 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; BCH1, 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.




DISCUSSION

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 BCH1 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.

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 BCH1 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.

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)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 ) and the Second Donor Site (5`D2) Are Poor Competitors

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 BCH1 cells, we changed the strength of these processing sites.

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.

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to this work.

A research assistant for the National Fund for Scientific Research (NFWO, Belgium). To whom correspondence should be addressed. Tel.: 32-16-34-58-34; Fax: 32-16-34-59-91; E-mail: Luc.Mertens@med.kuleuven.ac.be.

**
A postdoctoral researcher for the National Fund for Scientific Research (NFWO, Belgium).

§§
A research associate for the National Fund for Scientific Research (NFWO, Belgium).

The abbreviations used are: SERCA, sarco/endoplasmic reticulum Ca-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.


ACKNOWLEDGEMENTS

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.


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