Natural Trans-spliced mRNAs Are Generated from the Human Estrogen Receptor-α (hERα) Gene

The human estrogen receptor-α (hERα) gene is a complex genomic unit exhibiting alternative splicing and promoter usage in a tissue-specific manner. During the investigation of new hERα mRNA variants by rapid amplification of 5′ cDNA ends, we identified a cDNA in which the acceptor site of exon 1A, into which the different leader exons are normally alternatively spliced, was spliced accurately the 3′ extremity of exon 1A (scrambled 1A→1A hERα cDNA). Reverse transcription-PCR and S1 nuclease mapping analysis revealed that 1A→1A hERα transcripts were not circular RNAs constituted by exon 1A only but corresponded to linear polyadenylated hERα RNAs composed of the eight coding exons of the hERα gene and characterized by a duplication of exon 1A. Genomic Southern blot experiments excluded the hypothesis of duplication of hERα exon 1A in the human genome. Therefore, these data suggested that 1A→1A hERα transcripts were likely generated by trans-splicing. The production of such transcripts by trans-splicing of pre-mRNAs generated from a chimeric gene formed by a single hERα exon 1A, exon 2, and their flanking intronic regions was demonstrated in transient transfection experiments. Therefore, in addition to the alternative cis-splicing, the hERα gene is also subject to natural trans-splicing.

The estrogen receptor-␣ (ER␣) 1 is a ligand-inducible transcription factor that belongs to the steroid, thyroid hormone, and retinoic acid receptor family (1)(2)(3). As all members of this family, it modulates transcription of specific sets of genes by interacting either in a protein/DNA manner with cognate DNA sequences called responsive elements or in a protein/protein manner with other transcriptional factors (1)(2)(3)(4)(5).
ER␣ is a key component of a wide range of biological processes. Its main role is in the control of the reproductive functions such as the establishment and maintenance of female sex differentiation characteristics, reproductive cycle, and pregnancy (6,7). ER␣ is also involved in liver, fat, and bone cell metabolism, cardiovascular and neuronal activity, and embry-onic and fetal development (6,7). Finally, due to the mitogenic effect of its ligand, ER␣ is intimately associated with the biology of endometrium and breast cancers (8 -10).
ER status is used clinically both as a prognostic factor and as a target in the therapy of breast cancers (9). Patients with ER-positive tumors have a better prognosis than those with tumors that lack ER expression. The benefits of the anti-estrogen therapy are almost limited to these patients, although quite a number of ER-positive tumors do not respond to endocrine therapy (8,9). The resistance to hormonal therapy has often been associated with genetic defects within ER biology (11,12). Thus, the identification of the molecular mechanisms controlling ER␣ expression and function and those that may impair ER␣ biology turned out to be a crucial step for understanding the involvement of the estrogen receptor into several physiological and pathological processes.
Mapped to the long arm of chromosome 6 (13), the human ER␣ gene is over 140 kb in length with a coding region split into eight exons (14). Our laboratory has recently shown that this gene is in fact a complex genomic unit exhibiting alternative splicing and promoter usage in a tissue-specific manner (15,16). Using the rapid amplification of cDNA ends (RACE) methodology, we have isolated and characterized several new hER␣ cDNA isoforms and demonstrated that the hER␣ transcripts are produced from a single gene by the use of multiple promoters (16). Most of these hER␣ transcripts (A-F) encode a common ER␣ protein, hER␣ 66, but differ in their 5Ј-untranslated region as a consequence of an alternative splicing of several upstream exons (1B-1F) to a common acceptor site located in exon 1A, 5Ј to the initiation of translation codon. A new class of hER␣ transcripts that lack the first coding exon (exon 1A) of the ER␣ gene was also identified (17). These ⌬1A hER␣ transcripts originate from the E and F hER␣ promoters and encode the new N-terminal 173-amino acid truncated hER␣ 46 isoform (17).
During the RACE investigation, we amplified a hER␣ cDNA fragment in which the 3Ј extremity of exon 1A was spliced directly to the acceptor site of the same exon 1A that normally receives the alternative upstream exons 1B-1F. In this present study, we demonstrate that this RACE product was not an artifact but rather results from the amplification of a hER␣ cDNA with a duplication of exon 1A. The new hER␣ transcripts correspond to trans-spliced mRNA.
RNA Isolation-Total RNA from cell lines and tissues was extracted with TRIzol (Invitrogen) as described by the manufacturer. Total RNA from human mammary gland, human endometrium, human brain, human liver, and human skeletal muscle were purchased from CLON-TECH. Human pituitary RNA was kindly provided by Professor J. Duval (Université de Rennes, Rennes, France).
Plasmid Construction-The pCR-hER␣ Luc plasmid was constructed as follows: the coding region of the luciferase gene was amplified from the pGL2 vector (Promega) using flanking primers with BamHI restriction sites and was then inserted in the BamHI site of pCR 3.1 (Invitrogen) to obtain the pCR 3.1 Luc plasmid. The genomic fragments, a, b, and c (see Fig. 6), were amplified from the GHER 1 and 3 clones in Bluescript (14) using the following primers: XbaI-a5Ј (5Ј-ACGTTCTA-GATCGCGTTTATTTTAAGCCCAGTCTT-3Ј) and XhoI-a3Ј (5Ј-ACGTC-TCGAGCAGGTAGTAGGGCACCTGCTG-3Ј) for fragment a; XhoI-b5Ј (5Ј-ACGTCTCGAGGAGAACGAGCCCAGCGCCTAC-3Ј) and M13 primer for fragment b; and M13 primer and KpnI-c3Ј (5Ј-ACGTGGTA-CCAGCATAGTCATTGCACACTGC-3Ј) for fragment c. Fragment a was digested by XbaI and XhoI and subcloned into the XbaI/XhoI site of pST 1 Blue vector to form pST 1 blue vector a. Fragment b was digested by XhoI and EcoRI (site contained in Bluescript sequences) and subcloned into the XhoI/EcoRI site of pST 1 Blue vector a to form pST 1 blue vector aϩb. Fragment c was digested by EcoRI and KpnI and subcloned into the EcoRI/KpnI site of pST 1 Blue vector aϩb to form pST 1 blue vector aϩbϩc. Finally, pST 1 blue vector aϩbϩc was digested by NheI and KpnI, and the fragment NheI-aϩbϩc-KpnI was then inserted in NheI/KpnI site of the pCR 3.1 Luc plasmid to form the pCR-hER␣ Luc plasmid.
RACE-The trans-spliced hER␣ mRNA (1A31A) was cloned by an inverse PCR method (18). Reverse transcription of MCF7 total RNA (10 g) and second-strand synthesis were performed using a commercial kit (Invitrogen) as recommended by the manufacturer except that the hER␣ gene-specific primer IV (5Ј-CTCACAGGACCAGACTCCATAAT-GGTA-3Ј) located in exon 2 was used instead of the usual oligo(dT) primer (see Fig. 1). Subsequently, the cDNA was circularized in the presence of T4 DNA ligase and submitted to 35 rounds of PCR amplification using the sense primer X (5Ј-ACTCAACAGCGTGTCTCCGAG-3Ј) and the antisense primer VI (5Ј-TTGGATCTGATGCAGTAGGGC-3Ј) (see Fig. 1). The main PCR product was subcloned in the TA cloning vector pCR 2.1 (Invitrogen) and then sequenced by the dideoxy chain termination method.
2.5 l of the reverse transcriptase reactions resulting from primer I were used in two rounds of 30-cycle PCR amplification (see Fig. 5B). The 5Ј primer and nested primer used were X and VIII, respectively. The 3Ј primer II (5Ј-ATTATCTGAACCGTGTGGGAG-3Ј) and the nested primer III (5Ј-CGTGAAGTACGACATGTCTAC-3Ј) were from the 3Јuntranslated region of hER cDNAs (exon 8). Both rounds of amplification were performed using the Expand TM long template PCR system (Roche Molecular Biochemicals) as recommended by the manufacturer.
Finally, single-stranded cDNAs reverse-transcribed from primer L1 were subjected to either a 30-cycle PCR amplification using the 5Ј primer VIII and the 3Ј primer L2 (5Ј-CGGGCCTTTCTTTATGTTTT-3Ј) (see Fig. 6A) or two rounds of 30-cycle PCR amplification using the 5Ј primer X and nested primer XI with the 3Ј primer L2 and nested primer VI (see Fig. 6B).
Modified S1 Nuclease Mapping-Biotinylated single-stranded DNA templates were used to prepare highly labeled single-stranded DNA probes by extension from a specific primer with T7 DNA polymerase in the presence of [␣-32 P]dCTP (3000 Ci/mmol) (19). The origin of probe 1A31A (see Fig. 2A) template was an RT-PCR product that was amplified using the upstream primer XII (5Ј-GGCCCGCCGGCATTCTA-CAG-3Ј, located in exon 1A) with the downstream primer V and then subcloned downstream of T7 in the TA cloning vector pCR 2.1 (Invitrogen). A PCR reaction was performed using a biotinylated T7 primer with M13 reverse primer to obtain probe 1A31A template. To prepare the template used to make the probe 1A31A-2 (see Fig. 4B), an RT-PCR reaction was performed with the 5Ј primer XIII (5Ј-GGCCCGC-CGGCATTCTACAGGTGGCCCGCCGGTTTCTGAC-3Ј, the primer mapping the splice junction 1A31A) and the 3Ј primer XIV (5Ј-CAGAT-TCCATAGCCATAC-3Ј, located in exon 2). The RT-PCR product was subcloned downstream of T7 in the TA cloning vector pCR 2.1 (Invitrogen), and a PCR reaction was performed using a biotinylated T7 primer with M13 reverse primer.
Biotinylated PCR products were bound to streptavidin-coated magnetic beads (Dynal) as recommended by the manufacturer, and the nonbiotinylated DNA strands were removed by denaturation with 0.1 M NaOH. 1A31A and 1A31A-2 S1 single-stranded DNA probes were obtained by extending the respective V (in exon 1A), and XIV (in exon 2) primers annealed to the corresponding biotinylated single-stranded template. After elution of the single-stranded DNA probes by alkaline treatment and magnetic separation, the probe was then purified on a sequencing gel. 10 5 cpm of probe were coprecipitated with 30 g of total RNA and then dissolved in 20 l of hybridization buffer (80% formamide, 40 mM PIPES, pH 6.4, 400 mM NaCl, 1 mM EDTA, pH 8) denatured at 70°C for 10 min and hybridized overnight at 55°C. Digestion with S1 digestions were then carried out as described previously (20), and the samples were electrophoresed through a denaturing polyacrylamide/ urea gels.
Southern Blot-20 g of human genomic DNA (CLONTECH) were digested with EcoRI and BamHI restriction enzymes (Roche Molecular Biochemicals), resolved on 0.8% agarose gel, transferred to a nylon membrane (Hybond Nϩ, Amersham Biosciences), and hybridized with the random primed 32 P-labeled probe 1A, as recommended by the manufacturers. Probe 1A is a genomic fragment from exon 1A (ϩ171 to ϩ610 (21)) obtained by PCR amplification.

RESULTS
Evidence for the Existence of 1A31A hER␣ Transcripts with the Donor Site of Exon 1A Joined to the Acceptor Site of Exon 1A-To amplify new 5Ј mRNA extremities of the hER␣ gene, a 5Ј RACE approach based on a variation of the inverse PCR technique was performed on MCF7 hER␣ cDNA synthesized from primer IV located in exon 2 (Fig. 1A). Sequence analysis of the main RACE product (282 bp) showed that it corresponded to scrambled 1A31A hER␣ transcripts with the donor site of exon 1A joined to the acceptor site of exon 1A (Fig. 1B), a site where the alternative upstream exons 1B-1F are normally spliced (16). It should be noted that the hER␣ cDNA circularization step in the 5Ј RACE approach was not required to amplify the 1A31A hER␣ RACE product, which might explain its abundance. To confirm the existence of such hER␣ transcripts, an S1 nuclease mapping experiment was performed on total RNA from various tissues or cell lines. The singlestranded DNA S1 probe 1A31A was prepared from a 1A31A hER␣ RT-PCR product as described under "Experimental Procedures." This probe included 3Ј end exon 1A sequences spliced to 5Ј end exon 1A sequences and thus would not be completely protected if the standard transcripts were the only species presents. After hybridizing probe 1A31A with the RNA samples and S1 nuclease digestion, two protected fragments of 296 and 316 nucleotides were detected ( Fig. 2A). As expected, the smallest fragment corresponded to normal A-F hER␣ mRNAs, which remained homologous to probe 1A31A as far as the acceptor splice site of exon 1A and then diverged in their 5Ј ends from probe complementary to 1A31A sequences (16). The level and pattern of distribution of these hER␣ mRNAs were as described previously (16). The second protected fragment corresponded in size to a full protection of a hER␣-specific se- Open boxes indicate the unique (1A-1F) and the two first common (1A, 2) exons encoding each hER␣ mRNA isoform. The initiation codon AUG and the acceptor splice site position in exon 1A are indicated. The approximate locations of primers used for the RACE are shown by short arrows. Primer IV, located in exon 2, was used to prime hER␣ cDNA synthesis by reverse transcriptase. After cDNA circularization, the 5Ј RACE products were amplified with the sense primer X and the antisense primer VI located in the coding part of exon 1A. The oligonucleotide probe (P1) from the common part of exon 1A was used to confirm the specificity of the PCR products. In B, the hER␣ RACE products were amplified from MCF7 total RNA as described above. Yeast total RNA was used as a negative control. PCR products were electrophoresed through an agarose gel and transferred by Southern blot to a membrane that was then hybridized with the oligonucleotide probe P1 as described under "Experimental Procedures." Positions of migration of the molecular size markers are shown on the left side of the figure. In C, the sequence of the main RACE product revealed a hER␣ cDNA with the donor site of exon 1A joined to the acceptor site of exon 1A.
FIG. 2. 1A31A hER␣ transcript distribution analysis. In A, S1 nuclease mapping analysis was performed as described under "Experimental Procedures" with the single-stranded probe 1A31A and 30 g of total RNA from various sources as indicated at the top of each lane. Yeast total RNA was used as a negative control. The location and the size of the single-stranded probe 1A31A and the protected fragments obtained after S1 digestion are indicated. The probe was specific for 1A31A hER␣ transcripts but was also able to partially protect the A/F hER␣ mRNA isoforms (⌺-(1A31A hER␣ transcripts)) up to the splice site position. B, RT-PCR analysis. Approximate locations of primers are shown by short arrows. Primer V, located in exon 1A, was used to prime hER␣ cDNA synthesis by reverse transcriptase using total RNA from various sources as indicated at the top of each lane. Yeast total RNA was used as a negative control. The PCR amplification of 1A31A hER␣ cDNA was performed with the sense primer X and the antisense primer VI, both located in exon 1A. An oligonucleotide probe P1 was used to confirm the specificity of the PCR products. quence of the probe and therefore resulted from a hybridization with 1A31A hER␣ transcripts ( Fig. 2A). It was only weakly detected from MCF7 and T47D RNA samples. To study the tissue distribution of 1A31A hER␣ transcripts by a more sensitive approach, an RT-PCR analysis was performed on the RNA samples reverse-transcribed from a hER␣ gene-specific primer (V) chosen in exon 1A (Fig. 2B). This study showed that 1A31A hER␣ transcripts were detected by RT-PCR in tissues and cell lines expressing a relatively high level of normal hER␣ transcripts, for instance the mammary gland and the cell lines MCF7, T47D, and ZR75, which derive from this tissue, the endometrium, and the liver (Fig. 2B). It should be noted, however, that no amplification of 1A31A hER␣ transcripts was obtained from ovary despite the detection of normal hER␣ transcripts by S1 nuclease mapping in this tissue.
1A31A hER␣ Transcripts Likely Result from a Trans-splicing Reaction-To determine whether a hER␣ exon 1A duplication is present in the genome, human genomic DNA was digested with EcoRI and BamHI restriction enzymes and hybridized with an exon 1A probe (Fig. 3). The results of the Southern blot of both genomic digestions revealed a single hybridizing band, the size of which was in total agreement with the restriction enzyme map of the GHER␣ clones published previously (14). Therefore, exon 1A of the hER␣ gene was not duplicated.
Two other mechanisms might explain the detection of 1A31A hER␣ transcripts: 1) the formation of circular 1A31A hER␣ transcripts constituted by exon 1A only or 2) a transsplicing reaction occurring naturally between two hER␣ pre-mRNAs (Fig. 4A). In this last case, 1A31A hER␣ transcripts should contain additional exons of the hER␣ gene. To discriminate between these two hypotheses, an S1 nuclease mapping experiment was carried out using a probe designed to protect trans-spliced hER␣ transcripts with a 1A31A-2 exon organization. Thus, if a trans-splicing reaction occurs for the hER␣ gene, then the corresponding protected fragment would be 624 nucleotides in size. On the other hand, the protection of a circular 1A31A hER␣ transcript by probe 1A31A-2 would give rise to a fragment of the size of exon 1A, 521 nucleotides. The S1 nuclease mapping analysis of MCF7 total RNA by probe 1A31A-2 is shown in Fig. 4B. In addition to the 604-nucleotide fragment that results from a protection of probe 1A31A-2 by normal hER␣ transcripts up to the acceptor splice site of exon 1A, the results also showed a protected fragment of 624 nucleotides in size, thus demonstrating the trans-splicing origin of the scrambled 1A31A hER␣ transcripts. This result was strengthened by the detection of the 1A31A-2 protected fragment in the RNA poly(A ϩ ) fraction, which indicated that 1A31A hER␣ transcripts are polyadenylated molecules. Finally, no protected fragment corresponding in size to circular 1A31A hER␣ transcripts was seen in this S1 nuclease mapping experiment.
Since 1A31A hER␣ transcripts should contain the remaining exonic segments of the hER␣ gene that a trans-splicing process would be likely to generate, the exonic organization of 1A31A hER␣ transcripts was investigated. Firstly, to verify that a full exon 1A was present in 5Ј to the 1A31A junction, hER␣ transcripts from various sources of RNA were reversetranscribed from primer V in exon 1A, and two rounds of PCR were then performed to amplify a fragment of 1A31A hER␣ cDNAs containing the anticipated sequences as illustrated in Fig. 5A. A PCR product of the expected size was amplified from the tissues or the cell lines in which the 1A31A hER␣ transcript was detected previously by RT-PCR (Fig. 5B). The specificity of this product was further confirmed by Southern blot using the exon 1A-specific oligonucleotide probe P2. Secondly, to demonstrate that full-length 1A31A hER␣ transcripts had hER␣ sequences from exon 1A through to exon 8 (3Ј to the 1A31A junction), PCR analysis was performed on singlestrand cDNAs synthesized using a hER␣ gene-specific primer (I) chosen from the hER␣ mRNA 3Ј-untranslated region sequences (exon 8, Fig. 5B). 1A31A hER␣ cDNAs were amplified by two rounds of PCR using the 3Ј primer II and nested primer III located upstream from primer I in exon 8 in combination with the 5Ј primer X and nested primer VIII (Fig. 5B). It should be noted that the first round of PCR amplified both 1A31A and normal hER␣ cDNAs. Only the second round allowed to be specifically amplified 1A31A hER␣ cDNAs. Results showed that the size of the amplified cDNAs was as expected, and after Southern blotting, the hybridization of these PCR products with various oligonucleotide probes recognizing specifically the different eight coding exons of the hER␣ gene demonstrated that sequences from exon 1A to exon 8 were present in 1A31A hER␣ transcripts (Fig. 5B only shows the results obtained with the exon 1A-specific oligonucleotide probe P2). In conclusion, these data clearly demonstrated the existence of a new class of hER␣ mRNAs that presents a duplication of exon 1A and which is likely generated by a trans-splicing event between two hER␣ pre-mRNAs.
A Chimeric Gene Containing hER␣ Exon 1A, the 5Ј Part of Exon 2, and Their Flanking Intronic Sequences Generate Trans-spliced 1A31A Transcripts-To further define the mechanism generating 1A31A hER␣ transcripts, a chimeric gene called pCR-hER␣ Luc was constructed and analyzed for its ability to generate 1A31A trans-spliced transcripts after transient expression in the MCF7 cell line. pCR-hER␣ Luc was formed by the cytomegalovirus promoter, the hER␣ genomic region from exon 1B to an EcoRI restriction site in the 3Јflanking intronic region of exon 1A, a part of hER␣ exon 2 and its 5Ј-flanking sequence to an EcoRI restriction site, the luciferase coding region, and the 3Ј-untranslated region of the bo- vine growth hormone (see "Experimental Procedures" for the construction of pCR-hER␣ Luc) (Fig. 6A). To discriminate 1A31A hER␣ cDNAs generated from the chimeric gene from those arising from the standard hER␣ gene expression in MCF7 cells, an XhoI restriction site was created in the 3Ј extremity of exon 1A of pCR-hER␣ Luc gene. Thus, total RNA prepared from MCF7 transiently transfected with pCR-hER␣ Luc was used to reverse-transcribe hER␣ Luc mRNA from primer L1 located in the 5Ј end of the luciferase coding region. As illustrated in Fig. 6A, hER␣ Luc mRNA was accurately matured since the size of the hER␣ Luc cDNA PCR-amplified between exon 1A and the luciferase coding region indicated that exon 1A was spliced as expected to exon 2 with the removal of the ϳ2.5-kb intronic region. Then, in attempt to amplify trans-spliced 1A31A hER␣ Luc cDNAs, two rounds of PCR were performed on hER␣ Luc cDNAs reverse-transcribed from primer L1 as described in Fig. 6B. The result showed one main PCR product, the size of which was in agreement with the one expected from the amplification of 1A31A cDNAs (Fig.  6B). This result was confirmed by Southern blotting and by hybridization of the PCR product with two oligonucleotide probes, P1 and P3, that recognized the 5Ј and 3Ј extremities of exon 1A, respectively. Finally, the digestion of the amplified cDNA by the restriction enzyme XhoI generated two fragments of the expected size, which were selectively identified by the two probes P1 and P3, thus confirming the pCR-hER␣ Luc chimeric gene origin of the amplified trans-spliced 1A31A cDNA. Used as a control, trans-spliced 1A31A hER␣ cDNAs generated from the endogenous hER␣ gene were also analyzed in parallel. These data demonstrated that a single model gene FIG. 4. 1A31A hER␣ transcripts are linear polyadenylated molecules. A, schematic diagram of the two hypotheses proposed to explain the detection of 1A31A hER␣ transcripts: 1) circular RNAs constituted by exon 1A only or 2) linear RNAs formed by a trans-splicing reaction occurring naturally between two hER␣ pre-mRNAs. B, S1 nuclease mapping experiment carried out to discriminate between these two hypotheses. It was performed as described under "Experimental Procedures" with the single-stranded probe 1A31A-2, designed to protect linear trans-spliced hER␣ transcripts with a 1A31A-2 exon organization, and 30 g of MCF7 total RNA, 30 g of MCF7 poly(A) Ϫ RNA, or 0.1 g of MCF7 poly(A) ϩ RNA mixed with 30 g of yeast total RNA. Yeast total RNA (30 g) was used as a negative control. The location and the size of the single-stranded probe 1A31A-2 and the protected fragments obtained after S1 digestion are indicated. The probe was specific for 1A31A hER␣ transcripts but was also able to partially protect the A/F hER␣ mRNA isoforms (⌺-(1A31A hER␣ transcripts)) up to the splice site position. The probe was designed to contain vector sequence in its extremity (denoted by the thinner black line) to discriminate between undigested probes (Ͻ) and specific protected fragments. Positions of migration of the molecular size markers are shown on the left side of the figure.
formed by hER␣ exon 1A, exon 2, and their flanking intronic regions is able to generate trans-spliced 1A31A hER␣ transcripts and therefore contains all information required for this process.

DISCUSSION
In this investigation, we have demonstrated that the human estrogen receptor-␣ gene is able to generate novel hER␣ mRNAs by trans-splicing. The new class of hER␣ mRNAs presents a duplication of exon 1A and is referred to as transspliced 1A31A hER␣ transcripts.
Heterogeneity in the 5Ј ends of mRNAs generated by alternative promoter usage and splicing is a common feature among the members of the steroid/thyroid hormone/retinoic acid receptor gene family (22)(23)(24)(25). For the human, mouse, rat, or chicken ER␣ genes, several 5Ј end variants of ER␣ mRNAs produced by the splicing of alternative untranslated upstream exons to the first translated exon were reported (16, 26 -28). Most of these ER␣ mRNA variants were identified by 5Ј RACE. Surprisingly, when applied to human ER␣ mRNAs, this approach allowed us to amplify a new type of hER␣ cDNA. This variant, in contrast to the other amplified hER␣ cDNAs (A-F hER␣ cDNAs (16)), presented the 3Ј part of exon 1A in 5Ј to the acceptor of exon 1A, where normally the alternative upstream exons 1B-1F are spliced. The scrambled 1A31A hER␣ cDNA showed an accurate junction between the donor site of exon 1A and the acceptor site of exon 1A, which might indicate that it arises from a natural phenomena rather than a RACE artifact.
The existence of the 1A31A hER␣ transcripts in estrogen target cells was further confirmed by RT-PCR and S1 nuclease mapping experiments. In contrast to a previous report on a genomic rearrangement in an estrogen-independent subclone of the MCF7 human breast cancer cell line in which hER␣ exons (exons 6 and 7) were duplicated in an in-frame fashion (29), the hypothesis of a duplication of hER␣ exon 1A in the human genome was ruled out after a genomic Southern blot experiment. Results always revealed a single hybridizing band, demonstrating that this segment of the hER␣ gene is not duplicated. Furthermore, 1A31A hER␣ transcripts were detected in several healthy tissues, excluding a genomic rearrangement origin associated with a pathological process.
Exon scrambling is an event that has often been also associated with circular RNA molecules (30,31). Such RNAs were described for the testis-determining gene Sry in adult mouse testis (32), the human ets-1 gene (33), the human cytochrome p-450 2C18 gene in epidermis, and the rat androgen-binding protein gene in testis (31). Exons skipped during alternative pre-mRNA processing could be indeed present in a circular molecule that has the donor site of the 3Ј exon joined to the acceptor site of the 5Ј exon. Accordingly, if 1A31A hER␣ transcripts are the result of such a process, it would be expected that they are circular molecules composed of one single exon, exon 1A, joined at the 5Ј and 3Ј splice junctions, as in the case of the circular sry transcript in adult mouse testis (32). However, the presence in the transcripts of the other coding exons FIG. 5. Exonic organization of 1A31A hER␣ transcripts. A, RT-PCR analysis of the exonic organization in 5Ј to the 1A31A junction. Primer V, located in exon 1A, was used to prime hER␣ cDNA synthesis by reverse transcriptase using total RNA from various sources as indicated at the top of each lane. Yeast total RNA was used as a negative control. Primer VIII, which is located between the acceptor splice site of exon 1A and the ATG, was then used in a first round of PCR amplification with primer VI, which is nested to primer V in exon 1A. This PCR reaction should give rise to two hER␣ products as indicated on the schematic diagram. The shortest product corresponds to an amplification inside of exon 1A. To specifically reamplify 1A31A hER␣ cDNAs, a second round of PCR reaction was performed with primers IX and VII. B, RT-PCR analysis of the exonic organization in 3Ј to the 1A31A junction. Primer I, located in exon 8, was used to prime hER␣ cDNA synthesis by reverse transcriptase using total RNA from various sources. Yeast total RNA was used as a negative control. Primer X, located in the 3Ј part of exon 1A, was used in a first round of PCR amplification with primer II in exon 8. As mentioned previously for panel A, the first PCR reaction should give rise to two hER␣ products (see the schematic diagram). To specifically reamplify 1A31A hER␣ cDNAs, a second round of PCR reaction was performed with primers VIII and III. An oligonucleotide probe from exon A1 (P2) was used to confirm the specificity of the PCR products in panels A and B. Positions of migration of the molecular size markers are shown on the left side of the figures. of the hER␣ gene as well as the fact that they are polyadenylated molecules clearly demonstrates that 1A31A hER␣ transcripts are not circular RNAs but rather linear molecules that probably result from a trans-splicing event between two hER␣ pre-mRNAs.
Trans-splicing is a post-transcriptional process occurring during the mRNA maturation in which RNA segments of two independent transcripts are spliced together to generate a new mRNA specie. This mechanism was first demonstrated in trypanosomes (34) and subsequently reported in nematodes (35), flatworms (36), and plant cell organelles (37). In mammalian cells, trans-splicing events were suggested by computer analysis (38) and then by in vitro and in vivo experiments (39 -42). Mammalian cell extracts have been demonstrated to have the ability to join RNA segments together by trans-splicing (39). More recently, trans-splicing reactions between synthetic pre-mRNA substrates were shown in in vitro studies and require either a downstream 5Ј splice site or exonic enhancers (40,41). Finally, SV40 transcripts trans-spliced to each other were detected in cells transformed by an early SV40 DNA fragment (42). Naturally occurring pre-mRNA trans-splicing in mammalian cells has not been frequently reported. The first indications of its existence were based on cDNA sequencing experiments, but alternative cis-splicing could not be excluded. Recently, additional reports strengthened the idea that transsplicing events occur in mammalian cells and contribute to mRNA generation. In rat liver cells, Caudevilla et al. (43) have identified carnitine octanoyltransferase mRNA variants with a duplication of exon 2 or exons 2 and 3, which is not found in genomic DNA. Splicing experiments carried out in vitro with exon 2 plus the 5Ј-and 3Ј-adjacent intronic sequences indicated that accurate joining of two exons 2 occurs by the trans-splicing mechanism. An mRNA variant of the acyl-CoA:cholesterol acyltransferase was also shown to derive from two discontinuous precursor RNAs produced from different chromosomes (44).
Corroborating these data, we demonstrated in the present FIG. 6. A chimeric gene pCR-hER␣ Luc containing hER␣ exon 1A, the 5 part of exon 2, and their flanking intronic sequences generates trans-spliced 1A31A transcripts. In A, the plasmid pCR-hER␣ Luc was obtained after insertion of the PCR-amplified hER␣ genomic fragments a-c in pCR-Luc between the promoter CMC and the luciferase coding region, as described under "Experimental Procedures." The XhoI restriction site created in the 3Ј extremity of exon 1A is indicated by an asterisk. To test the maturation of hER␣ Luc transcripts, total RNA prepared from MCF7 transitively transfected with pCR-hER␣ Luc was used to reverse-transcribe hER␣ Luc mRNA from primer L1 located in the luciferase coding region. A 35-cycle PCR reaction was then performed with primers L2 and VIII on hER␣ Luc cDNA as well as on the pCR hER␣ Luc DNA used as a control. B, RT-PCR amplification of trans-spliced 1A31A transcripts generated from the pCR-hER␣ Luc chimeric gene. Total RNA prepared from MCF7 transitively transfected with pCR-hER␣ Luc was used to reverse-transcribe hER␣ Luc mRNA from primer L1. Primer X, which is specific for exon 1A, was then used in a first round of PCR amplification with primer L2, which is nested to primer L1. A second round of PCR reaction was performed with the primers XI and VI as illustrated on the schematic diagram. As a control, the endogenous trans-spliced 1A31A hER␣ transcript was also reverse-transcribed from primer IV and PCR-amplified using the primer XI and VI. After purification, the PCR products were or were not digested with the restriction enzyme XhoI, electrophoresed through an agarose gel, and transferred by Southern blot to a membrane, which was then hybridized with the oligonucleotide probes P1 and P3 specific for the 5Ј and 3Ј regions of exon 1A, respectively. study that natural trans-spliced 1A31A mRNA is generated from the hER␣ gene, and such a process can be mimicked in vivo with a chimeric gene containing hER␣ exon 1A, the 5Ј part of exon 2, and their flanking intronic sequences.
If 1A31A hER␣ transcripts result from a trans-splicing event, it is likely that this process would generate two products: a long mRNA having a duplication of exon 1A (1A31A hER␣ mRNA) and a short mRNA lacking exon 1A (⌬1A hER␣ mRNA). RT-PCR analysis clearly confirmed, in several ER␣positive tissues or cell lines, the presence of the long 1A31A trans-spliced hER␣ mRNA composed by the eight coding exons of the hER␣ gene including a duplication of the first coding exon, exon 1A. Upstream to the main open reading frame that encodes hER␣ 66, 1A31A hER␣ mRNA presents a second open reading frame, shared by the trans-spliced exon 1A and the 5Ј part of the acceptor exon 1A, which would encode for a protein of 156 amino acid residues, equivalent to the A/B domain of hER␣. Such a protein will be unable to bind directly DNA but would contain the transactivation domain AF1, which could constitutively act by interacting with coactivators such as SRC-1 or the p68 and p72 RNA helicases (45,46) and as a result could act independently or could modulate the binding of the hER␣ protein to its target sites. Short mRNAs lacking exon 1A have been reported previously (17). They were shown to originate from the E and F hER␣ promoters and to be produced by the splicing of exon 1E directly to exon 2. The underlying mechanism was assumed to be alternative cis-splicing, but trans-splicing cannot be excluded. The ⌬1A hER␣ transcripts encode an isoform of hER␣, hER␣ 46, lacking the first 173 amino acids present at the N terminus of hER␣ 66 and therefore devoid of the A/B domain having the AF1 transactivation function. Detected in the ER␣-positive breast carcinoma cell line MCF7, hER␣ 46 acts as an AF1-competitive inhibitor of hER␣ 66 (17).
In conclusion, the hER␣ gene was already known to be a genomic unit exhibiting significant alternative cis-splicing activities between the different untranslated first exons (16) and the coding exons (47). In this report, we demonstrate for the first time that the hER␣ gene is also subject to trans-splicing, a mechanism that generates hER␣ mRNA variants with a different exonic organization and potentially encoding new ER␣ proteins. In light of the central role that the ER␣ gene plays in the physiology of several tissues, in particular those involved in the reproductive function, such as endometrium and breast tissue, it is obvious that an increase in the occurrence of such a process might have physiological and/or pathological consequences for these tissues. The mechanisms that may modulate the trans-splicing activity of the ER␣ gene remain to be defined.