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Originally published In Press as doi:10.1074/jbc.M611126200 on March 1, 2007

J. Biol. Chem., Vol. 282, Issue 16, 11648-11657, April 20, 2007
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A Sequence Motif in the Simian Virus 40 (SV40) Early Core Promoter Affects Alternative Splicing of Transcribed mRNA*Formula

Elisenda Gendra1, Diana F. Colgan12, Brendan Meany, and Maria M. Konarska3

From the Rockefeller University, New York, New York 10021

Received for publication, December 4, 2006 , and in revised form, February 26, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
To identify new sequence elements in the promoter that affect splicing patterns of pre-mRNAs, we analyzed effects of different promoters on alternative splicing of model reporter genes. We compared the E1a alternative splicing pattern in transcripts expressed from the full-length cytomegalovirus, SV40 early, or a hybrid cytomegalovirus/SV40 early promoter and found that the hybrid promoter improved selection of the suboptimal E1a 5'SS-1. Expressing RNA from the hybrid promoter also enhanced selection of suboptimal splice sites in other alternatively spliced reporter genes, demonstrating the generality of this effect. Unlike previously defined promoter elements shown to affect alternative splicing, which were located in the enhancer/upstream activating sequences, the motif identified in this work is positioned within the core promoter; it is comprised of eight T-residues directly upstream of the SV40 early TATA box. This motif was previously implicated in DNA bending and negative regulation of transcription. Together, these results suggest that the identity of transcription complex assembled in the core promoter-dependent fashion can affect splice site selection during pre-mRNA splicing, perhaps by influencing the processivity of transcription elongation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transcription initiation, elongation, and termination, as well as mRNA capping, splicing, and polyadenylation, are all inter-connected, yet the specific mechanisms underlying these connections are not well understood. Such transcription-splicing connections were first demonstrated by different alternative splicing patterns observed depending on whether pre-mRNA splicing substrates were presynthesized or spliced co-transcriptionally (1, 2). Splicing patterns can also be affected by the rate of transcription elongation (e.g. 3–5 and reviewed in Ref. 6). A kinetic model was proposed to explain these findings; in this model, a slow RNA polymerase II (RNAPII)4 elongation rate facilitates selection of suboptimal splice sites by the spliceosome, favoring inclusion of alternative exons (typically flanked by suboptimal splice signals), whereas a rapid RNAPII elongation rate favors skipping of alternatively spliced exons. Furthermore, these studies suggested that the RNAPII elongation rate is affected by the promoter identity (4 and reviewed in Ref. 6).

Eukaryotic promoters typically contain a core segment that recruits general transcription factors (GTFs) and an upstream activating sequence (UAS) that provides binding sites for transcriptional activators (7). Transcription activation also involves the Mediator complex thought to facilitate interactions between activators and the preinitiation complex (PIC) as they assemble at the promoter (8). PIC assembly starts with binding of the GTF TFIID (composed of the TATA-binding protein TBP and associated TAFs) to the TATA box. The UAS and activators modulate TAF recruitment, which then affect subsequent recruitment of TFIIB, RNAPII, TFIIF, TFIIE, and TFIIH (9). Chromatin immunoprecipitation analyses suggest alternative pathways of PIC assembly, yielding complexes of varied composition (10). Differential expression of TBP-like and TAF-like factors in various cell types or tissues can result in a broad spectrum of functional TFIID complexes (10). Furthermore, specific sequence motifs in core promoters, the TFIIB recognition element, initiator, and downstream promoter element, can affect transcription by altering binding of GTFs and/or transcriptional activators (11). Similarly, the identity of the core promoter can influence selection of enhancer (11). Thus, core promoter sequence elements can affect transcription by altering the PIC composition and/or function.

All stages of transcription, i.e. the initiation, elongation, and termination, are functionally connected. For example, mutations in the elongation factors FACT or Spt6 also affect initiation (12–14), and deletion of the elongation factor ATPase CHD1 also affects termination of transcription (15, 16). Likewise, transcription initiation, promoter clearance, and elongation are all affected by the PAF complex, which interacts with both initiation and elongation factors and moreover affects 3' end processing and mRNA surveillance; this is consistent with a role for PAF in modulating multiple aspects of co-transcriptional events during mRNA biogenesis (17).

At all stages of transcription, the RNAPII complex directly associates with mRNA processing machineries (18, 19). A number of splicing factors are known to influence transcription. For example, in vitro, the association of small nuclear ribonucleo-protein particles with the elongation factor TAT-SF1 stimulates transcription elongation (20), and the association of U1 snRNA with TFIIH stimulates transcription initiation (21). Moreover, alternative splicing patterns can be affected in vivo if the transcription elongation rate is altered by an RNAPII mutant with a slow elongation rate, by the presence of transcriptional activators, or by pause sites in the DNA template. This reveals a functional connection between transcription elongation and splice site selection (6). In coordinating all these processes, the C-terminal domain of RNAPII is thought to serve as a binding platform for multiple transcription and processing regulators (22–24). The identity of the promoter can also affect the splicing patterns of the resulting transcript (2, 25–27). One mechanism proposed to explain these observations involves altered recruitment of splicing factors to the promoter at the stage of PIC formation. For example, the transcription factors Spi/PU.1 (28) and WT1 (29) interact with splicing factors {alpha}-CAPER and beta-CAPER (members of the U2AF65 family), affecting both transcription and splicing (30). Multiple studies suggest connections between transcription initiation and 3' end processing (31, 32). Additional connections between pre-mRNA processing events and initiation, elongation, and termination of transcription are likely to exist.

To further investigate the promoter dependence in pre-mRNA processing, we analyzed the effect of different promoters on splicing of several alternatively spliced minigene reporters. A hybrid CMV/SV40 early (C/S) promoter, in which the upstream region of cytomegalovirus (CMV) promoter was fused to the simian virus early promoter (SV40) core promoter, alters the splicing pattern of E1a, improving recognition of the suboptimal 5'SS-1 and thus increasing the levels of 11 S, 10 S and 9 S mRNA. Moreover, the CMV and SV40 early core promoters differently affect alternative splicing; the SV40 core promoter strongly improves selection of suboptimal splice sites as compared with the CMV core promoter. Mutations within the T-stretch element directly upstream of the TATA box in SV40 core promoter change these alternative splicing patterns, identifying this sequence motif as responsible for the observed promoter effect. These results suggest that the core promoter can modulate pre-mRNA expression at the level of splicing.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
E1a Minigene Constructs—The pCMV-E1a was generated by replacing the KpnI-HindIII fragment of pBSV-E1a (containing the SV40 early promoter) with the KpnI-HindIII fragment of pGL2-CMV (containing the CMV promoter). The pGL2-CMV contains the 681-nt MluI-HindIII fragment of pCDNA3.1 (Invitrogen) inserted into pGL2 (Invitrogen) between MluI-HindIII sites. The EcoRI-StuI fragment of pBSV-E1a was replaced by the 442-bp EcoRI-PvuII fragment of pBSAd22 (33) to produce pMLP-E1a. The pRSV-E1a was generated by inserting the 396-bp MluI-HindIII fragment of pRC-RSV (Invitrogen) into pCMV-E1a between MluI-HindIII sites. In p-{alpha}-globin-E1a, the KpnI-HindIII fragment of pBSV-E1a was replaced by a 554-bp fragment of human {alpha}-globin promoter amplified from genomic DNA. The pCMV-E1a U5G 5'SS-1 was generated by mutational overlapping PCR. The pC/S-E1a contains the 379-bp KpnI-NcoI fragment of pCMV-E1a (from –614 to –235) inserted into pBSV-E1a between KpnI-NcoI sites. In pC/S-E1a U5G 5'SS-1, the KpnI-HindIII fragment of pC/S replaced the KpnI-HindIII fragment of pCMV-E1a U5G 5'SS-1. For pSVEcore-E1a (containing the SV40 early promoter pos. from 5171 to 37), pBSV-E1a was cut with EcoRV-NcoI, blunt-ended, and religated. pCMVcore-E1a contained the CMV core promoter sequence from pos. +93 to –41.

Collagen Constructs—Human collagen COL1A1 gene (exons 12–16) was amplified from genomic DNA and cloned into pCMV-E1a, yielding pCMV-collagen. The plasmids pC/S-collagen, pCMVcore-collagen, and pSVEcore-collagen were generated by inserting individual promoter KpnI-HindIII fragments in pCMV-collagen.

pSXN Minigene Constructs—pCMV-ESE, the exonic splicing enhancer (ESE)-containing fragment of wild type (WT) or mutant pSXN-ESE (34) was cloned into pCMV-E1a. pSVE-ESE, pC/S-ESE, pCMVcore-ESE, and pSVEcore-ESE plasmids were generated by inserting the corresponding promoters between KpnI-HindIII sites in the pCMV-ESE construct.

pCMV-E1a Deletion Constructs—pCMV-E1a deletion constructs were generated by partial digestion of the pCMV-E1a construct with AatII followed by religation.

pCIS, pSIC, and SVEcore Promoter Mutants—pCIS, pSIC, and SVEcore promoter mutants (supplemental Fig. S1) were obtained by overlapping PCR (56).

Luciferase Constructs—Luciferase constructs were generated by subcloning promoters between KpnI and HindIII sites in pGL2 (Promega).

Transfections and Reporter Assays—Human cervical epithelial carcinoma (HeLa) and mouse hematopoietic RAW 264 cells were transfected by electroporation with reporter plasmid (10 µg) and with green fluorescent protein (1 µg, to calculate transfection efficiency) and analyzed after 48 h. For reporter assays, HeLa cells were co-transfected with 10 µg of the indicated promoter-luciferase and 1 µg of green fluorescent protein, and after 36 h, cells were counted, lysed, and assayed for luciferase activity. For each construct, three independent transfections were carried out, and all luciferase assays were performed in triplicates. Luminescence was measured by a luminometer, and the values were normalized to the efficiency of transfection.

RNA Extraction and RT-PCR Reactions—At 48 h after transfection, cells were harvested, and total RNA was isolated using TRIzol reagent (Invitrogen) and treated with DNase (Invitrogen). First strand cDNA was synthesized from 4 µg of DNase-treated RNA using Superscript reverse transcriptase III (Invitrogen). Primer sequences are: E1a-RT1 forward, 5'-CCGAAGAAATGGCCGCCAGTC-3'; E1a-RT2 reverse, 5'-GGACGCCGGGTAGGTCTTGC-3'; Col1 forward, 5'-CTTTCAAGCTTTAGGGTTTCAGTGGTTTGG-3'; Col2, reverse 5'-GCCTCTAGACCTTAGCACCAACAGCACCAG-3'; SXN1 forward, 5'-GTGGGGCAAGGTGAACG-3'; SXN2 reverse, 5'-GCCTTTGACCTACTAGTGTGGC-3'. PCR reaction products were separated in 2% agarose gels, dried, and exposed. Densitometry was performed using GE Healthcare PhosphorImager.


Figure 1
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  		FIGURE 1.
Expression of adenovirus E1a from different promoters. A, schematic representation of adenovirus type 2 E1a mRNAs. The initiation site and 5' and 3' splice sites used are indicated (position numbering according to adenovirus type 2 genome). Primers used for RT and RT-PCR are represented by black arrows. 9 S, 10 S, and 11 S mRNAs use the 5'SS-1 (pos. 636), which is skipped in 12 S and 13 S transcripts. polyA, polyadenylation signal. B, RT-PCR analysis of total RNA isolated from HeLa and RAW 264 cells transiently transfected with E1a reporters under the control of CMV (lanes 1 and 6 and 7); SVE (lane 2); RSV (lane 3); MLP (lanes 4 and 8); and human {alpha}-globin (lane 5) promoters. The position of unspliced RNA is indicated. In all experiments, the efficiency of 12 S + 13 S and 9 S + 10 S + 11 S formation (in %) was calculated from three independent transfections. Ad, adenovirus.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Promoter Identity Affects Splicing Patterns of Adenovirus E1a mRNA—To better understand the mechanism by which promoter elements affect processing of nascent transcripts, we analyzed several well characterized promoters for effects on alternative splicing patterns. An adenovirus E1a reporter was selected because its transcript is short enough to be tested in its entirety yet can be alternatively spliced to produce at least five mRNAs (13 S, 12 S, 11 S, 10 S, and 9 S). These five isoforms arise from selection of various combinations of splice sites: 5'SS-1, 5'SS-2, 5'SS-3, and 3'SS-1 or 3'SS-2 (Fig. 1A) (35).

We initially tested the human {alpha}-globin and CMV promoters because these were reported to exhibit different effects on alternative exon selection (4). We also analyzed three other promoters: Rous Sarcoma Virus long terminal repeat (RSV), adenovirus major late promoter (MLP), and SV40. Each promoter was inserted upstream of an E1a reporter that was transiently transfected into HeLa cells; after 24–48 h, total RNA was isolated and analyzed by RT-PCR, using semiquantitative PCR with a 5' end-labeled primer (Fig. 1B; see "Experimental Procedures").

For every promoter tested, RT-PCR analysis detected the five E1a isoforms; all promoters similarly expressed high levels of the isoforms derived from selecting optimal 5'SS-2 or -3 (yielding 13 S or 12 S), whereas mRNAs derived from selecting the suboptimal 5'SS-1 (11 S, 10 S, and 9 S) were less abundant (Fig. 1B) (35). Although the observed effects were subtle, quantitation of the isoform levels revealed reproducible differences between promoters. The {alpha}-globin promoter stimulated production of the 11 S, 10 S, and 9 S isoforms up to 16% of the E1a mRNAs (Fig. 1B, lane 5). By contrast, RSV and MLP promoters enabled selection of the 5'SS-1 in only 5–8% (lanes 3 and 4), whereas CMV and SV40 early promoters exhibited intermediate patterns, resulting in 5'SS-1 selection in ~11% (lanes 1 and 2). This intermediate effect of the CMV promoter is consistent with previous reports (4, 25). In parallel, mouse hematopoietic RAW 264 cells were transfected with the same set of reporter constructs, yielding similar splicing patterns (Figs. 1B and 2B, and data not shown). Thus, the described promoter effects on E1a splicing are not restricted to HeLa cells. However, in agreement with previous reports (4, 25), promoter effects on alternative splicing were rather limited with the strongest ({alpha}-globin, 16% selection of 5'SS-1) and the weakest (RSV and MLP, 5–8% selection of 5'SS-1) effects differing only by 2-fold.

Hybrid CMV/SV40 (C/S) Promoter Alters the E1a Splicing Pattern by Improving Selection of the Suboptimal 5'SS-1—The E1a 5'SS-1 at position 636 deviates from the 5'SS consensus; the non-consensus U at intron position +5 destabilizes 5'SS:U6 snRNA pairing, affecting positioning of the 5'SS for the first step of splicing (36, 37). Indeed, a 5'SS-1 mutation (U5G) that restores the consensus G at the intron position +5 significantly increased CMV-driven expression of 11 S, 10 S, and 9 S mRNAs. 5'SS-1 selection increased from 9% (U5, WT) to 26% (U5G) (Fig. 2B, cf. lanes 1 and 2). Thus, the non-consensus U at pos. +5 contributes to the skipping of 5'SS-1. However, even when 5'SS-1 was mutated to match the consensus, it was mostly skipped (74%) when expressed from the fulllength CMV promoter.

5'SS-1 selection was substantially improved when expressed from a hybrid CMV/SV40 early promoter (C/S). The SV40 promoter (366 bp) consists of two tandem copies of a 72-bp repeat (which acts as an enhancer), three copies of the 21-bp repeat, and the 17-bp AT-rich region that spans the TATA box (pos. 15–31). We generated a hybrid promoter (C/S) in which the 72- and 21-bp repeat segments of SV40 were replaced by an upstream fragment of the CMV promoter. Specifically, in the C/S construct, the 109-bp minimal SV40 early core promoter sequence (pos. 5171 to –37) was fused to the 379-bp segment of the CMV enhancer/promoter (pos. –614 to –235 relative to the CMV +1 start site) (Fig. 2A and supplemental Fig. S1). Expression of E1a from the hybrid C/S promoter yielded splicing patterns substantially different from those observed using the CMV promoter; transcripts utilizing the suboptimal 5'SS-1 (11 S, 10 S, and 9 S) collectively represented 35% of the total (Fig. 2B, lane 3), in contrast to only 9% when expressed from the full-length CMV promoter (lane 1). This difference was also evident when the U5G 5'SS-1 E1a mutant was expressed from the C/S promoter; in this case, the 5'SS-1 was selected in 84% of total mRNA, in contrast to 26% when expressed from the full-length CMV promoter. Again, similar promoter effects were observed in RAW 264 cells (Fig. 2B and data not shown). Thus, the hybrid C/S promoter substantially enhances selection of the 5'SS-1; this effect could result either from stimulation of the suboptimal 5'SS or from repression of other sites.


Figure 2
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  		FIGURE 2.
Effect of the hybrid C/S promoter and deletions within CMV promoter on E1a expression. A, schematic diagram of CMV, SVE, and C/S promoters. The TATA box motifs are shown as open boxes, and the positions of SV40 early (SVE EES1 and EES2) and CMV transcription starts sites are indicated by arrows. Dark boxes represent the 72- and 21-bp repeats of SV40. The positions are numbered relative to the transcription start site (+1) for CMV or according to the SV40 system (41). B, RT-PCR analysis of E1a transcripts from HeLa and RAW 264 cells: CMV (lanes 1, 2, 5, and 6) and C/S hybrid (lanes 3, 4, and 7) promoters. The 5'SS-1 was WT (U+5) in lanes 1, 3, 5, and 6 or mutated (U+5G) in lanes 2, 4, and 7. C, deletion analysis of the CMV promoter. The indicated segments in the CMV core promoter were deleted, and the resulting constructs were tested for E1a expression. D, RT-PCR analysis of total RNA from HeLa cell transfected with the CMV promoter deletion constructs, as indicated. The positions of 13 S, 12 S, 11 S, 10 S, and 9 S mRNAs are indicated.

 
The upstream CMV promoter region (pos. –514 to –138) contains a complex array of repeated sequence motifs exhibiting enhancer activity (38). The CMV fragment in the C/S promoter (pos. –416 to –37) contains only a portion of this enhancer, raising the possibility that disruption of the intact CMV enhancer element altered the E1a splicing patterns. To test this possibility, we analyzed a series of deletions in the CMV enhancer region (Fig. 2, C and D). None of the eight promoter deletions tested affected the E1a splicing pattern, suggesting that the upstream CMV promoter sequences do not contribute to splicing effects exhibited by the hybrid C/S promoter. Therefore, the observed C/S promoter-dependent effects likely arise from sequences in the SV40 early core promoter.

SV40 Core Promoter Sequences Generally Enhance Selection of Suboptimal 5' Splice Sites—The minimal core promoters of CMV (pos. –31 to +93, CMV core) or SV40 early (pos. 5171 to –37; SV40 early core, supplemental Fig. S1), which encompass the TATA box, initiator (Inr), and downstream sequences, yielded readily detectable levels of E1a RNA (Fig. 3, A and B). The SV40 core promoter displayed a splicing pattern similar to that obtained with the C/S promoter; 11 S, 10 S, and 9 S represented 41% of total E1a mRNA as compared with 10% observed using the SV40 early and CMV full-length promoters (Fig. 3B, cf. lane 3 with lanes 1 and 2). The SV40 early core promoter displayed an even stronger effect than the C/S promoter (41% versus 35%). Thus, in the absence of upstream enhancer sequences, the SV40 early core promoter facilitates selection of the suboptimal 5'SS-1. Expression from the CMV core promoter affected 5'SS-1 selection such that 11 S, 10 S, and 9 S represented 15% of total E1a mRNA as compared with 10% for the full-length CMV promoter (cf. lanes 2 and 4).

To investigate the generality of these effects, two other alternatively spliced reporters were analyzed. First, we tested the pSXN reporter (34) that contains human beta-globin exons 1 and 2 separated by a short, 34-nt alternatively spliced exon (E2) (Fig. 3C). In the absence of an ESE, the E2 is predominantly skipped due to its small size and the presence of a suboptimal 5'SS (intron positions +3 and +4 are T and G, instead of consensus A/G and A, respectively) (34). The pSXN reporters containing either a functional WT or a mutant ESE under the control of different promoters were transiently transfected into HeLa cells, and alternative splicing of E2 was monitored by RT-PCR. As a control, splicing patterns were compared with those of pSXN expressed from the RSV promoter (34) where E2 is mostly included (85% E2 inclusion) in transcripts containing a WT ESE but mostly skipped (72% E2 skipping) in the presence of a mutant ESE (Fig. 3C, lanes 1 and 2). A similar pattern was observed using pSXN-WT ESE expressed from the CMV core, hybrid C/S, or SV40 core promoters (83–87% E2 inclusion, cf. lanes 3, 5, and 7 with lane 1). However, promoter-dependent differences in splicing patterns were observed for pSXN-mutant ESE reporters; although the level of E2 inclusion was similar for RSV (28%; lane 2) and CMV (27%; lane 4) promoters, E2 inclusion was significantly improved by the C/S (80%; lane 6) and SV40 core (83%; lane 8) promoters. Therefore, as shown above for the E1a reporter, the hybrid C/S and SV40 early core promoters facilitate inclusion of a suboptimal exon in pSXN reporter.


Figure 3
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  		FIGURE 3.
The SVE core promoter affects alternative splicing patterns of E1a, pSXN, and collagen reporters. A, schematic diagram of the CMV-core and SVE-core promoters. B, RT-PCR analysis of E1a reporters expressed from SVE (lane 1); CMV (lane 2); SVE core (lane 3); and CMV core (lane 4) promoters. The efficiency of 12 S + 13 S and 9 S + 10 S + 11 S formation (in %) was calculated from three independent transfections. C, RT-PCR analysis of pSXN mRNAs expressed from RSV (lanes 1 and 2), CMV core (lanes 3 and 4), hybrid C/S (lanes 5 and 6), and SVE core (lanes 7 and 8) promoters. HeLa cells transformed with constructs containing WT ESE (lanes 1, 3, 5, and 7) or mutant (mut) ESE (lanes 2, 4, 6, and 8) were analyzed. The efficiency of ESE+ (E2 inclusion) and ESE– (E2 skipping) signals was calculated from three independent experiments. D, inclusion of COL1A exon 14 was monitored by RT-PCR using the primers indicated by arrows. Collagen mRNAs expressed from CMV (lanes 1 and 2), hybrid C/S (lanes 3 and 4), CMV core (lane 5), and SVE core (lane 6) promoters were analyzed by RT-PCR. The 5'SS flanking exon 14 was WT (lanes 1 and 3) or mutant (lanes 2 and 4–6). The efficiency of E14 inclusion and skipping was calculated from at least three independent transfections.

 
We also tested the promoter effects on alternative splicing of a collagen reporter, which comprises exons E12–16 with the intervening full-length introns of human COL1A1 collagen gene (Fig. 3D). In E14, a disease-associated 5'SS point mutation (intron position G+5 mutated to an A; G5A) results in exon skipping (39). As expected, under the control of the full-length CMV promoter, WT exon E14 is mostly included (86% E14 inclusion), but E14-5'SS G5A mutant exon is mostly skipped (16% E14 inclusion) (Fig. 3D, lanes 1 and 2). Expression of the collagen E14-G5A mutant from the CMV core promoter also yielded increased E14 inclusion (25%; lane 5). Thus, the CMV core promoter improved selection of the G5A 5'SS as compared with the full-length CMV promoter. Expression of the collagen mutant reporter from the SV40 early core promoter showed a significant improvement of the G5A 5'SS selection, yielding 48% inclusion of E14 (lane 6). However, expression from the hybrid C/S promoter yielded splicing patterns similar to those observed using the CMV full-length or CMV core promoters; E14 was predominantly skipped (23% E14 inclusion, lane 4). Thus, the C/S promoter facilitates selection of the suboptimal E1a 5'SS-1 (Fig. 2B) but not the collagen E14 G5A mutant 5'SS. Although these results do reveal some variation in promoter effects on splicing patterns, in general, the CMV core and full-length CMV promoters affected 5'SS selection only minimally, whereas selection of suboptimal 5'SS was most affected by SV40 early core and C/S hybrid promoters. Similar promoter-dependent changes of splicing patterns were observed for three different alternatively spliced reporters, arguing for the generality of these effects.

Splicing Patterns Do Not Directly Correlate with Transcription Activity of the Promoter—To examine a possible influence of the promoter strength on alternative splicing, we analyzed a series of reporters in which a luciferase gene was placed under the control of the full-length CMV and SV40 early promoters, core CMV and SV40 promoters, or the hybrid C/S promoter. Consistent with their well established, strong activity in mammalian cells, both CMV and SV40 early full-length promoters yielded high levels of luciferase activity with the CMV promoter exhibiting 8-fold higher activity than the SV40 early promoter (Fig. 4 D). As expected, CMV and SV40 core promoters displayed the lowest levels of luciferase activity, consistent with the absence of enhancer elements in these constructs, and the hybrid C/S promoter exhibited an intermediate activity (8- and 53-fold lower than full-length SV40 and CMV promoters but 426- and 24-fold higher than the SV40 and CMV core promoters, respectively).


Figure 4
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  		FIGURE 4.
The region of SVE core promoter upstream of the transcription start site is responsible for the modulation of splicing patterns in the resulting transcripts. A, schematic diagrams of the CMV, CIS, CMV core, SVE core, C/S, and SIC promoters. B, RT-PCR analysis of E1a transcripts expressed from CMV (lane 1), CIS (lane 2), CMV core (lane 3), SVE core (lane 4), hybrid C/S (lane 5), and SIC (lane 6) promoters. The efficiency of 13 S + 12 S and 9 S + 10 S + 11 S was calculated from three independent experiments. C, RT-PCR analysis of pSXN transcripts expressed from various promoters, as in B. For each construct, the efficiency of mRNAs including and skipping exon 2 was calculated from three independent transfections. D, the activity of luciferase expressed from various promoters (as indicated) was measured as described under "Experimental Procedures." The data represent the average of three independent experiments performed in triplicate. E, titration of E1a RNA concentration in RT-PCR assays. RNA from HeLa cells transfected with the E1a reporter was serially diluted with total HeLa RNA from mock-transfected cells as indicated and analyzed using the standard RT-PCR protocol.

 
Thus, alternative splicing patterns do not directly correlate with transcriptional activity of the promoter. Although CMV and SV40 core promoters have different effects on alternative splicing patterns, they both exhibit low levels of luciferase expression. The improved selection of suboptimal splice sites is not due to a simple inhibition of transcription because the full-length CMV and CMV core promoters, which differ 1284-fold in transcription activity, yield similar splicing patterns, both resulting in skipping of suboptimal splice sites. Similarly, whereas SV40 core and C/S promoters differ in transcription activity by 426-fold, they display a similar splicing pattern, both improving recognition of suboptimal sites.

RNA expression levels could influence splicing patterns by affecting the RT-PCR amplification of different RNAs. Therefore, we carried out a serial dilution of E1a RNA with a mock-transfected total HeLa RNA and analyzed E1a splicing patterns using the standard RT-PCR protocol. E1a RNAs were detected up to 10–3 dilution without any detectable change in the distribution of individual isoforms (Fig. 4E). Thus, the observed promoter-dependent differences in splicing patterns seem to reflect differences not due to the overall transcription activity but rather due to some other features conferred by the promoter.


Figure 5
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  		FIGURE 5.
The T-stretch upstream of the TATA box in the SV40 core promoter affects splicing patterns of pSXN, E1a, and collagen transcripts. A, schematic representation of SV40 early core promoter mutants. Mutations are clustered in four different groups according to their location: downstream of TATA box (M1–5); within the TATA box (pos. 14–21, M6–8); within the T-stretch (pos. 22–28, M9–11); and upstream of the T-stretch (pos. 29–35, M12–14). B, RT-PCR analysis of pSXN mRNA expressed from SVE mutant promoters, as indicated. The efficiency of 12 S + 13 S and 9 S + 10 S + 11 S formation (in %) was calculated from three independent transfections. C and D, HeLa cells were transfected with E1a (C) and collagen (D) reporters containing the SVE core promoter mutations (M9–11). The positions of spliced RNA signals are indicated. The efficiency of inclusion/skipping was calculated from three independent experiments.

 
Alternative Splicing Patterns Are Not Affected by the Nature of 5'-untranslated Regions—To test whether the sequence of the 5'-untranslated regions affects the splicing patterns, we generated two additional E1a reporter constructs: CIS and SIC (Fig. 4A and supplemental Fig. S1). In the CIS construct, the CMV core promoter, spanning the TATA box and Inr site, was linked to a 63-nt segment of SV40 sequences between its Inr element and the HindIII site. In the SIC construct, the SV40 early core promoter, including its TATA box and Inr, was linked to a 67-nt segment of CMV sequences between its Inr and HindIII site. Thus, identical pre-mRNA transcripts are produced from CMV or SIC promoters and SV40 early or CIS promoters. The splicing pattern of E1a expressed from the SIC promoter was no different from that observed using SV40 core or C/S promoters (Fig. 4B), yielding increased levels of 11 S, 10 S, and 9 S (39%; lane 6 and 34%; lane 4). By contrast, E1a expressed from the CIS, CMV core, or full-length CMV promoters utilized 5'SS-1 in only 10–15% of transcripts (Fig. 4B, cf. lanes 1–3 and 4–6).

Analogous results were obtained using the pSXN-mutant ESE reporter (Fig. 4C); under the control of the SIC, hybrid C/S, or SV40 core promoters, exon E2 was efficiently included (85–88% E2 inclusion, lanes 4–6), whereas under the CIS, CMV core, or full-length CMV promoters, E2 was mostly skipped (73–76% E2 skipping, lanes 1–3). These results indicate that the 5'-untranslated sequences are not responsible for the observed differences in splicing patterns.

To compare their relative transcriptional activities, the CIS and SIC promoters were used to express luciferase (Fig. 4D). No significant differences in the luciferase expression levels were detected between the CIS and CMV core promoters and the SIC and SV40 early core promoters, respectively. Thus, the 5'-untranslated regions do not substantially affect transcription levels from CMV and SV40 early core promoters.

Mutations of the T-stretch in the SV40 Core Promoter Affect Alternative Splicing of pSXN, E1a, and Collagen Transcripts—To identify promoter sequences that affect selection of suboptimal 5'SS, we carried out a mutational analysis of the SV40 core promoter. Sequence comparison of the SV40 early and CMV core promoters revealed two major differences. First, the region between the TATA box and Inr in the CMV and SV40 early promoters consists of 52 and 75% G/C residues, respectively. Second, the CMV TATA box represents an exact match to the consensus, whereas the SV40 promoter contains a TATA-like motif (TATTTAT, pos. 15–21) directly preceded by eight consecutive T residues (nt 21–28) (Fig. 5A). This T-stretch has been shown to induce DNA bending and affect both early transcription and DNA replication (40, 41). Therefore, using pSXN as a reporter, we focused our initial mutational analysis on these regions of the SV40 core promoter.

We generated five SV40 promoter mutants in the region between the TATA and Inr sites (Fig. 5A, M1–5). Three additional mutations were introduced within the TATA box (M6–8); in M6, the TATA box (TATTTAT) was changed to improve its match to the consensus (TATaTAT), and in M7 (gATaTAT) and M8 (TATaTAc), additional T to G or C mutations were introduced. In addition, three mutants within the T-stretch upstream of the TATA-like box contained T to G or C mutations (M9–10; TTgcgTTT, TTgcgTcT, respectively) or lacked this segment (M11). Three other mutations were introduced in the upstream sequence (M12–14).

Mutations within (Fig. 5B, cf. M6–M8) and downstream of the TATA box (cf. WT with M1–M5) did not affect the splicing pattern. By contrast, a triple point mutation of the T-stretch directly upstream of the TATA box (M9) reduced inclusion of alternative exon E2 (67% E2 inclusion in M9 as compared with 83% in WT SV40 early core). An even stronger effect was observed for the M10 mutant, where E2 was skipped in almost all transcripts (13% E2 inclusion). Deletion of the T-stretch (M11) exhibited a similar effect, resulting in only 11% of E2 inclusion. By contrast, mutations immediately upstream of the T-stretch (M12–14) did not affect splicing patterns.

To test the generality of these promoter effects, the T-stretch mutants (M9–11) were also tested with E1a and E14-mutant collagen reporters (Fig. 5, C and D). All three promoter mutants improved 12 S and 13 S RNA production by inhibiting 5'SS-1 selection as compared with the WT SV40 core promoter (Fig. 5C), whereas under the control of the M9 promoter, 12 S and 13 S RNAs represented 71% (lane 2), and the M10 and M11 mutants further increased 12 S and 13 S levels (83 and 85% respectively, as compared with 58% 12 S and 13 S in WT SV40 core). Similar results were obtained using the G5A mutant collagen reporter (Fig. 5D); although the use of the M9 promoter only modestly increased exon E14 skipping (56% for M9 as compared with 52% using the WT SV40 early core), the M10 and M11 mutant promoters resulted in predominant skipping of E14 (81 and 86%, respectively; lanes 3 and 4). In parallel, luciferase activity from the M9, M10, and M11 promoter mutants was also slightly increased (data not shown), consistent with previous results demonstrating that mutations within the T-stretch slightly improved transcription in vitro (41). Together, these results suggest that the T-stretch in the SV40 early promoter plays an important role in regulation of gene expression, affecting both transcription and selection of suboptimal splice sites in the transcript.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although different promoters have been shown to differently affect splicing of primary transcripts, the specific promoter elements responsible for these effects are only partially characterized. To identify additional promoter sequences that can affect pre-mRNA splicing, we set out to analyze promoter-dependent effects on alternative splicing of several minigene reporters. E1a transcripts initiated from a hybrid C/S promoter exhibited altered splicing patterns as compared with transcripts initiated from full-length CMV or SV40 early promoters. These promoter-specific effects were not due to motifs either in the UAS or in 5'-untranslated sequences but rather depended on properties of the core promoter sequence itself.

A number of previous studies identified sequences within enhancers or UAS regions that affected mRNA processing events (Refs. 2, 4, 25, 26, and 42–44 and reviewed in Ref. 45); our results demonstrate that sequences within the core promoter can similarly alter pre-mRNA splicing patterns. In our studies, expression from the SV40 early core promoter, in the absence of any UAS, affected patterns of alternatively spliced transcripts. Specifically, point mutations (M9 and M10) and a deletion (M11) within the T-stretch immediately upstream of the TATA box altered selection of suboptimal splice sites in SXN, E1a, and collagen reporters. Moreover, our experiments show that sequences adjacent to the core promoter can modulate its effects on splicing; although the full-length SV40 promoter (i.e. containing the SV40 enhancer) facilitated skipping of suboptimal splice sites, the same SV40 core promoter sequence in the hybrid C/S construct (containing the CMV enhancer) favored their inclusion. Thus, the observed promoter-dependent splicing effects depend on specific combinations of enhancer and core promoter sequences, suggesting that selective recruitment of TAFs by enhancer/UAS sequences affects transcription from these promoters (46), indirectly affecting pre-mRNA splicing. In fact, many cell type-specific TFIID components (e.g. TBP-like and TAFs-like factors) have been identified that result in a broad spectrum of functional PIC complexes (10, 47, 48).

Core promoters play a central role in initiation of transcription; however, less is known about their effects on the elongation phase. Previous in vitro analysis of the SV40 early promoter (41) detected a limited but reproducible enhancement of transcription caused by mutations within the T-stretch. Although these results were interpreted as indicative of improved transcription initiation, it is possible that these T-stretch mutations also improved elongation. Measurements of luciferase activity indicate that the differences in splicing patterns exhibited by the full-length, core, and hybrid CMV and SV40 promoters are not proportional to the efficiency of initiation (promoter strength) alone. Clearly, further studies are required to measure transcription elongation rates as a function of different promoters. At present, we can only speculate that transcripts initiated from the SV40 (full-length) or CMV (full-length or core) promoters are elongated more efficiently than those initiated from the SV40 core or C/S hybrid promoters and that changes in alternative splicing patterns can serve as a convenient marker of changes in transcription elongation.

By what mechanism(s) might the T-stretch adjacent to the SV40 TATA box slow elongation and, in turn, facilitate selection of suboptimal splice sites? One possibility is that the T-stretch affects recruitment of factors (through either sequence-specific or structure-specific interactions) that bind to PIC complex and remain associated with the elongation complex, ultimately affecting splicing either by altering the elongation rate or by interacting with the spliceosome and/or the nascent pre-mRNA. A number of mRNA processing factors and RNA-binding proteins have been implicated in interactions with promoter-associated complexes (20, 21, 27, 33, 49, 50). Among these, the splicing factor U2AF65 associates with early transcription complexes and cross-links to nascent transcripts (51). In these studies, U2AF65 was found to improve transcription elongation by reducing RNAPII pausing when bound to specific RNA recognition sites in vitro. In addition, elongation factors P-TEFb and TFIIS were found associated with the promoter complex, suggesting that they are recruited already at the stage of transcription initiation (52, 53). These observations suggest that processivity of elongation by RNAPII affects spliceosome function, altering selection of suboptimal splice sites. In further support of this model, connections between transcription initiation and 3' end processing have been described (31, 32).

A related possibility is that sequences flanking the TATA box (i.e. the T-stretch in the SV40 promoter) affect binding of GTFs and thus formation of the PIC complex, resulting in subsequent changes in elongation, indirectly affecting alternative splicing. The T-stretch located immediately upstream of the SV40 TATA box is known to induce DNA bending, and point mutations in this region were shown to affect both DNA bending and the efficiency of transcription (40, 41, 54). Biochemical and structural studies indicate that sequences flanking the TATA box on either side are contacted by TFIIB (55, 56) and TFIIF (57, 58). The geometry of DNA in this region affects PIC formation, modulating binding of TBP to TATA box and consequently altering transcriptional activity. Thus, proteins that modulate TFIIB binding would be expected to affect transcription from suboptimal promoters. Indeed, yeast Sub1 and Ssu72 factors, initially identified as suppressors of TFIIB defects (59, 60), bind to TFIIB and are present at the promoter (61). Moreover, both proteins bind to the 3'-end mRNA processing machinery (62, 63) and associate with and affect the function of the elongation complex (61, 64), suggesting a link between initiation, elongation, and termination of transcription and mRNA processing events. Recently, Sub1 (a transcription coactivator PC4 in humans) was shown to bind yeast polyadenylation factor Ran15 (human CstF-64), and this interaction persists through both initiation and elongation of transcription (59, 62). Similarly, the Ssu72 component of the polyadenylation complex interacts with TFIIB and affects its binding at the promoter, influencing PIC assembly and affecting selection of transcription start site (32). Ssu72 is a Ser-5 phosphatase of the RNAPII C-terminal domain that plays a direct role in transcription elongation (64, 65). Thus, Sub1 and Ssu72 that link initiation, elongation, and termination of transcription also affect pre-mRNA processing events. Additional factors connecting pre-mRNA processing events with initiation, elongation, and termination of transcription are likely to exist. These proteins would play an important role during the transition from initiation to elongation, modulating association of processing factors with the transcription complex. The observation that Sub1 affects binding of TBP to TFIIB would favor this model (59). It remains to be tested whether SV40 T-stretch mutations (M9-M11) improve binding of TFIIB, resulting in a global improvement of transcription initiation and elongation and thus decreased selection of suboptimal splice sites.

Our results illustrate an important role of core promoter elements in control of alternative splicing and, in general, gene expression. Such regulation would be especially important in higher eukaryotes, characterized by widespread existence of alternative splicing, where even subtle variations in RNAPII elongation rates could have important phenotypic effects. Additionally, our results suggest that during SV40 viral infection, gene expression from the SV40 early promoter may be modulated through PIC assembly at its core, producing different spliced forms (small, middle, and large T antigens) in different cell environments.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM49044 (to M. M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure. Back

1 Both authors contributed equally to this work. Back

2 Supported by a Charles H. Revson/Norman and Rosita Winston Foundation fellowship and National Institutes of Health Grant T32 CA009673. Back

3 To whom correspondence should be addressed: The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8432; Fax: 212-327-7174; E-mail: konarsk{at}mail.rockefeller.edu.

4 The abbreviations used are: RNAPII, RNA polymerase II; CMV, cytomegalovirus; SV40, simian virus 40; UAS, upstream activating sequence; TF, transcript factor; GTF, general TF; PIC, preinitiation complex; ESE, exonic splicing enhancer; RSV, Rous Sarcoma Virus long terminal repeat; MLP, major late promoter; RT, reverse transcription; TBP, TATA-binding protein; SS, splice site; nt, nucleotide; Inr, initiator; WT, wild-type; pos., position. Back


   ACKNOWLEDGMENTS
 
We are grateful to Charles Query and the members of the laboratory for helpful discussions and comments on the manuscript.



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