©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Multiple Splicing Signals Control Alternative Intron Retention of Bovine Growth Hormone Pre-mRNA (*)

(Received for publication, November 8, 1994)

Wessel P. Dirksen Qiang Sun Fritz M. Rottman (§)

From the Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4960

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A fraction of bovine growth hormone (bGH) pre-mRNA undergoes alternative splicing in which the last intron is retained and transported to the cytoplasm. Our goal was to characterize the cis-acting signals in bGH pre-mRNA that collectively determine the distribution between intron splicing and intron retention. We now demonstrate that the balance between splicing and intron retention in cytoplasmic mRNA is primarily determined by the interaction of three splicing signals and the degree to which these signals deviate from consensus splicing signals. Intron retention requires the presence of both suboptimal 5`- and 3`-splice sites. Mutation of either splice site toward consensus leads to complete splicing of the intron. In the presence of both wild-type, suboptimal splice sites, efficient splicing of this intron is ensured by the presence of a third splicing element, a purine-rich exonic splicing enhancer (ESE). Although strong ESEs can be contained within very small sequences, the bGH ESE activity appears to be composed of multiple sequences spread throughout a 115-nucleotide region of exon 5. Consequently, the final ratio of splicing to intron retention depends on the balance between the relative strengths of each of these three splicing signals, which still allow intron-containing coding sequences to be transported to the cytoplasm.


INTRODUCTION

Alternative splicing of pre-mRNAs, including the retention of an entire intron, is a common mechanism whereby cells produce multiple proteins from a single pre-mRNA sequence(1, 2, 3, 4) . Once thought to be an exception to the rule (one gene, one protein), alternative splicing is now estimated to occur in at least 1 of every 20 genes(5) . The mechanism of splice site selection and the interaction between multiple cis-acting elements and corresponding protein factors during alternative splicing events remain to be determined.

Several examples of alternative splicing involving intron retention have been described. In the case of the Drosophila P element (6, 7) , an intron may either be spliced or retained, depending on the cell type in which the gene is expressed. In other cases(8, 9, 10, 11) , a defined ratio between intron retention/splicing may be desired within the same cell type. Such alternative splicing events are even more complex since intron-containing mRNAs, which are usually restricted to the nuclear compartment(1, 12, 13, 14) , must now be transported to the cytoplasmic compartment. Our goal was to determine how different splicing signals act in a coordinated fashion to achieve a fixed ratio of splicing and intron retention and to allow the intron-containing mRNA to be transported to the cytoplasm.

We have pursued the study of intron retention using the splicing of the last intron (intron D) of bovine growth hormone (bGH) (^1)pre-mRNA as a model system. This intron is retained in a small fraction of the cytosolic mRNA in bovine anterior pituitary somatotrophs(10) . Intron retention in the cytosol is also observed when bGH is transfected into a variety of tissue culture cell lines. The percentage of intron retention in pituitary and transfected cells varies from 0.1 to 20%. This suggests that a common pathway may exist that allows certain introns to escape the splicing pathway and be transported to the cytoplasm.

In previous studies(15) , we identified a 115-nucleotide, FspI-PvuII restriction fragment (FP sequence) within the last exon (exon 5) of bGH pre-mRNA that is required for efficient splicing of intron D upon expression in transfected cells. Removal of this sequence (DeltaFP) leads to almost complete retention of intron D, both in vivo and in vitro, suggesting that the FP sequence functions to enhance the splicing efficiency of this intron. The FP sequence appears to activate splicing through interaction with transacting factors, including the general splicing factor SF2/ASF(16) . The activity of this exonic splicing enhancer (ESE) was shown to require purine-rich sequences(17) . Furthermore, the ESE was no longer required for efficient splicing when the 5`-splice site of intron D was mutated to match consensus.

In this study, we report how different cis-acting signals act together to control the balance between splicing and intron retention. Retention of bGH intron D depends on the presence of both suboptimal 5`- and 3`-splice sites. Mutation of either site to match consensus splice sites eliminates intron retention. These two suboptimal splice sites, incapable of supporting splicing by themselves, are counterbalanced by a third signal, the ESE in exon 5. The final ratio of intron D splicing to retention is determined by a balance of the relative strengths of each of these three signals in a way that still allows an intron-containing mRNA to escape the nuclear compartment.


MATERIALS AND METHODS

DNA Transfection of CHO Cells and Nuclease S1 Mapping of RNA

CHO cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, non-essential amino acids, and antibiotics (penicillin and streptomycin). Cells were transfected using Lipofectin with conditions recommended by the manufacturer (Life Technologies, Inc.). Cells were harvested, and either polysomal RNA was prepared 48 h after transfection or nuclear RNA was prepared at various times after transfection, as described elsewhere(15, 18) . Polyadenylated RNA was prepared by oligo(dT)-cellulose chromatography(19) .

Probes for nuclease S1 protection experiments were 3` end-labeled with T4 DNA Polymerase and [alpha-P]dCTP. S1 nuclease analysis of poly(A) RNA was carried out as described earlier(15) . Scanning of S1-protected fragments was carried out using a PhosphorImager scanner (Molecular Dynamics Inc.), and the resulting peaks were integrated by area.

RNA Synthesis and in Vitro Splicing Reactions

Capped RNAs were synthesized from linearized vectors pbGH-4D5, pbGH-4D5/DeltaFP, pbGH-4D5/CSD, pbGH-4D5/CSD/DeltaFP, pbGH-4D5/CSA, pbGH-4D5/CSA/DeltaFP, pbGH-4D5/CSD/CSA, and pbGH-4D5/CSD/CSA/DeltaFP as previously described(20) , using T3 RNA polymerase and [alpha-P]GTP to label the RNAs. These labeled RNAs were then incubated in HeLa cell nuclear extract, and the spliced products were separated on polyacrylamide gels as described (20) .

Plasmid Construction

The parent expression plasmid, pSVB3/Ba, contains the entire structural region of the bGH gene driven by the SV40 late promoter(21) . Plasmid derivatives of pSVB3/Ba, pDeltaFP (previously DeltaB and pFPD), pCSD, pCSD/DeltaFP (previously pCSD/FPD), pbGH-4D5, pbGH-4D5/DeltaFP (previously pbGH-4D5/FPD), pG(3)A(2), pG(4)A(3), pG(5)A(4), and pG(7)A(6) are described elsewhere(15, 17, 20) . Plasmids pCSA, pCSA/DeltaFP, pCSD/CSA, pCSD/CSA/DeltaFP, pMSA, pMSA/DeltaFP, pCSD/MSA, and pCSD/MSA/DeltaFP were constructed by subcloning polymerase chain reaction-amplified fragments of bGH between two Bsu36I sites in intron D and exon 5 of pSVB3/Ba, pDeltaFP, pCSD, or pCSD/DeltaFP, using oligonucleotide Bsu/ex5 (5`-GGTACGTCTCCGTCTT-3`) as downstream primer and either oligonucleotide CSA (5`-CTTCCCTTCTTCCTCTCTTTCTCTCCCTCCCTTCCCAGG-3`) or oligonucleotide MSA (5`-GGGCC(C/G)TTCGGCC(G/T)CTCTGTCTCGCCCTC(C/G)CTTGGC-3`) as upstream primers. Plasmids pbGH-4D5/CSD, pbGH-4D5/CSD/DeltaFP, pbGH-4D5/CSA, pbGH-4D5/CSA/DeltaFP, pbGH-4D5/CSD/CSA, and pbGH-4D5/CSD/CSA/DeltaFP were constructed by subcloning polymerase chain reaction-amplified fragments of pCSD, pCSD/DeltaFP, pCSA, pCSA/DeltaFP, pCSD/CSA, or pCSD/CSA/DeltaFP, respectively, into the PstI and EcoRI sites of pBS-M13(+) using oligonucleotides ML1 (5`-CCGGAATTCTGGGAGTGGCACCTTC-3`) and ML2 (5`-GAGCTGCTTCGCATCTC-3`). Plasmids pU1-8/9, pU1-8/9DeltaFP, pU1-3/9, and pU1-3/9DeltaFP were constructed by creating polymerase chain reaction-derived fragments using either oligonucleotide U1-8/9 (5`-TGTCTATGAGAAGCTGAAGGACCTGCAGGAAAGTATCCTG-3`) or oligonucleotide U1-3/9 (5`-TGTCTATGAGAAGCTGAAGGACCTGGACCAAGGCATCCTG-3`) as upstream primers and oligonucleotide bGH3`19 (5`-TGCGATGCAATTTCCTCAT-3`) as downstream primer. These fragments were then subcloned into the Tth111I and BspEI sites of pSVB3/Ba, followed by deletion of the FspI-PvuII fragment in exon 5 to create the DeltaFP versions. Plasmids pDeltaGA1-2 and pDeltaGA1-3 were constructed by removing either the Tth111I-PstI or Tth111I-StyI exon 4 fragments from pU1-8/9 and pU1-3/9, respectively. Plasmids pG(4)A(4), pG(6)A(4), pG(6)A(5), pG(6)A(6), and pG(8)A(6) were constructed by subcloning polymerase chain reaction-amplified fragments of bGH between a BsmI site in intron D and the PvuII site in exon 5 using oligonucleotide 223 (5`-CCTCGGACCGTGTCTATGAG-3`) as upstream primer and oligonucleotides G(4)A(4) (5`-TTCCTTCCGCATGTTTGTG-3`), G(6)A(4) (5`-CCTTCCTTCCGCATGTTTGTG-3`), G(6)A(5) (5`-TCCTTCCTTCCGCATGTTTGTG-3`), G(6)A(6) (5`-TTCCTTCCTTCCGCATGTTTGTG-3`), and G(8)A(6) (5`-CCTTCCTTCCTTCCGCATGTTTGTG-3`) as downstream primers, respectively. Plasmids pG(2)A(2) and pG(4)A(2) were obtained accidentally when small deletions occurred on the ends of the polymerase chain reaction-amplified fragments during construction of the above plasmids. Plasmids pFP1, pFP2, pFP3, pDeltaFP1, pDeltaFP2, and pDeltaFP3 were constructed by removing the appropriate restriction fragments from the wild-type exon 5 using the available restriction sites (see Fig. 6A). All plasmids were confirmed by sequencing.


Figure 6: The ESE is diffuse within the FP sequence. A, schematic representation of exon 5 of bGH constructs containing deletions within the FP sequence. FP subfragment 1 (FspI-BspEI) is 46 nucleotides in length. FP subfragment 2 (BspEI-RsaI) is 25 nucleotides in length. FP subfragment 3 (RsaI-PvuII) is 40 nucleotides in length. B, S1 nuclease mapping of mRNA isolated from CHO cells transiently expressing the constructs depicted in A. Lane1, pDeltaFP1; lane2, pDeltaFP2; lane3, pDeltaFP3; lane4, pFP1; lane5, pFP2; lane6, pFP3. C, purine-rich sequences found within each FP subfragment. D, quantification of S1 nuclease mapping analysis of these constructs. Values represent the mean ± S.D. from at least three transfection experiments.




RESULTS

Role of 5`- and 3`-Splice Sites in bGH Intron D Retention

In this report, we were interested in determining which splicing signals contribute to the level of intron D retention in cytoplasmic bGH mRNA. Earlier studies demonstrated that a suboptimal 5`-splice site is essential for bGH intron D retention in transfected cells(17) . It was not known what role, if any, the 3`-splice site plays in determining the final level of intron D retention. Inspection of the 3`-splice site sequence, which is composed of the branch point, polypyrimidine tract, and 3`-acceptor site, revealed minor deviations from the 3`-splice site consensus sequence (Fig. 1). Reed and Maniatis (22) have shown that the preferred nucleotide 3`-adjacent to the branch point is a cytosine, while bGH contains a guanosine at this position(20) . In addition, the bGH polypyrimidine tract has several purine insertions, which are known to affect splicing efficiency(23, 24, 25) . To determine the contribution of these deviations to the ratio of splicing to intron retention, the 3`-splice site in intron D was mutated toward either a stronger or weaker consensus sequence (Fig. 1). The mutation toward consensus (CSA, consensus splice acceptor) involved correcting the last position of the branch point and substitution of purines in the polypyrimidine tract to pyrimidines. The mutation away from consensus (MSA, mutant splice acceptor) involved substitution of four pyrimidines to purines in the polypyrimidine tract. These mutations were analyzed, in conjunction with 5`-splice site mutations and downstream FP element deletions, by transfection into CHO cells. Following RNA purification, S1 analysis was used to determine the ratio of spliced to unspliced bGH mRNA.


Figure 1: Sequence of wild-type and mutant splice sites in bGH intron D. Sequences shown represent the wild-type sequences for the 5`- and 3`-splice sites (branch point, polypyrimidine tract and acceptor site) in bGH intron D, as well as mutations made in these sites and the consensus sequences derived for mammalian splice sites (1, 36) . Mutations were designed to make the wild-type sequence either more like consensus (CSD, CSA) or less like consensus (MSA). Slashes represent splicing cleavage sites. The shadowed ``A's'' represent the branch site as previously mapped(20) . Underlinednucleotides represent mutations made to the wild-type sequence.



As previously observed, intron D in wild-type bGH mRNA is largely spliced, while deletion of the FP element leads to mostly unspliced bGH mRNA (Fig. 2B, lanes1 and 2). When the 5`-splice site is changed to consensus (CSD, consensus splice donor), splicing of bGH intron D is close to 100%, independent of the FP element (Fig. 2B, lanes3 and 4). Interestingly, mutation of the 3`-splice site toward consensus (CSA) also leads to improved splicing, independent of the FP element (lanes5 and 6), just as observed with the consensus 5`-splice site (CSD). Therefore, it appears that the bGH 3`-splice site is also suboptimal and acts in concert with the attenuated 5`-splice site to allow for retention of intron D. However, the relative strength of the wild-type 3`-splice site appears to be adjusted to provide for a given level of intron retention. Mutation of the wild-type 3`-splice site further away from consensus (MSA) leads to even higher levels of intron D retention in the presence of the FP element (Fig. 2B, lane9) and abolishes splicing in the absence of the FP element (lane10). Interestingly, this very weak 3`-splice site (MSA) can still be rescued by concomitant strengthening of the wild-type 5`-splice site (lanes11 and 12), although the overall cytoplasmic levels of bGH mRNA in CSD/MSA/DeltaFP are decreased 10-fold (data not shown). Therefore, intron D retention would not be possible if either of the two splice sites matched consensus sequence. We conclude that both the 5`- and 3`-splice sites are suboptimal and are required to establish a defined level of intron D retention in cytoplasmic bGH mRNA.


Figure 2: Intron retention requires the presence of two suboptimal splice sites. A, schematic representation of exons 4 and 5 of bGH constructs used in the experiment containing splice site mutations. The open and closedcircles represent the wild-type and CSD 5`-splice sites, respectively. The open, closed, and stripedovals represent the wild-type, CSA, and MSA 3`-splice sites, respectively. The darklyshadedboxes denote the bGH FP element in exon 5. The lightlyshadedboxes denote other exon 5 sequences downstream of the SmaI site. B, S1 nuclease mapping of polysomal mRNA isolated from CHO cells transiently expressing the constructs depicted in A. Probelane, untreated probe; tRNAlane, probe mock-hybridized with 10 µg of tRNA; lane1, pSVB3/Ba; lane2, pDeltaFP; lane3, pCSD; lane4, pCSD/DeltaFP; lane5, pCSA; lane6, pCSA/DeltaFP; lane7, pCSD/CSA; lane8, pCSD/CSA/DeltaFP; lane9, pMSA; lane10, pMSA/DeltaFP; lane11, pCSD/MSA; lane12, pCSD/MSA/DeltaFP. Unspliced indicates intron D-containing bGH mRNA. Spliced indicates bGH mRNA where intron D has been removed by splicing. Lanenumbers correspond to the schematic diagrams in A.



In Vitro Splicing of Splice Site Mutants

We were interested in confirming and extending these results by testing the above constructs in vitro. The above mutations were cloned into a vector to allow synthesis of radiolabeled transcripts. In vitro splicing of bGH pre-mRNAs was performed, and the spliced products were analyzed on polyacrylamide gels as described(20) . The results obtained (Fig. 3) essentially confirmed the in vivo results. The wild-type substrate splices inefficiently in vitro, which is not surprising because of the suboptimal splice sites (Fig. 3B, lane1). The DeltaFP mutant does not splice, consistent with the requirement of the FP element for splicing (lane2). The mutations CSD and CSA each lead to a significant increase in splicing (Fig. 3B, lanes3 and 5), and the CSD/CSA double mutant splices extremely well compared with wild type (lane7). This result corroborates the hypothesis that in wild-type bGH intron D, both the 5`- and 3`-splice sites are suboptimal. When the FP element is deleted, CSA/DeltaFP and CSD/CSA/DeltaFP pre-mRNAs are still able to be spliced (Fig. 3B, lanes6 and 8). However, one major difference was observed in comparing in vitro spliced products to the results obtained in vivo. Deletion of the FP element in CSD/DeltaFP pre-mRNA does not result in significant levels of spliced product in vitro (Fig. 3B, lane4). This is in contrast to in vivo results in which CSD/DeltaFP pre-mRNA splices well in the absence of the FP element (Fig. 2B, lane4).


Figure 3: The bGH ESE partially compensates for a weak 3`, but not a weak 5`, splice site in vitro. A, schematic representation of bGH pre-mRNAs containing splice site mutations. Symbols are as described in Fig. 2A legend. B, in vitro splicing of bGH pre-mRNAs containing splice site mutations. P, precursors; S, spliced RNAs; L, lariats containing the intron and the exon; I, lariats containing the intron only; E, free 5`-exon. Lane1, bGH-4D5; lane2, bGH-4D5/DeltaFP; lane3, bGH-4D5/CSD; lane4, bGH-4D5/CSD/DeltaFP; lane5, bGH-4D5/CSA; lane6, bGH-4D5/CSA/DeltaFP; lane7, bGH-4D5/CSD/CSA; lane8, bGH-4D5/CSD/CSA/DeltaFP.



CSD/DeltaFP Pre-mRNA Splices Well in Vivo

One possible explanation for this difference is that the CSD/DeltaFP mutant may not splice well in vivo, but because the strong 5`-splice site leads to formation of early spliceosome commitment complexes, the precursor mRNA is trapped in the nucleus(1, 12, 13, 14) , allowing only the spliced mRNA into the cytoplasm. Since we only analyzed cytoplasmic RNA, we would have missed any in vivo accumulation of nuclear intron D-containing RNA, and CSD/DeltaFP would have appeared to splice well in vivo. We tested this possibility by analyzing nuclear RNA at various times after transfection (Fig. 4). Both wild-type (lanes1-4) and DeltaFP (lanes 5-8) exhibit slightly higher levels of unspliced bGH mRNA in the nucleus than in the cytoplasm during the time course of the experiment. However, there was no significant nuclear accumulation of precursor RNA in either the CSD (lanes 9-12) or CSD/DeltaFP (lanes 13-16) mutants. In addition, when internal controls were used, no significant decrease in the total cytoplasmic levels of CSD/DeltaFP mRNA was observed compared with CSD mRNA levels (data not shown), which would also have been indicative of trapping of the mRNA in the nucleus. Thus, it appears that CSD/DeltaFP pre-mRNA splices well in vivo.


Figure 4: CSD/DeltaFP pre-mRNA containing intron D does not accumulate in the nucleus. S1 nuclease mapping analysis of nuclear RNA isolated from CHO cells transiently expressing wild-type (WT), DeltaFP, CSD, and CSD/DeltaFP bGH constructs. Nuclear RNA was isolated 8, 12, 18, and 24 h following transfection. Lanes 1-4, pSVB3/Ba; lanes 5-8, pDeltaFP; lanes 9-12, pCSD; lanes 13-16, pCSD/DeltaFP.



The discrepancy observed in the splicing of CSD/DeltaFP pre-mRNA in vivo compared with in vitro may be due to inherent differences between the two systems regarding the substrate RNAs. The complete bGH pre-mRNA sequence is available to the splicing machinery in vivo, whereas in vitro a smaller pre-mRNA substrate is used. This raises the possibility that the larger in vivo substrate is able to stimulate splicing of the CSD/DeltaFP intron D through intron cooperativity. Intron cooperativity occurs when the splicing efficiency of a given intron is improved by the concomitant splicing of the other introns in the pre-mRNA(26, 27) . To test this possibility, introns A, B, and C were removed from the wild-type, DeltaFP, CSD, and CSD/DeltaFP bGH vectors. While the splicing efficiency of intron D in wild type and DeltaFP was diminished, there was no effect on the splicing of intron D in the CSD and CSD/DeltaFP vectors (data not shown). Additionally, longer substrates tested did not improve CSD/DeltaFP pre-mRNA splicing in vitro (data not shown), suggesting that the inherent differences between the in vivo and in vitro systems are not due to differences in RNA substrate length.

Exon 4 Sequences Are Not Involved In Intron C or D Splicing

Inspection of the exon 4 sequence revealed three short purine-rich sequences near the 5`-splice site, one of which (sequence 3) also exhibited partial homology to a 5`-splice site (Fig. 5A). The purine-rich nature of these sequences suggested the possibility that they may be acting as exonic splicing enhancers for either intron C and/or intron D. On the other hand, the partial homology of sequence 3 to a 5`-splice site suggested the possibility that it may strengthen definition of exon 4(28, 29) and/or it may be acting as a pseudo-5`-splice site (7, 30) that inhibits the use of the intron D 5`-splice site. We decided to dissect this exon 4 sequence by making point mutations that either increased or decreased the complementarity of sequence 3 to U1 snRNA (Fig. 5A, U1-8/9 toward consensus and U1-3/9 away from consensus) and by deleting two or all three of the purine-rich sequences (Fig. 5A, DeltaGA1-2 and DeltaGA1-3).


Figure 5: Exon 4 sequences not normally involved in splicing of introns C or D can be made to inhibit intron D splicing. A, sequence shown is that of the 3` portion of exon 4 and beginning of intron D. Sequences underlined and numbered1-3 represent the purine-rich/U1-like sequences referred to in the text. Nucleotides in smallcaseletters represent mutations made to the wild-type sequence, and gaps refer to deletions in the sequence. Slashes represent the exon/intron boundary. B, S1 nuclease mapping of mRNA isolated from CHO cells transiently expressing the constructs depicted in A. Probelane, untreated probe; tRNAlane, probe mock-hybridized with 10 µg of tRNA; lane1, pU1-8/9; lane2, pU1-8/9DeltaFP; lane3, pU1-3/9; lane4, pU1-3/9DeltaFP; lane5, pDeltaGA1-2; lane6, pDeltaGA1-3.



Mutation away from U1 consensus (U1-3/9) did not change the pattern of intron C and D splicing normally observed in vivo with or without the FP element present (cf. Fig. 5B, lanes3 and 4, with Fig. 2B, lanes1 and 2). This suggests that this purine-rich sequence 3 is not an inhibitory pseudo-5`-splice site interfering with the wild-type 5`-splice site nor is it a positive signal required for intron C splicing. However, mutation toward U1 consensus (U1-8/9) resulted in complete inhibition of intron D splicing, whether or not the FP element was present (Fig. 5B, lanes1 and 2). This suggests that, although such a sequence is not normally involved in intron D splicing, it can be artificially created to function as a pseudo-5`-splice site, thereby inhibiting the use of the wild-type 5`-splice site in a similar fashion to that observed with the naturally occurring Drosophila P element intron 3(7) .

To test the potential effect of the purine-rich sequences in this same region of exon 4, the purine sequences were removed by several small deletions. The first mutation (DeltaGA1-2) deleted the first two purine sequences, and the second mutation (DeltaGA1-3) deleted all three of these sequences (Fig. 5A). S1 analysis revealed that both mutants, DeltaGA1-2 (Fig. 5B, lane5) and DeltaGA1-3 (lane6), gave rise to the same pattern of intron C and D splicing as parent clones U1-8/9 (Fig. 5B, lane1) and U1-3/9 (lane3), respectively. Thus, these purine sequences, located upstream of intron D, do not function as exonic splicing enhancers in the splicing of either intron C or D. We conclude that these sequences in exon 4 are not involved in intron D splicing nor are they required to support a suboptimal 5`-splice site in the definition of exon 4 (28) and splicing of intron C.

Characterization of Different ESEs

The above results demonstrate that both the 5`- and 3`-splice sites of bGH intron D are weak and are incapable of supporting splicing of intron D. We had previously shown that a purine-rich ESE is present in exon 5, which allows intron D to be spliced(17) . We wished to further define the nature of the purine sequences in the FP element that function as splicing enhancers and the extent to which they influence the ratio of intron splicing and retention. For this purpose, a series of mutants were constructed in which the entire 115-nucleotide FP element was replaced with a short purine motif contained within the FP element. Each subsequent construct contained a single nucleotide addition to this motif, thereby generating a series of purine insertions of increasing length, containing from 4 to 14 purines. Each mutant was analyzed in transfected cells, and the level of intron splicing/retention was measured. The results obtained ranged from 20 to 80% splicing (Table 1). Although there was a general increase in splicing efficiency with increasing length of the purine insert, this increase was not strictly linear. Interestingly, a drop-off in splicing efficiency was observed whenever two adjacent guanosines were present at the 3`-end of the purine insert (mutants G(4)A(2), G(6)A(4), and G(8)A(6)). This may reflect the fact that two adjacent guanosines at the end may be inherently inhibitory to splicing or that sequences immediately 3` to the purine insertions may be required at a specific distance for splicing stimulation. Another interesting feature of these simple purine sequences is that they give rise to a surprisingly wide range of splicing efficiencies, and their ESE activity can be contained in a small RNA sequence.



The FP element in bGH pre-mRNA, which gives wild-type levels of intron D splicing, is 115 nucleotides long, whereas the best sequence from the purine series was only 13 nucleotides long. We wished to determine if the ESE in bGH was confined to a small sequence or loosely contained throughout the FP element. To address this question, the FP element was divided into three subfragments (Fig. 6A), one of which contained only one short purine stretch. The other two subfragments contained a number of purine stretches similar to those shown to affect splicing (Fig. 6C). These subfragments were either deleted individually from exon 5 (Fig. 6B, lanes1-3) or reinserted into the FP-deleted exon (lanes 4-6) and tested for their ability to enhance intron D splicing in transfected cells. Surprisingly, all three subfragments significantly enhanced splicing (Fig. 6D), even though subfragment 1 contains only one 5-nucleotide purine sequence (Fig. 6C). This result suggests that the ESE contained within the FP element may represent the cumulative effect of multiple sequences. Nevertheless, a simple repeat containing only three copies of GGAA restores splicing to near wild-type levels (Table 1).


DISCUSSION

Intron-containing mRNA sequences are normally restricted to the nuclear compartment due to formation of prespliceosome complexes that commit the pre-mRNA to the splicing pathway(1, 31) . This poses a problem for alternative processing events where retention of an intact intron is required to produce a new protein isoform. The mechanism whereby these intron-containing mRNAs escape the splicing pathway and exit the nucleus is unclear. We have used the alternative splicing of the last intron of bGH pre-mRNA as a model system to study how intron-containing mRNAs are allowed into the cytoplasm and the interplay of cis-acting signals that leads to a given level of unspliced cytoplasmic mRNAs. Several conclusions are apparent from this study. First, two suboptimal splice sites, which together are unable to support splicing on their own, are required for intron retention and transport. Second, strong splice sites commit pre-mRNAs to intron removal in the nucleus. Third, an additional nucleotide signal sequence located in the downstream exon, called the exonic splicing enhancer, is required for splicing of the weakened intron. Finally, a fine balance between the strength of these three different splicing signals establishes a defined level of intron retention.

We have determined that intron retention can only occur in the presence of both suboptimal 5`- and 3`-splice sites. If either site is strengthened by mutation toward consensus, no intron retention is observed. One possible explanation is that two competing reactions may occur, one to commit pre-mRNAs to the splicing pathway, which is kinetically more favorable, and the other to transport mRNAs to the cytoplasm. Presence of either a strong 5`- or 3`-splice site favors direction of the mRNA into the splicing pathway, where it can either be spliced or the intron-containing sequence is degraded. When both 5`- and 3`-splice sites are weak, pre-mRNAs may form the commitment complex (31) more slowly and inefficiently, resulting in some of the intron-containing pre-mRNAs escaping the splicing pathway and, by default, allowing their transport to the cytoplasm.

Our results are consistent with the above hypothesis. Strengthening of either 5`- or 3`-splice sites of bGH intron D resulted in no intron retention. Furthermore, splicing of these pre-mRNAs did not result in an accumulation of precursor mRNA in the nucleus (Fig. 4) nor a decrease in the amount of bGH mRNA in the cytoplasm (data not shown), compared with wild-type bGH mRNA. This suggests that strong 5`- and 3`-splice sites directly enhanced splicing efficiency. The in vivo result contrasted somewhat with the in vitro result (Fig. 3), which suggested that while the strong 5`-splice site improves splicing efficiency in the presence of the ESE, it was not able to compensate for the absence of the ESE in vitro (this effect was also observed by Tian and Maniatis(32) ), as it did in vivo. This discrepancy was not resolved but may reflect differences in levels of certain splicing factors between the in vivo and in vitro systems.

The strong 5`-splice site in conjunction with the weakened 3`-splice site (CSD/MSA/DeltaFP) lead to only spliced mRNA (Fig. 2B, lane12), but this mRNA accumulated in the cytoplasm to a 10-fold lower amount compared with wild-type mRNA (data not shown). This result suggests that even though a strong 5`-splice site may commit pre-mRNAs to the splicing pathway, it cannot compensate for an excessively weakened 3`-splice site to provide for efficient intron removal. This result is consistent with a two-site model, similar to one proposed by Grabowski et al.(29) , in which the strength of two splice sites must each surpass a threshhold to allow for splicing and further indicates that, although intron retention requires two suboptimal splice sites, these sites cannot be too weak.

At least in the case of bGH pre-mRNA, the effect of a strong 3`-splice site is essentially the same as a strong 5`-splice site. The strong 3`-splice site increases the efficiency of splicing and compensates for the absence of the FP element ( Fig. 2and Fig. 3). However, we do not know if a strong 3`-splice site commits pre-mRNA to the splicing pathway, regardless of intron strength.

We also tested sequences in exon 4 for their involvement in splicing of upstream intron C and downstream intron D. Purine-rich sequences present just upstream of the intron D 5`-splice site resembled U1 small nuclear ribonucleoprotein binding sites. These purine-rich sequences do not function as ESEs for either intron C or D, since their deletion does not significantly alter the pattern of bGH pre-mRNA splicing (Fig. 5B). Thus, sequences in the 3`-half of exon 4 are apparently not involved or required for intron D retention.

We have shown that intron retention requires the presence of both suboptimal 5`-and 3`-splice sites. Under these conditions virtually no splicing is observed. Thus, a third signal is required to produce a reasonable level of intron splicing while still allowing a portion of the intron-containing sequence to be transported to the cytoplasm where it is translated. In the case of bGH pre-mRNA, this ESE resides within the FP element in exon 5(15, 17) . We had previously shown that short purine-rich motifs form the core of this ESE, but the nature of the extended ESE element was undefined. We show here that ESEs of differing strengths can be achieved by simply altering or increasing the purine content of a relatively short sequence replacing the FP element (Table 1). Increasing the length of this purine sequence one nucleotide at a time increases the strength of the ESE, although this increase is not completely linear. This suggests that ESEs of various strengths can be obtained by simply changing their length and purine composition. One possibility is that different splicing factors bind to different classes of ESEs. This would be consistent with recent observations that show that splicing factors, such as SR proteins, have distinct functions with different pre-mRNAs(33, 34) .

In contrast to these short purine-rich sequences, the ESE within the FP element is likely to be diffuse and composed of multiple sequences, possibly including a few non-purine residues. Subdividing the FP element into three fragments revealed that each fragment had ESE activity (Fig. 6), even though subfragment 1 contained only one short purine stretch. Although the reason for the diffuse nature of bGH ESE is not fully understood, it could be due to restrictions set by other pre-mRNA functions, including the protein-coding capacity of this sequence in wild-type bGH.

In conclusion, we have defined the elements in bGH pre-mRNA that establish a defined level of intron retention in an alternative splicing event. Three separate splicing signals are involved in this process. Both 5` and 3` suboptimal splice sites are required to decrease splicing efficiency and allow for intron retention, presumably by preventing efficient formation of commitment splicing complex(31) . Since these suboptimal splice sites are unable to support efficient splicing, the ESE is required to stimulate splicing of the bulk of the bGH pre-mRNA. These three signals act in concert to establish a defined level of intron retention. This mechanism may be a common and simple way to achieve intron retention and to modulate the level of retention through the action of constitutive trans-acting factors and ESEs(35) .


FOOTNOTES

*
This work was supported by Public Health Service Grant DK32770 from the National Institutes of Health (to F. M. R.). 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.

§
To whom correspondence should be addressed. Tel.: 216-368-3420; Fax: 216-368-3055.

(^1)
The abbreviations used are: bGH, bovine growth hormone; CHO, Chinese hamster ovary; ESE, exonic splicing enhancer; FP, FspI-PvuII; DeltaFP, FspI-PvuII deletion; CSD, consensus splice donor; CSA, consensus splice acceptor; MSA, mutant splice acceptor.


ACKNOWLEDGEMENTS

We thank Joseph A. Bokar, Robert K. Hampson, Rachael L. Ludwiczak, and James P. Bruzik for critically reading the manuscript.


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