(Received for publication, November 8, 1994)
From the
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.
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) ()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 (FP) 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.
Probes for nuclease S1 protection
experiments were 3` end-labeled with T4 DNA Polymerase and
[-
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.
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, pFP1; lane2, p
FP2; lane3, p
FP3; 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.
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/FP 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, pFP; lane3, pCSD; lane4, pCSD/
FP; lane5, pCSA; lane6, pCSA/
FP; lane7, pCSD/CSA; lane8, pCSD/CSA/
FP; lane9,
pMSA; lane10, pMSA/
FP; lane11, pCSD/MSA; lane12,
pCSD/MSA/
FP. 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.
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/FP; lane3, bGH-4D5/CSD; lane4, bGH-4D5/CSD/
FP; lane5,
bGH-4D5/CSA; lane6, bGH-4D5/CSA/
FP; lane7, bGH-4D5/CSD/CSA; lane8,
bGH-4D5/CSD/CSA/
FP.
Figure 4:
CSD/FP 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),
FP, CSD, and CSD/
FP bGH constructs. Nuclear
RNA was isolated 8, 12, 18, and 24 h following transfection. Lanes
1-4, pSVB3/Ba; lanes 5-8, p
FP; lanes
9-12, pCSD; lanes 13-16,
pCSD/
FP.
The
discrepancy observed in the splicing of CSD/FP 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/
FP
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,
FP, CSD, and
CSD/
FP bGH vectors. While the splicing efficiency of intron D in
wild type and
FP was diminished, there was no effect on the
splicing of intron D in the CSD and CSD/
FP vectors (data not
shown). Additionally, longer substrates tested did not improve
CSD/
FP 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.
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, pU1-8/9FP; lane3, pU1-3/9; lane4,
pU1-3/9
FP; lane5, p
GA1-2; lane6,
p
GA1-3.
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
(GA1-2) deleted the first two purine sequences, and the
second mutation (
GA1-3) deleted all three of these sequences (Fig. 5A). S1 analysis revealed that both mutants,
GA1-2 (Fig. 5B, lane5)
and
GA1-3 (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.
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).
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/FP) 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) .