BACKGROUND
pre-mrna splicing is a fundamental process that joins exons by catalyzing the removal of intervening sequences  from mrna precursors.
pre-mrnas are spliced in a two-step pathway catalyzed by the spliceosome, a dynamic macromolecular machinery consisting of five small nuclear ribonucleoproteins  and many non-snrnp proteins.
in the first step, pre-mrna is cleaved at the 5' splice site  generating two splicing intermediates: a linear first exon, and an intron-second exon in a lariat configuration.
in the second step, the 3'-hydroxyl group of the last nucleotide in first exon makes a nucleophillic attack at the phosphodiester bond separating the intron and the second exon , enabling the joining of two exons and release of the intron as a lariat .
the differential joining of 5' and 3' ss of a single pre-mrna, a phenomenon known as alternative splicing, can generate variant mrnas with diverse and often antagonistic functions  <cit> .
alternative splicing of pre-mrna is now considered to be the most important source of protein diversity in vertebrates  <cit> .
it is estimated that 35–60% of human genes generate transcripts that are alternatively spliced  <cit> , and 70–90% of alternative splicing events affect coding capacity of genes  <cit> .
importantly, deregulated splice variant expression has been identified as the cause of a number of genetic disorders  <cit> , and certain forms of cancer have been linked to unbalanced isoform expression from genes involved in cell cycle regulation or apoptosis  <cit> .
given the critical role of alternative splicing in a variety of cellular processes, strategies that could influence pre-mrna splicing decisions will have far-reaching effects in biotechnology and medicine.
initial efforts aimed at controlling pre-mrna splicing exploited the intrinsic property of nucleic acids to bind specific pre-mrna sequence and inhibit splicing  <cit> .
however, susceptibility of antisense oligonucleotides to nuclease digestion, off-target effects, difficulty with delivery and localization led to the realization that improved methods are required  <cit> .
bifunctional molecules that function like an antisense oligonucleotide, but carry the binding site for a known splicing factor have proved to be useful in reprogramming pre-mrna splicing  <cit> .
while bifunctional molecules have overcome some of the problems of the original antisense-based approach, requirements of chemical modifications limit their utility.
in addition, these approaches function in a constitutive manner, i.e., an antisense oligonucleotide directed to inhibit the splicing of a pre-mrna will continue to do so as long as the oligonucleotide is available.
since alternative splicing of many pre-mrnas is controlled by splicing regulators  <cit> , a small molecule ligand that could mimic the function of a splicing regulator would be of broad general application in controlling gene expression  <cit> .
we have previously shown that insertion of a theophylline-responsive riboswitch into the 3' ss region of a model pre-mrna enables its splicing to be repressed during the second step specifically by theophylline  <cit> .
since the vast majority of human genes are alternatively spliced and splice site pairing in general occurs at early stages of the splicing pathway, we reasoned that sequestering of bps in a theophylline aptamer would be a better approach for controlling alternative splicing.
in this study, we tested this idea by constructing a series of pre-mrnas in which bps was inserted within theophylline aptamer.
we show that theophylline-induced sequestering of bps is highly specific and inhibits pre-mrna splicing both in vitro and in vivo in a dose dependent manner.
our results indicate that the thermodynamic stability of rna-theophylline complex and the location of bps within this complex affect the efficiency of splicing inhibition.
finally, we show that sequestering of bps within rna-theophylline complex can modulate alternative splicing both in vitro and in cultured cells.
RESULTS
insertion of bps within theophylline-responsive riboswitch confers ligand dependent control of splicing
to investigate whether theophylline-induced sequestering of bps would allow conditional control of splicing, we constructed a series of adml pre-mrna derivatives in which bps was embedded within high-affinity theophylline binding aptamer  .
these pre-mrnas differ in terms of the distance of aptamer sequence from the 3' ss ag.
32p-labeled pre-mrnas were transcribed as runoff transcripts using t <dig> rna polymerase and gel purified rnas were incubated in hela nuclear extract under the conditions that support in vitro splicing.
the results presented in fig.
1b demonstrate that these substrates underwent normal splicing, albeit with lower efficiency compared with the parent pre-mrna .
significantly, splicing reactions performed in the presence of theophylline gave rise to lower yields of spliced mrna, suggesting that theophylline-mediated sequestering of branchpoint inhibits pre-mrna splicing .
quantitation of these data indicate that theophylline inhibited the splicing of adbpt pre-mrnas by ~65–90%, but had no effect on the splicing of adml 21ag, a pre-mrna that does not contain theophylline-binding aptamer .
we chose adbpt15ag for further experiments because of its better splicing efficiency in the absence of theophylline; while 17% of adbpt15ag pre-mrna was converted into mrna, only ~7% and 9% of adbpt12ag and adbpt18ag pre-mrnas gave rise to spliced product, respectively.
to determine theophylline concentration that may be optimum for inhibiting pre-mrna splicing, 32p-labeled adbpt15ag pre-mrna was subjected to in vitro splicing in the presence of increasing concentrations of theophylline.
results shown in fig.
1d indicate that theophylline inhibited the splicing of adbpt15ag pre-mrna in a dose-dependent manner with an ic <dig> value  <dig>  ±  <dig>  mm.
a slight increase in the splicing efficiency at lower concentration of theophylline may be the rna stabilization rather than inhibitory effect of the ligand.
splicing inhibition through theophylline-induced sequestering of bps is specific
our model of conditional control of splicing specifies that theophylline inhibits pre-mrna splicing by binding to its cognate sequence, thereby sequestering the bps within rna-theophylline complex.
to confirm that the splicing inhibition shown in fig.
1b is specific, we performed the following experiments.
first, we constructed madbpt15ag pre-mrna that harbors mutations in the core of tba, but otherwise is identical to adbpt15ag .
on the basis of previously published reports  <cit> , which suggest that the 15-nucleotide motif  is essential for high-affinity theophylline binding, we predicted the splicing of madbpt15ag to remain unaltered in the presence of theophylline.
indeed, results shown in fig.
2b demonstrate that theophylline failed to inhibit the splicing of madbpt15ag pre-mrna.
in contrast, a similar concentration of theophylline was found to inhibit the splicing of adbpt15ag .
relatively low splicing efficiency of madbpt15ag pre-mrna in the absence of theophylline suggests that mutations in the core of tba although prevented theophylline binding, apparently this did not completely abolish secondary structure of the aptamer.
support for this interpretation comes from a web-based rna-folding program indicating that the mutated aptamer can fold into a secondary structure .
next, we generated adt+ <dig> pre-mrna in which tba was relocated upstream of bps and analyzed its splicing in vitro using hela nuclear extract.
as illustrated in fig.
2c, this pre-mrna underwent normal splicing in the presence of theophylline, suggesting that sequestering of bps within rna-theophylline complex is necessary for splicing inhibition.
finally, we examined the splicing of adbpt15ag pre-mrna in the presence of caffeine, a purine that is similar in shape and size to theophylline.
consistent with our previously published observation  <cit> , caffeine failed to exert any influence on the splicing of adbpt15ag pre-mrna .
taken together, we conclude that theophylline-dependent inhibition of pre-mrna splicing is highly specific.
insertion of bps in theophylline-binding aptamer does not activate cryptic branchpoint
previous studies have shown that cryptic branchpoint activation allows accurate in vitro splicing of mammalian pre-mrnas albeit with lower efficiency  <cit> .
to determine if relatively lower splicing efficiency of adbpt15ag pre-mrna is due to the activation of a cryptic branchpoint, we mapped the position of the rna branch.
preparative amounts of lariat intermediates were isolated from in vitro splicing reaction of adbpt15ag pre-mrna performed in the absence or presence of theophylline.
the branchpoint was mapped by the primer extension of untreated or s <dig> treated lariat intermediate, which allows debranching of the lariat rna  <cit> .
the position of reverse transcriptase stop site was assigned by using a dideoxy-sequencing ladder from the parental dna.
the primer extension analysis of adbpt15ag pre-mrna derived lariat intermediate showed a single stop immediately before the branchpoint adenosine .
this band was not detected with unspliced pre-mrna  or the lariat intermediate that was treated with s <dig> extract .
in contrast, the primer extension of s <dig> treated lariat species generated a  <dig> nt band corresponding to the size of linear intron-exon  <dig> rna .
importantly, the lariat intermediate isolated from theophylline treated splicing reaction gave rise to a single reverse transcriptase stop signal corresponding to  <dig> nt .
in addition, this lariat intermediate was s <dig> sensitive, and the primer extension of debranched lariat intermediate produced a  <dig> nt species .
the bands marked with asterisk are the primer extension products of s <dig> rna likely generated due to the non-specific binding of the primer.
this interpretation is supported by the fact that primer extension of rna isolated from s <dig> extract resulted in identical reverse transcriptase stops .
we conclude that theophylline-dependent sequestering of bps does not activate cryptic branchpoint.
thermodynamic stability of lower theophylline aptamer stem and location of bps affects splicing inhibition
biochemical and structural studies showed that the lower theophylline aptamer stem is not critical for ligand binding  <cit> , but affects the stability of rna-theophylline complex.
if true, then altering the length of lower theophylline aptamer stem is expected to affect splicing suppression.
to test this idea, we constructed two adbpt15ag derivatives in which the length of lower theophylline aptamer stem was either decreased to a single bp  or increased to eight bp  .
to assess the consequences of varied lower theophylline aptamer stem on pre-mrna splicing, 32p-labeled substrates were incubated in hela nuclear extract in the absence or presence of theophylline under splicing conditions.
we found that compared to ~60% splicing repression with adbp15ag substrate, theophylline inhibited the splicing of adbpt15ag-8s and adbpt15ag-1s pre-mrnas by ~75% and 25%, respectively .
these results indicate a direct correlation between the thermodynamic stability of lower theophylline aptamer stem and efficiency of splicing repression.
it is worth mentioning here that although increasing the length of the lower aptamer stem resulted in stronger inhibition of splicing, stem > <dig> bp could inhibit splicing even in the absence of theophylline .
to examine whether the location of bps within the aptamer affects efficiency of splicing inhibition, we constructed a model pre-mrna in which bps was inserted in the lower aptamer stem .
results presented in fig.
5b demonstrate that relocation of bps to the lower stem rendered host pre-mrna less responsive to theophylline-mediated inhibition of splicing .
quantitation of the data indicate that compared to ~75% splicing inhibition with adbpt15ag-8s, theophylline inhibited adbpt15ag-ls pre-mrna splicing by ~40%.
the efficiency of splicing repression by theophylline is not significantly different between adbp15ag and adbpt15ag-ls pre-mrnas, even though the latter contains a thermodynamically more stable lower stem .
based on these results, we propose a direct correlation between the thermodynamic stability of lower theophylline aptamer stem and efficiency of repression, provided the branchpoint sequence is present in the upper stem.
theophylline-dependent control of alternative splicing in vitro
naturally occurring rna structure elements  <cit>  as well as artificial stem-loop structures  <cit>  are known to influence splice site choice.
to determine whether theophylline-induced secondary structure could influence alternative splicing, we constructed a series of pre-mrnas comprising of three exons interrupted by two introns .
the sequence encompassing exon  <dig>  intron  <dig> and exon  <dig> is identical to adbpt15ag-8s, whereas sequence downstream of exon  <dig> 5' ss is from prg <dig>  an adenovirus major late pre-mrna derivative  <cit>  .
in addition, these pre-mrnas differ from each other in terms of the strength of exon  <dig> 5' ss, which increases in the order: abt0m< abt2m< abt4m< abt6m  <cit> .
we hypothesized that sequestering of intron  <dig> branchpoint by theophylline would allow intron  <dig> branchpoint to choose between the 5' ss of exon  <dig> and  <dig> for the first step of splicing.
thus, depending upon which of the two 5' ss is utilized will determine the amount of exon  <dig> included/excluded mrna.
to test this hypothesis, radioactively labeled abt0m-6m pre-mrnas were transcribed and incubated in hela nuclear extract in the absence or presence of theophylline as described earlier.
splicing of abt0m substrate gave rise to two spliced products , a slower migrating band of ~ <dig> nt and an additional band of ~ <dig> nt .
to determine the identity of these mrnas, representative bands were excised and rna was subjected to rt-pcr followed by dna sequencing.
the sequencing results  indicated that the slower migrating band represents full-length mrna, whereas faster moving band represents exon 2-skipped mrna generated as a result of alternative splicing.
notably, theophylline shifted the splicing of abt0m pre-mrna in favor of exon  <dig> excluded isoform by repressing the activation of intron  <dig> branchpoint.
this is evident by a sharp decline in the amount of full-length mrna and concomitant increase of intron 1-retained mrna .
further examination of these results across abt0m to abt6m panel indicate theophylline-dependent increase in the amount of intron 1-retained mrna, and progressively weak suppression of exon 2-included mrna .
while an increase in the amount of intron 1-containing mrna is due to the inhibition of intron  <dig> excision, increasingly weak suppression of exon  <dig> included mrna is the result of a gradual increase in the strength of exon  <dig> 5' ss, which promotes exon  <dig> inclusion.
these data indicate 2– <dig> fold increase in exon  <dig> excluded: included mrna ratio across the panel .
we also observed the presence of intron  <dig> containing mrna in reactions where theophylline was excluded.
this could simply be the result of lower efficiency of intron  <dig> excision, which was also observed in case of constitutively spliced substrate .
theophylline-dependent control of alternative splicing in vivo
to determine whether theophylline-induced sequestering of branchpoint can control splicing in living cells, we inserted the dna that encodes abt4m pre-mrna into the mammalian expression vector pcdna  <dig> / myc-his c yielding pcabt4m.
a mutant  that does not bind to theophylline was also constructed.
next, hela cells were transfected transiently with pcabt4m, pcabt4mmu or empty vector and treated with theophylline or buffer.
after  <dig> h incubation, cells were harvested, and total rna was isolated.
reverse transcription followed by pcr shown in figure 7a demonstrates that theophylline can regulate the alternative splicing of abt4m pre-mrna.
in contrast, theophylline had a less significant effect on the splicing of abt4mmu pre-mrna, suggesting that binding of theophylline to its cognate rna is necessary for controlling alternative splicing .
we note that theophylline-dependent decrease in exon  <dig> included mrna is not accompanied by a corresponding increase in exon  <dig> skipping.
this suggests that although a block in the assembly of components involved in proximal 3' ss recognition prevented exon inclusion, the effect was not strong enough to influence a switch in splice site choice.
discussion
we have previously shown that insertion of 3' ss ag within a theophylline aptamer inhibits pre-mrna splicing at the second step specifically by theophylline  <cit> .
since selection of bps is made during early stages of the splicing pathway, we reasoned that sequestering of bps within rna-theophylline complex might be an effective strategy for controlling alternative splicing.
to test this hypothesis, we constructed a series of model pre-mrnas in which bps was engineered to be part of theophylline-binding aptamer .
we found that these substrates underwent normal splicing, albeit with lower efficiency, when incubated with hela nuclear extract.
significantly, splicing of these substrates was inhibited specifically by theophylline .
the observed low efficiency of splicing of these substrates could be due to a relatively long bps-to-poly- distance and/or the effect of purine residues that separate bps from the poly- tract; both of these factors have been shown to affect lariat formation and exon ligation  <cit> .
indeed, relocation of bps into lower aptamer stem, which decreased bps-to-poly- distance as well as excluded purine residues, improved splicing efficiency .
in addition, unavailability of a competing bps likely forced the splicing machinery to select a structured branchpoint, thus affecting the splicing efficiency.
support for this explanation comes from a previously reported study in which sequestering of a splice site within artificial hairpin sequence was found to be less detrimental to the splicing provided an alternative splice site is available  <cit> .
given the affinity  of theophylline for cognate rna aptamer is ~ <dig>  mm  <cit> , an incomplete splicing inhibition at  <dig>  mm theophylline is intriguing .
we can offer the following explanations to this observation.
first, the kinetic effect likely prevented rna-theophylline complex from complete inhibition of splicing.
it has been previously reported that the association kinetics of theophylline-aptamer binding is more than 1000-times slower than a diffusion controlled rate, which makes the conformational change in aptamer the rate limiting step for the formation of rna-theophylline complex  <cit> .
thus, even though  <dig>  mm theophylline is expected to saturate rna aptamer, the slow association kinetics likely prevented complete splicing inhibition.
second, differential requirement of mg2+ for binding of theophylline to its cognate rna and in vitro splicing may be the reason for incomplete splicing repression.
whereas efficient binding of theophylline to rna aptamer requires  <dig>  mm mg2+  <cit> , ~ <dig>  mm mg2+ has been found optimum for in vitro splicing  <cit> .
therefore, the residual splicing could be the result of a thermodynamically less stable rna-theophylline-complex.
third, it is possible that theophylline also binds to other cellular factor, which may not affect splicing, but could decrease the amount of available theophylline.
nevertheless, theophylline dependent inhibition of splicing is highly specific  and is carried out within the range of concentrations that has been reported in other studies.
for example, an et al showed that 1– <dig> mm theophylline is required to affect the cleavage of an aptamer-fused shrna by dicer  <cit> .
similarly, translation repression of a cat reporter containing theophylline-binding aptamer in wheat germ extracts required  <dig> mm theophylline  <cit> .
our data suggest that the size of lower theophylline aptamer stem as well as the location of bps within aptamer affects the degree of splicing inhibition.
for example, increasing the length of lower theophylline aptamer stem from a single to eight bp improved the efficiency of splicing inhibition by ~3-fold , suggesting a linear relationship between the length of the stem and the percent splicing inhibition.
the insertion of bps within lower theophylline aptamer stem although improved splicing efficiency, it also rendered pre-mrna to be less responsive to theophylline-dependent splicing inhibition .
the most straightforward explanation to this observation is that when present in the aptamer lower stem, the accessibility of bps to the spliceosome is energetically more favorable compared to its presence in the upper stem whose unwinding may require additional ~ <dig>  kcal/mole  <cit> , the binding energy of theophylline to rna aptamer.
differential bps recognition plays an important role in the regulation of alternative splicing  <cit> .
for example, a splicing suppressor element that binds to sr proteins, 3re, has been shown to regulate adenovirus major late iiia pre-mrna alternative splicing by preventing the binding of u <dig> snrnp to the bps  <cit> .
here in this study we have shown that theophylline induced sequestering of bps can influence alternative splicing both in vitro and in cultured cells .
our data suggest that theophylline-mediated exon  <dig> skipping is due to intron  <dig> branchpoint sequestration, which allows intron  <dig> branchpoint to attack two alternative 5' splice sites for step i of the splicing.
two lines of evidence support this interpretation.
first, splicing of abt0m-6m pre-mrnas yielded intron 1-retained mrnas whose amount increased in the presence of theophylline .
second, theophylline shifted splicing in favor of exon  <dig> excluded mrna by enabling intron  <dig> branchpoint to attack exon  <dig> 5' ss.
this effect is more pronounced in abt4m where the strength of exon  <dig> 5' ss is moderate; a very weak and a very strong exon  <dig> 5' ss is expected to be skipped or included, respectively.
an alternative explanation for theophylline-mediated exon  <dig> skipping is the process of exon definition.
in this scenario sequestering of intron  <dig> branchpoint may interfere in the recognition of exon  <dig> 3' ss, which in turn may compromise u <dig> snrnp binding to the downstream 5' ss resulting in exon skipping.
quantitative analysis of data indicated that theophylline shifted exon  <dig> excluded/exon  <dig> included mrna ratio between  <dig> to 3-fold in all of the tested substrates.
it is important to point out that alternative splicing typically involves relatively small  changes in isoform ratio, and the observed changes in the splice variant ratios may prove to be ideal for controlling the splicing of many biologically important pre-mrnas.
although the present work represents only a proof-of-principle study, this system can be further fine-tuned to influence alternative splicing.
for example, simultaneous sequestration of intron  <dig> branchpoint sequence and the 5' splice site of exon  <dig> has the potential to alter the splice variant ratio by several fold.
a previous report by solnick and lee analyzing the splicing pattern of artificial rna secondary structure indicates that inverted repeat inhibits splicing in vitro, but failed to exert any effect in vivo unless the size of the stem is more than  <dig> nucleotides of perfect complementarity  <cit> .
how, then, does theophylline induced secondary structure with a significantly short stem size compared with the one employed by these authors could influence splicing in vivo?
this could be attributed to the intricate network of hydrogen bonds and stacking interactions that locks theophylline into the aptamer core thereby providing stability to the rna secondary structure  <cit> .
since rna secondary structure formed in solnick and lee study does not appear to be bound by a small molecule or a protein, the cellular rna unwinding activity could have destabilized rna secondary structure allowing normal splicing.
alternatively the use of larger loop size by solnick and lee could have allowed assembly of splicing factors on the nascent rna while the other half of the inverted repeat is undergoing transcription  <cit> .
CONCLUSIONS
in conclusion, we have demonstrated that the rna affinity of theophylline can be exploited to develop a system for conditional control of splicing.
the modulation of mrna splicing by theophylline offers several advantages.
for example, theophylline-mediated control of splicing is flexible and can regulate splicing before or after the completion of the first step  <cit> .
the fact that theophylline is a known drug with favorable pharmacokinetics and cellular uptake properties, this provides the possibility for controlling splicing of a trans gene in a gene therapy setting where the target gene expression could be controlled in a ligand-dependent manner.

 pre-mrna splicing is a fundamental process that joins exons by catalyzing the removal of intervening sequences from mrna precursors. pre-mrnas are spliced in a two-step pathway catalyzed by the spliceosome, a dynamic macromolecular machinery consisting of five small nuclear ribonucleoproteins and many non-snrnp proteins. in the first step, pre-mrna is cleaved at the 5' splice site generating two splicing intermediates: a linear first exon, and an intron-second exon in a lariat configuration. in the second step, the 3'-hydroxyl group of the last nucleotide in first exon makes a nucleophillic attack at the phosphodiester bond separating the intron and the second exon, enabling the joining of two exons and release of the intron as a lariat. the differential joining of 5' and 3' ss of a single pre-mrna, a phenomenon known as alternative splicing, can generate variant mrnas with diverse and often antagonistic functions. alternative splicing of pre-mrna is now considered to be the most important source of protein diversity in vertebrates. it is estimated that 35–60% of human genes generate transcripts that are alternatively spliced, and 70–90% of alternative splicing events affect coding capacity of genes. importantly, deregulated splice variant expression has been identified as the cause of a number of genetic disorders, and certain forms of cancer have been linked to unbalanced isoform expression from genes involved in cell cycle regulation or apoptosis. given the critical role of alternative splicing in a variety of cellular processes, strategies that could influence pre-mrna splicing decisions will have far-reaching effects in biotechnology and medicine. initial efforts aimed at controlling pre-mrna splicing exploited the intrinsic property of nucleic acids to bind specific pre-mrna sequence and inhibit splicing. however, susceptibility of antisense oligonucleotides to nuclease digestion, off-target effects, difficulty with delivery and localization led to the realization that improved methods are required. bifunctional molecules that function like an antisense oligonucleotide, but carry the binding site for a known splicing factor have proved to be useful in reprogramming pre-mrna splicing. while bifunctional molecules have overcome some of the problems of the original antisense-based approach, requirements of chemical modifications limit their utility. in addition, these approaches function in a constitutive manner, i.e., an antisense oligonucleotide directed to inhibit the splicing of a pre-mrna will continue to do so as long as the oligonucleotide is available. since alternative splicing of many pre-mrnas is controlled by splicing regulators, a small molecule ligand that could mimic the function of a splicing regulator would be of broad general application in controlling gene expression. we have previously shown that insertion of a theophylline-responsive riboswitch into the 3' ss region of a model pre-mrna enables its splicing to be repressed during the second step specifically by theophylline. since the vast majority of human genes are alternatively spliced and splice site pairing in general occurs at early stages of the splicing pathway, we reasoned that sequestering of bps in a theophylline aptamer would be a better approach for controlling alternative splicing. in this study, we tested this idea by constructing a series of pre-mrnas in which bps was inserted within theophylline aptamer. we show that theophylline-induced sequestering of bps is highly specific and inhibits pre-mrna splicing both in vitro and in vivo in a dose dependent manner. our results indicate that the thermodynamic stability of rna-theophylline complex and the location of bps within this complex affect the efficiency of splicing inhibition. finally, we show that sequestering of bps within rna-theophylline complex can modulate alternative splicing both in vitro and in cultured cells.  insertion of bps within theophylline-responsive riboswitch confers ligand dependent control of splicing to investigate whether theophylline-induced sequestering of bps would allow conditional control of splicing, we constructed a series of adml pre-mrna derivatives in which bps was embedded within high-affinity theophylline binding aptamer. these pre-mrnas differ in terms of the distance of aptamer sequence from the 3' ss ag. 32p-labeled pre-mrnas were transcribed as runoff transcripts using t rna polymerase and gel purified rnas were incubated in hela nuclear extract under the conditions that support in vitro splicing. the results presented in fig. 1b demonstrate that these substrates underwent normal splicing, albeit with lower efficiency compared with the parent pre-mrna. significantly, splicing reactions performed in the presence of theophylline gave rise to lower yields of spliced mrna, suggesting that theophylline-mediated sequestering of branchpoint inhibits pre-mrna splicing. quantitation of these data indicate that theophylline inhibited the splicing of adbpt pre-mrnas by ~65–90%, but had no effect on the splicing of adml 21ag, a pre-mrna that does not contain theophylline-binding aptamer. we chose adbpt15ag for further experiments because of its better splicing efficiency in the absence of theophylline; while 17% of adbpt15ag pre-mrna was converted into mrna, only ~7% and 9% of adbpt12ag and adbpt18ag pre-mrnas gave rise to spliced product, respectively. to determine theophylline concentration that may be optimum for inhibiting pre-mrna splicing, 32p-labeled adbpt15ag pre-mrna was subjected to in vitro splicing in the presence of increasing concentrations of theophylline. results shown in fig. 1d indicate that theophylline inhibited the splicing of adbpt15ag pre-mrna in a dose-dependent manner with an ic value ± mm. a slight increase in the splicing efficiency at lower concentration of theophylline may be the rna stabilization rather than inhibitory effect of the ligand. splicing inhibition through theophylline-induced sequestering of bps is specific our model of conditional control of splicing specifies that theophylline inhibits pre-mrna splicing by binding to its cognate sequence, thereby sequestering the bps within rna-theophylline complex. to confirm that the splicing inhibition shown in fig. 1b is specific, we performed the following experiments. first, we constructed madbpt15ag pre-mrna that harbors mutations in the core of tba, but otherwise is identical to adbpt15ag. on the basis of previously published reports, which suggest that the 15-nucleotide motif is essential for high-affinity theophylline binding, we predicted the splicing of madbpt15ag to remain unaltered in the presence of theophylline. indeed, results shown in fig. 2b demonstrate that theophylline failed to inhibit the splicing of madbpt15ag pre-mrna. in contrast, a similar concentration of theophylline was found to inhibit the splicing of adbpt15ag. relatively low splicing efficiency of madbpt15ag pre-mrna in the absence of theophylline suggests that mutations in the core of tba although prevented theophylline binding, apparently this did not completely abolish secondary structure of the aptamer. support for this interpretation comes from a web-based rna-folding program indicating that the mutated aptamer can fold into a secondary structure. next, we generated adt+ pre-mrna in which tba was relocated upstream of bps and analyzed its splicing in vitro using hela nuclear extract. as illustrated in fig. 2c, this pre-mrna underwent normal splicing in the presence of theophylline, suggesting that sequestering of bps within rna-theophylline complex is necessary for splicing inhibition. finally, we examined the splicing of adbpt15ag pre-mrna in the presence of caffeine, a purine that is similar in shape and size to theophylline. consistent with our previously published observation, caffeine failed to exert any influence on the splicing of adbpt15ag pre-mrna. taken together, we conclude that theophylline-dependent inhibition of pre-mrna splicing is highly specific. insertion of bps in theophylline-binding aptamer does not activate cryptic branchpoint previous studies have shown that cryptic branchpoint activation allows accurate in vitro splicing of mammalian pre-mrnas albeit with lower efficiency. to determine if relatively lower splicing efficiency of adbpt15ag pre-mrna is due to the activation of a cryptic branchpoint, we mapped the position of the rna branch. preparative amounts of lariat intermediates were isolated from in vitro splicing reaction of adbpt15ag pre-mrna performed in the absence or presence of theophylline. the branchpoint was mapped by the primer extension of untreated or s treated lariat intermediate, which allows debranching of the lariat rna. the position of reverse transcriptase stop site was assigned by using a dideoxy-sequencing ladder from the parental dna. the primer extension analysis of adbpt15ag pre-mrna derived lariat intermediate showed a single stop immediately before the branchpoint adenosine. this band was not detected with unspliced pre-mrna or the lariat intermediate that was treated with s extract. in contrast, the primer extension of s treated lariat species generated a nt band corresponding to the size of linear intron-exon rna. importantly, the lariat intermediate isolated from theophylline treated splicing reaction gave rise to a single reverse transcriptase stop signal corresponding to nt. in addition, this lariat intermediate was s sensitive, and the primer extension of debranched lariat intermediate produced a nt species. the bands marked with asterisk are the primer extension products of s rna likely generated due to the non-specific binding of the primer. this interpretation is supported by the fact that primer extension of rna isolated from s extract resulted in identical reverse transcriptase stops. we conclude that theophylline-dependent sequestering of bps does not activate cryptic branchpoint. thermodynamic stability of lower theophylline aptamer stem and location of bps affects splicing inhibition biochemical and structural studies showed that the lower theophylline aptamer stem is not critical for ligand binding, but affects the stability of rna-theophylline complex. if true, then altering the length of lower theophylline aptamer stem is expected to affect splicing suppression. to test this idea, we constructed two adbpt15ag derivatives in which the length of lower theophylline aptamer stem was either decreased to a single bp or increased to eight bp. to assess the consequences of varied lower theophylline aptamer stem on pre-mrna splicing, 32p-labeled substrates were incubated in hela nuclear extract in the absence or presence of theophylline under splicing conditions. we found that compared to ~60% splicing repression with adbp15ag substrate, theophylline inhibited the splicing of adbpt15ag-8s and adbpt15ag-1s pre-mrnas by ~75% and 25%, respectively. these results indicate a direct correlation between the thermodynamic stability of lower theophylline aptamer stem and efficiency of splicing repression. it is worth mentioning here that although increasing the length of the lower aptamer stem resulted in stronger inhibition of splicing, stem > bp could inhibit splicing even in the absence of theophylline. to examine whether the location of bps within the aptamer affects efficiency of splicing inhibition, we constructed a model pre-mrna in which bps was inserted in the lower aptamer stem. results presented in fig. 5b demonstrate that relocation of bps to the lower stem rendered host pre-mrna less responsive to theophylline-mediated inhibition of splicing. quantitation of the data indicate that compared to ~75% splicing inhibition with adbpt15ag-8s, theophylline inhibited adbpt15ag-ls pre-mrna splicing by ~40%. the efficiency of splicing repression by theophylline is not significantly different between adbp15ag and adbpt15ag-ls pre-mrnas, even though the latter contains a thermodynamically more stable lower stem. based on these results, we propose a direct correlation between the thermodynamic stability of lower theophylline aptamer stem and efficiency of repression, provided the branchpoint sequence is present in the upper stem. theophylline-dependent control of alternative splicing in vitro naturally occurring rna structure elements as well as artificial stem-loop structures are known to influence splice site choice. to determine whether theophylline-induced secondary structure could influence alternative splicing, we constructed a series of pre-mrnas comprising of three exons interrupted by two introns. the sequence encompassing exon intron and exon is identical to adbpt15ag-8s, whereas sequence downstream of exon 5' ss is from prg an adenovirus major late pre-mrna derivative. in addition, these pre-mrnas differ from each other in terms of the strength of exon 5' ss, which increases in the order: abt0m< abt2m< abt4m< abt6m. we hypothesized that sequestering of intron branchpoint by theophylline would allow intron branchpoint to choose between the 5' ss of exon and for the first step of splicing. thus, depending upon which of the two 5' ss is utilized will determine the amount of exon included/excluded mrna. to test this hypothesis, radioactively labeled abt0m-6m pre-mrnas were transcribed and incubated in hela nuclear extract in the absence or presence of theophylline as described earlier. splicing of abt0m substrate gave rise to two spliced products, a slower migrating band of ~ nt and an additional band of ~ nt. to determine the identity of these mrnas, representative bands were excised and rna was subjected to rt-pcr followed by dna sequencing. the sequencing results indicated that the slower migrating band represents full-length mrna, whereas faster moving band represents exon 2-skipped mrna generated as a result of alternative splicing. notably, theophylline shifted the splicing of abt0m pre-mrna in favor of exon excluded isoform by repressing the activation of intron branchpoint. this is evident by a sharp decline in the amount of full-length mrna and concomitant increase of intron 1-retained mrna. further examination of these results across abt0m to abt6m panel indicate theophylline-dependent increase in the amount of intron 1-retained mrna, and progressively weak suppression of exon 2-included mrna. while an increase in the amount of intron 1-containing mrna is due to the inhibition of intron excision, increasingly weak suppression of exon included mrna is the result of a gradual increase in the strength of exon 5' ss, which promotes exon inclusion. these data indicate 2– fold increase in exon excluded: included mrna ratio across the panel. we also observed the presence of intron containing mrna in reactions where theophylline was excluded. this could simply be the result of lower efficiency of intron excision, which was also observed in case of constitutively spliced substrate. theophylline-dependent control of alternative splicing in vivo to determine whether theophylline-induced sequestering of branchpoint can control splicing in living cells, we inserted the dna that encodes abt4m pre-mrna into the mammalian expression vector pcdna / myc-his c yielding pcabt4m. a mutant that does not bind to theophylline was also constructed. next, hela cells were transfected transiently with pcabt4m, pcabt4mmu or empty vector and treated with theophylline or buffer. after h incubation, cells were harvested, and total rna was isolated. reverse transcription followed by pcr shown in figure 7a demonstrates that theophylline can regulate the alternative splicing of abt4m pre-mrna. in contrast, theophylline had a less significant effect on the splicing of abt4mmu pre-mrna, suggesting that binding of theophylline to its cognate rna is necessary for controlling alternative splicing. we note that theophylline-dependent decrease in exon included mrna is not accompanied by a corresponding increase in exon skipping. this suggests that although a block in the assembly of components involved in proximal 3' ss recognition prevented exon inclusion, the effect was not strong enough to influence a switch in splice site choice. discussion we have previously shown that insertion of 3' ss ag within a theophylline aptamer inhibits pre-mrna splicing at the second step specifically by theophylline. since selection of bps is made during early stages of the splicing pathway, we reasoned that sequestering of bps within rna-theophylline complex might be an effective strategy for controlling alternative splicing. to test this hypothesis, we constructed a series of model pre-mrnas in which bps was engineered to be part of theophylline-binding aptamer. we found that these substrates underwent normal splicing, albeit with lower efficiency, when incubated with hela nuclear extract. significantly, splicing of these substrates was inhibited specifically by theophylline. the observed low efficiency of splicing of these substrates could be due to a relatively long bps-to-poly- distance and/or the effect of purine residues that separate bps from the poly- tract; both of these factors have been shown to affect lariat formation and exon ligation. indeed, relocation of bps into lower aptamer stem, which decreased bps-to-poly- distance as well as excluded purine residues, improved splicing efficiency. in addition, unavailability of a competing bps likely forced the splicing machinery to select a structured branchpoint, thus affecting the splicing efficiency. support for this explanation comes from a previously reported study in which sequestering of a splice site within artificial hairpin sequence was found to be less detrimental to the splicing provided an alternative splice site is available. given the affinity of theophylline for cognate rna aptamer is ~ mm, an incomplete splicing inhibition at mm theophylline is intriguing. we can offer the following explanations to this observation. first, the kinetic effect likely prevented rna-theophylline complex from complete inhibition of splicing. it has been previously reported that the association kinetics of theophylline-aptamer binding is more than 1000-times slower than a diffusion controlled rate, which makes the conformational change in aptamer the rate limiting step for the formation of rna-theophylline complex. thus, even though mm theophylline is expected to saturate rna aptamer, the slow association kinetics likely prevented complete splicing inhibition. second, differential requirement of mg2+ for binding of theophylline to its cognate rna and in vitro splicing may be the reason for incomplete splicing repression. whereas efficient binding of theophylline to rna aptamer requires mm mg2+, ~ mm mg2+ has been found optimum for in vitro splicing. therefore, the residual splicing could be the result of a thermodynamically less stable rna-theophylline-complex. third, it is possible that theophylline also binds to other cellular factor, which may not affect splicing, but could decrease the amount of available theophylline. nevertheless, theophylline dependent inhibition of splicing is highly specific and is carried out within the range of concentrations that has been reported in other studies. for example, an et al showed that 1– mm theophylline is required to affect the cleavage of an aptamer-fused shrna by dicer. similarly, translation repression of a cat reporter containing theophylline-binding aptamer in wheat germ extracts required mm theophylline. our data suggest that the size of lower theophylline aptamer stem as well as the location of bps within aptamer affects the degree of splicing inhibition. for example, increasing the length of lower theophylline aptamer stem from a single to eight bp improved the efficiency of splicing inhibition by ~3-fold, suggesting a linear relationship between the length of the stem and the percent splicing inhibition. the insertion of bps within lower theophylline aptamer stem although improved splicing efficiency, it also rendered pre-mrna to be less responsive to theophylline-dependent splicing inhibition. the most straightforward explanation to this observation is that when present in the aptamer lower stem, the accessibility of bps to the spliceosome is energetically more favorable compared to its presence in the upper stem whose unwinding may require additional ~ kcal/mole, the binding energy of theophylline to rna aptamer. differential bps recognition plays an important role in the regulation of alternative splicing. for example, a splicing suppressor element that binds to sr proteins, 3re, has been shown to regulate adenovirus major late iiia pre-mrna alternative splicing by preventing the binding of u snrnp to the bps. here in this study we have shown that theophylline induced sequestering of bps can influence alternative splicing both in vitro and in cultured cells. our data suggest that theophylline-mediated exon skipping is due to intron branchpoint sequestration, which allows intron branchpoint to attack two alternative 5' splice sites for step i of the splicing. two lines of evidence support this interpretation. first, splicing of abt0m-6m pre-mrnas yielded intron 1-retained mrnas whose amount increased in the presence of theophylline. second, theophylline shifted splicing in favor of exon excluded mrna by enabling intron branchpoint to attack exon 5' ss. this effect is more pronounced in abt4m where the strength of exon 5' ss is moderate; a very weak and a very strong exon 5' ss is expected to be skipped or included, respectively. an alternative explanation for theophylline-mediated exon skipping is the process of exon definition. in this scenario sequestering of intron branchpoint may interfere in the recognition of exon 3' ss, which in turn may compromise u snrnp binding to the downstream 5' ss resulting in exon skipping. quantitative analysis of data indicated that theophylline shifted exon excluded/exon included mrna ratio between to 3-fold in all of the tested substrates. it is important to point out that alternative splicing typically involves relatively small changes in isoform ratio, and the observed changes in the splice variant ratios may prove to be ideal for controlling the splicing of many biologically important pre-mrnas. although the present work represents only a proof-of-principle study, this system can be further fine-tuned to influence alternative splicing. for example, simultaneous sequestration of intron branchpoint sequence and the 5' splice site of exon has the potential to alter the splice variant ratio by several fold. a previous report by solnick and lee analyzing the splicing pattern of artificial rna secondary structure indicates that inverted repeat inhibits splicing in vitro, but failed to exert any effect in vivo unless the size of the stem is more than nucleotides of perfect complementarity. how, then, does theophylline induced secondary structure with a significantly short stem size compared with the one employed by these authors could influence splicing in vivo? this could be attributed to the intricate network of hydrogen bonds and stacking interactions that locks theophylline into the aptamer core thereby providing stability to the rna secondary structure. since rna secondary structure formed in solnick and lee study does not appear to be bound by a small molecule or a protein, the cellular rna unwinding activity could have destabilized rna secondary structure allowing normal splicing. alternatively the use of larger loop size by solnick and lee could have allowed assembly of splicing factors on the nascent rna while the other half of the inverted repeat is undergoing transcription.  in conclusion, we have demonstrated that the rna affinity of theophylline can be exploited to develop a system for conditional control of splicing. the modulation of mrna splicing by theophylline offers several advantages. for example, theophylline-mediated control of splicing is flexible and can regulate splicing before or after the completion of the first step. the fact that theophylline is a known drug with favorable pharmacokinetics and cellular uptake properties, this provides the possibility for controlling splicing of a trans gene in a gene therapy setting where the target gene expression could be controlled in a ligand-dependent manner. 
several lines of evidence suggest that theophylline-dependent inhibition of splicing is highly specific, and thermodynamic stability of rna-theophylline complex as well as the location of bps within this complex affects the efficiency of splicing inhibition. finally, we have constructed an alternative splicing model pre-mrna substrate in which theophylline caused exon skipping both in vitro and in vivo, suggesting that a small molecule-rna interaction can modulate alternative splicing. we constructed a series of pre-mrnas in which the bps was inserted within theophylline aptamer. the present study was performed to investigate whether a theophylline-dependent "splice switch" that sequesters the branchpoint sequence within rna-theophylline complex can regulate alternative splicing. we show that theophylline-induced sequestering of bps inhibits pre-mrna splicing both in vitro and in vivo in a dose-dependent manner.
 despite tremendous progress in understanding the mechanisms of constitutive and alternative splicing, an important and widespread step along the gene expression pathway, our ability to deliberately regulate gene expression at this step remains rudimentary. the present study was performed to investigate whether a theophylline-dependent "splice switch" that sequesters the branchpoint sequence within rna-theophylline complex can regulate alternative splicing.  we constructed a series of pre-mrnas in which the bps was inserted within theophylline aptamer. we show that theophylline-induced sequestering of bps inhibits pre-mrna splicing both in vitro and in vivo in a dose-dependent manner. several lines of evidence suggest that theophylline-dependent inhibition of splicing is highly specific, and thermodynamic stability of rna-theophylline complex as well as the location of bps within this complex affects the efficiency of splicing inhibition. finally, we have constructed an alternative splicing model pre-mrna substrate in which theophylline caused exon skipping both in vitro and in vivo, suggesting that a small molecule-rna interaction can modulate alternative splicing.  these findings provide the ability to control splicing pattern at will and should have important implications for basic, biotechnological, and biomedical research. 