Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing - PubMed (original) (raw)
Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing
Dong-Suk Kim et al. BMC Mol Biol. 2008.
Abstract
Background: 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 (BPS) within RNA-theophylline complex can regulate alternative splicing.
Results: 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.
Conclusion: These findings provide the ability to control splicing pattern at will and should have important implications for basic, biotechnological, and biomedical research.
Figures
Figure 1
Insertion of BPS within theophylline-responsive riboswitch confers ligand-dependent control of splicing. (A) Schematic diagram of AdBPT pre-mRNAs. Underlined sequence and encircled adenosine residue represents the BPS and branch nucleotide, respectively. Open boxes represent exon sequences and horizontal lines between exons indicate introns. Arrow specifies 5' and 3' splice sites. The boxed residues within the secondary structure of theophylline binding aptamer are conserved for theophylline binding. The numbering system follows according to the reference [29]. (B) In vitro splicing of AdML 21AG and AdBPT pre-mRNAs. 32P-labeled pre-mRNAs were incubated in HeLa nuclear extracts at 30°C for 2 h in the absence or presence of theophylline (for experimental details, see methods section). Total RNA isolated from each reaction was fractionated on a 13% polyacrylamide denaturing gel. The bands corresponding to pre-mRNA substrates, intermediates and spliced products are indicated on the right. M, Century™-plus RNA size marker (Ambion). Asterisk (*) indicates degraded lariat. (C) Histogram depicting the effect of theophylline on the splicing of AdML 21AG and AdBPT pre-mRNAs. Splicing was calculated as the ratio of spliced mRNA to the total and normalized to the control (no theophylline). Data represent mean ± standard error of the mean (SEM) of three independent experiments. Asterisk represents significant change as compared to the control (*, P < 0.0005). (D) Theophylline inhibits the splicing of AdBPT15AG pre-mRNA in a dose-dependent manner. 32P-labeled AdBPT15AG pre-mRNA was incubated in HeLa nuclear extracts in the absence or with increasing concentration of theophylline for 2 h as described in (B). % Splicing calculated as described above and plotted against theophylline (mM). The values are expressed as mean ± SEM.
Figure 2
Theophylline-dependent inhibition of pre-mRNA splicing is specific. (A) The diagram of MAdBPT15AG pre-mRNA. The nucleotide residues shown in bold have been mutated to prevent the binding of theophylline. The RNA secondary structure is adapted from Zucker's M-FOLD program [62] (see Additional File 2). (B) Theophylline failed to inhibit the splicing of a substrate (MAdBPT15AG) that contains mutations within aptamer core. 32P-labeled MAdBPT15AG pre-mRNA was subjected to in vitro splicing in the absence (lane 2) or presence of theophylline (lanes 3) for 2 h. The extracted RNA was fractionated on a 13% polyacrylamide denaturing gel. The bands corresponding to splicing substrates, intermediates and spliced products are indicated on right. The % splicing calculated as in Fig. 1 and values are expressed as mean ± SEM. (C) AdT+10, a pre-mRNA in which theophylline-binding aptamer was inserted upstream of BPS was constructed as described in methods. 32P-labeled AdT+10 pre-mRNA was subjected to in vitro splicing in the absence (lane 1) or presence of theophylline (lane 2) for 2 h. The extracted RNA was fractionated on a 13% polyacrylamide denaturing gel (details as in Fig. 1B).
Figure 3
Insertion of BPS in theophylline-binding aptamer does not activate cryptic branchpoint. Lariat intermediate isolated from preparative splicing reactions of AdBPT15AG pre-mRNA performed in the absence (lanes 6 and 7) or presence of theophylline (lanes 8 and 9) was subjected to branchpoint mapping. Primer extension was performed on intact (lane 6, -S100) or debranched lariat intermediate (lanes 7 and 9, +S100). Asterisks (*) represent primer extension products from S100 total RNA (lane 10). The primer extension products were separated on a 13% denaturing polyacrylamide gel. The dideoxy-sequencing ladder from parental DNA is shown in lanes 2–5. M, 10 bp DNA ladder (Invitrogen).
Figure 4
Thermodynamic stability of lower theophylline aptamer stem affects splicing inhibition. (A) Schematic diagram of AdBPT pre-mRNAs exhibiting varying length of lower aptamer stem. The encircled adenosine residue represents branch nucleotide. The distorted hairpin loop structure is the simplified secondary structure of theophylline aptamer. The horizontal lines in aptamer lower stem represent hydrogen bonds between complementary bases. (B) The splicing of AdBPT15AG pre-mRNA with lower aptamer stem of 4 bp (shown in Fig. 1A) was compared with substrates in which the stem was either decreased to a single bp (AdBPT15AG-1S) or increased to eight bp (AdBPT15AG-8S). 32P-labeled pre-mRNAs were subjected to in vitro splicing for 2 h in the absence (lanes 1, 3 and 5) or presence of theophylline (lanes 2, 4 and 6) as described in Fig. 1. The extracted RNAs were fractionated on a 13% denaturing polyacrylamide gel. The % splicing calculated as in Fig. 1 and values are expressed as mean ± SEM.
Figure 5
The location of branchpoint sequence affects efficiency of splicing inhibition. (A) Schematic representation of AdBPT15AG-LS pre-mRNA. The branchpoint sequence is present in the lower aptamer stem and the length of stem is 8 bp. (B) 32P-labeled AdBPT15AG-LS pre-mRNA was incubated in HeLa nuclear extract for 2 h in the absence (lanes 1) or presence of theophylline (lanes 2) as described in Fig. 1. The extracted RNA was fractionated on a 13% polyacrylamide denaturing gel. Percent splicing calculated as in Fig. 1 and values are expressed as mean ± SEM.
Figure 6
Theophylline can modulate alternative splicing in vitro. (A) Schematic representation of three-exon pre-mRNA substrates with varying strength of exon 2 5' ss. The sequence of first two exons and the entire intron 1 is identical to AdBPT15AG-8S. The sequence downstream of intron 2 5' ss, including BPS and exon 3 is from pRG1 [44]. The relative strength of exon 2 5' ss described here was based on the published report [45]. (B) Splicing in vitro of the panel of substrates (shown in A) displaying the effect of theophylline on the alternative splicing of exon 2. 32P-labeled AdML0M-6M pre-mRNAs were subjected to in vitro splicing in the absence (lanes 1, 3, 5 and 7) or presence of theophylline (lanes 2, 4, 6 and 8) as described in Fig. 1. The extracted RNA was fractionated on a 13% polyacrylamide denaturing gel. Other labeling is as in Fig. 1. The internal exon 2 exclusion: inclusion ratio for each substrate in the absence or presence of theophylline is shown below each lane.
Figure 7
Theophylline-mediated modulation of alternative splicing in vivo. (A) HeLa cells were transiently transfected with empty vector, pcABT4M or pcABT4Mmu (containing mutations within core TBA). Cells were treated with buffer (lanes 4 and 6) or with 1 mM theophylline (lanes 5 and 7). The product of RT-PCR assays of total RNA isolated from each well was analyzed on a 2.5% agarose gel to estimate the effect of theophylline on the efficiency of exon 2 alternative splicing. The PCR amplified bands corresponding to exon 2 included or excluded mRNA is indicated. (B) The internal exon 2 exclusion: inclusion ratio for ABT4M or ABT4Mmu in the absence (open box) or presence of theophylline (shaded box) is shown. The data represent mean ± SEM. * P < 0.04 versus mutant is significant.
References
- Butcher SE, Brow DA. Towards understanding the catalytic core structure of the spliceosome. Biochem Soc Trans. 2005;33:447–449. -PubMed
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