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. 1998 Mar 3;95(5):2163-8.
doi: 10.1073/pnas.95.5.2163.

SR proteins are sufficient for exon bridging across an intron

J M Stark  1 , D P Bazett-Jones, M Herfort, M B Roth
Affiliations

SR proteins are sufficient for exon bridging across an intron

J M Stark et al. Proc Natl Acad Sci U S A. .

Abstract

We have developed a defined system to characterize the role of SR proteins and exonic enhancers in directly promoting splice-site interactions across an intron. Using RNA affinity chromatography, we find that SR proteins alone are sufficient to promote the specific association of the enhancer-containing exon 5 with the adjoining exon 6 from avian cardiac troponin-T. Direct visualization of this exon/exon association by electron spectroscopic imaging shows it to be highly specific. Furthermore, using in vivo characterized mutants of exon 5, we also show that this exon/exon association depends on the splicing enhancer within exon 5. These results suggest a model by which SR proteins may function through exonic enhancers to directly promote exon bridging.

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Figures

Figure 1
Figure 1
A subset of SR proteins associate with UP exon 5 and promote its association with the U1 snRNP. Immunoblotting signals from mAb 104 and mAb 16H3 are shown for RNA-affinity complexes isolated from HeLa nuclear extract without RNA lane 2 (A) and lane 3 (B), with the R17-UP RNA lane 3 (A) and lane 4 (B), and with R17-DOWN RNA lane 4 (A) and lane 5 (B). Fifty percent of a complex assembled with 5 μg of column RNA was loaded in each lane. The two bands marked with asterisks on the right of each blot, the lower of which is the R17-GST protein, are due to signal that is independent of primary antibody. Lane 1 of each blot contains 20 μg SR proteins. (B) Lane 2 shows immunoblotting signal from 3 μl HeLa nuclear extract. The outlined arrow to the right indicates a band that is abundant only in starting extract and not the complexes. The solid arrow to the right indicates a band that is present in both starting extract and purified complexes. (C) Northern blot hybridization signals from anti-U snRNA oligonucleotides are shown for R17-UP RNA-affinity complexes isolated from HeLa S100 with (lane 2) or without (lane 1) SR proteins. RNA was also extracted from supernatants of the incubation reactions and probed by Northern blotting with a DNA oligonucleotide antisense to the U1 snRNA. Shown are the resulting hybridization signals for supernatant fractions from the complexes shown in lanes 1 and 2 (lanes 3 and 4, respectively).
Figure 2
Figure 2
SR proteins, in a defined system, promote an association of exons that are spliced together in vivo that is specific with respect to intronic RNAs. (A) Association of exon 6 with R17-UP columns in the presence and absence of SR proteins. The relative amount of exon 6 and control RNA added to R17-UP columns is shown (lane 1). The signals from exon 6 and the control RNA are shown for complexes assembled in the absence (lane 2) or presence (lane 3) of SR proteins at a concentration similar to that in standard in vitro splicing reactions. Shown are the signals for supernatant fractions from the complexes shown in lanes 2 and 3 (lanes 4 and 5, respectively). (B and C) Association of intron fragments Int1 and Int2 with R17-UP columns in the presence and absence of SR proteins. (B) The relative amount of UP exon 5, exon 6, and Int1 RNA added to R17-UP affinity columns is shown (lanes 1, 2, and 3, respectively). The signals from the UP exon 5, exon 6, and Int1 RNAs are shown from complexes assembled in either the absence (B, lanes 4–6, respectively), or presence (lanes 7–9, respectively) of SR proteins. (C) The relative amount of UP exon 5 and Int2 RNA added to R17-UP affinity columns are shown (lanes 1 and 2, respectively). The signals from the UP exon 5 and Int2 RNAs are shown from complexes assembled in duplicate in the absence (lanes 3 + 4 and 5 + 6, respectively) and presence (lanes 7 + 8 and 9 + 10, respectively) of SR proteins. (D) The signal from the 5′ splice site RNA is shown from R17-UP affinity complexes assembled in the absence (lane 1) and presence (lane 2) of SR proteins. Because of the small size of the 5′ splice site, a larger control RNA was used in this experiment. In all experiments, one-third of each complex was loaded on each lane. The relative migration of the exon RNAs and the internal control RNAs is indicated to the right of each figure by arrows and Cs, respectively.
Figure 3
Figure 3
SR protein-mediated exon/exon associations are enhancer-dependent. Shown is relative association of UP exon 5, DOWN exon 5, and HET exon with R17-exon columns in the presence and absence of SR proteins. The relative amount of UP exon 5 and DOWN exon 5 RNA added to R17-exon 6 (A) or R17-UP affinity columns (B) is shown (lanes 1 and 2, respectively). The signals from the UP exon 5 and DOWN exon 5 RNAs are shown from complexes assembled either in the absence (lanes 3 and 4, respectively) or presence (lanes 5 and 6, respectively) of SR proteins. A darker exposure of the control RNA is shown for the complexes assembled with R17-exon 6 (lanes 3–6). (C) The relative amount of UP exon 5 and HET exon RNA added to the R17-UP affinity columns is shown (lanes 1 and 2, respectively). The signals from the UP exon 5 and HET exon RNAs are shown from complexes assembled either in the absence (lanes 3 and 4, respectively) or presence (lanes 5 and 6, respectively) of SR proteins. In all experiments, one-third of each RNA-affinity complex was loaded on each lane. The relative migration of the exon RNAs and the internal control RNAs is indicated to the right of each figure by arrows and Cs, respectively.
Figure 4
Figure 4
Electron spectroscopic images of SR protein/cTnT pre-mRNA incubation reactions show that SR proteins are able to create a loop in the RNA between the two functional exons. SR proteins and UP5/WT6 RNA were incubated under similar conditions as in Fig. 4, and fractions of these reactions were placed directly on EM grids and analyzed with a Zeiss EM 902 transmission electron microscope. The images were recorded at 120 eV. Linear molecules are shown in a, and structures compacted because of RNA secondary structure are shown in b. Loop structures involving the RNA with two functional exons are shown in c–f, one loop involving the RNA with the mutation in exon 5 is shown in g, and RNA with a mutation in exon 6 is shown in h. (Bar = 55 nm.)
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