Selection of alternative 5' splice sites: role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1

Abstract

The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.

FIG. 1

Effects of hnRNP A1 and SF2/ASF on selection of alternative 5′SSs and U1 snRNP binding. (A) Splicing in vitro of AdML WW in nuclear extract (NE) supplemented with SF2/ASF (SF2 [1.2 μM]) or hnRNP A1 (A1 [5.5 μM]) or both (SF2+A1). The splicing reaction mixtures contained AMT-psoralen and were incubated for the times shown (in minutes) above each lane before irradiation and electrophoresis of a portion of each reaction on an 8% denaturing polyacrylamide gel. (B) Ratios of splicing efficiency at the upstream (u/s) and downstream (d/s) 5′SSs. The signals from u/s and d/s mRNA in panel A at 60 min were measured, corrected for label incorporation, and expressed as the ratios of the two isoforms of mRNA produced in each reaction. (C) Analysis of U snRNA cross-links formed in the reactions. Portions of the reactions in panel A were analyzed by electrophoresis on a 5% denaturing polyacrylamide gel. (D) Abundance of the cross-linked adducts. The intensities of the cross-linked U1 snRNA bands at 15 min in panel C are shown as percentages of the pre-mRNA intensities and as a ratio for the two sites; the ratio of the U6 cross-links is plotted also. (E) Correlation between U1 snRNP binding and splicing at the downstream (d/s) site. The graph shows on the ordinate the fraction of splicing to the d/s site at 60 min (d/s mRNA/[u/s mRNA + d/s mRNA]) versus the values for d/s U1 cross-links at 15 min (as in the bar chart). Four points are derived from the experiment shown in panels A and C; four others are derived from an independent experiment (not shown).

FIG. 2

hnRNP A1 reduces binding of U1 snRNP to 5′SSs. (A) Effects of hnRNP A1 and SF2/ASF on U1 snRNP-dependent protection of a consensus 5′SS against RNase H cleavage. C175G pre-mRNA was incubated with RNase H and purified U1 snRNP (0.035 μM), recombinant hnRNP A1 (0.59 μM), or recombinant SF2/ASF (0.24 μM). The components in each reaction are shown by shaded boxes above each lane. Portions of each mixture were incubated for 15 min with one of two cleavage oligonucleotides (sites of cleavage shown by arrows). Reaction mixtures 1 through 8 were incubated with an oligonucleotide complementary to a consensus 5′SS, which is protected by U1 snRNP binding; reaction mixtures 9 through 16 were incubated with an oligonucleotide complementary to an unprotected site. The diagrams at the sides show the substrate, with black bars indicating the structure of the RNA fragment in the corresponding position of the gel. (B) Effects of hnRNP A1 and SF2/ASF on binding of RNA to and dissociation from immobilized U1 snRNP. The labeled RNA retained (cpm) is plotted against the approximate time of washing. Purified U1 snRNP was immobilized in microtiter plate wells (ca. 1 μg per well) and incubated in splicing buffer with ³²P-labeled C175G RNA and either hnRNP A1 or SF2/ASF at 0.5 μM. Parallel wells were treated identically but without U1 snRNP [curves labeled (−)].

FIG. 3

hnRNP A1 mutants that are unable to affect alternative splicing do not block U1 snRNP binding. C175G pre-mRNA was subjected to RNase H digestion at the consensus 5′SS for a fixed time after incubation in the presence of U1 snRNP (0.04 μM) and either hnRNP A1, mutant proteins, or other hnRNP A/B proteins (1.0 μM). The proportion of uncut (protected) RNA in each case is expressed relative to the high proportion protected by U1 snRNP in the absence of hnRNP A1. The values shown are the means of 12 determinations in each case, and the error bars represent the standard deviation. The proteins tested are represented to the right of the chart. The two RRMs (boxed) and the Gly-rich C-terminal domain (hexagon) are shown. X indicates mutations in the conserved RNP-1 submotif of an RRM; A1/RS contains the C-terminal RS domain of SF2/ASF (hatched star) instead of the Gly-rich domain; A1-B is an alternatively spliced variant of A1 with an insertion within the C-terminal domain.

FIG. 4

HnRNP A1 does not facilitate U1 snRNP dissociation from 5′SSs or form stable complexes with U1 snRNP or pre-mRNA. Time courses for oligonucleotide-directed RNase H cleavage at the consensus 5′SS in C175G are shown, with the proportion of uncut RNA plotted against the time of RNase H digestion. Components were incubated for some or all of a 20-min period, as shown at the right of the time courses, before RNase H cleavage was initiated by addition of the oligonucleotide complementary to the 5′SS. (A) Effect of hnRNP A1 on U1 snRNP dissociation rates. U1 snRNP was present at 0.04 μM, and hnRNP A1 was present at 1 μM. HnRNP A1 was added with the U1 snRNP (curve 1), after 10 min (curve 2) or just before the cleavage oligonucleotide was added (curve 3). U1 snRNP was added with the oligonucleotide in one sample (curve 5). In this case, RNase H cleavage was unaffected by the addition, indicating that the dissociation of U1 snRNP was irreversible once the oligonucleotides were added. (B) Addition of unlabeled RNA to test whether hnRNP A1 forms stable complexes with pre-mRNA or U1 snRNP. The components were added for the times shown in the diagram. “RNA” is pre-mRNA; “tRNA” is yeast tRNA. The final concentrations, before addition of the oligonucleotides, were as follows: U1 snRNP, 0.04 μM; hnRNP A1, 0.5 μM; yeast RNA, 20 ng/μl.

FIG. 5

HnRNP A1 does not reduce binding of short 5′SS RNA to immobilized U1 snRNP. Pieces of nitrocellulose were incubated with or without 0.5 μg of U1 snRNP. After blocking and washing, the filters were incubated in splicing buffer with four RNA sequences and either hnRNP A1, UP1 (both at 0.5 μM), or no protein. Reactions were done in triplicate. After washing, the RNA was eluted. (A) Analysis of the RNA on biphasic denaturing gels of 5 and 20% polyacrylamide. AdML MM and AdML CC were derived from AdML WW by mutation of the 5′SSs to respectively prevent recognition or produce consensus sequences (68). (B) Quantification of the results in panel A. The band intensities were expressed relative to the intensity of the AdML MM RNA in each lane, and the mean was determined for each set of triplicates. The standard deviations are shown.

FIG. 6

SF2/ASF and hnRNP A1 compete for binding to pre-mRNA. (A) Cross-linking at equilibrium of proteins mixed in various proportions to labeled C175G pre-mRNA. Each 10-μl reaction contained hnRNP A1 at 0.5 μM and SF2/ASF as indicated. After UV irradiation and RNase digestion, samples were analyzed by SDS-PAGE. The right panel shows the ratio of the signals from the cross-linked proteins (y axis) at the different SF2/ASF concentrations, compared with the molar ratios of the proteins added to the RNA. (B) Lack of effect of a high-affinity site for hnRNP A1 on competition for binding. Cross-linking was done with labeled CE1a RNA, containing either a high-affinity site (UAGAGU) or one of two mutant sites (UAGCGU and UAGCU). Reactions were in 5 μl with hnRNP A1 at 0.5 μM. (C) Reactions were done as in panel B but with mutant SF2ΔRS.

FIG. 7

Absence of the RS domain of SF2/ASF does not compromise the enhancement of U1 snRNP binding to 5′SSs. (A) Effects of SF2/ASF mutant proteins on binding of human β-globin IVS-1 pre-mRNA to immobilized U1 snRNP. (Left panel) U1 snRNP was immobilized in microtiter plate wells and, after blocking and washing, incubated with ³²P-labeled RNA in splicing buffer in the presence of SF2/ASF proteins at 0.5 μM; reactions were done in triplicate, and the mean value for labeled RNA retained was plotted, with the standard deviations shown; control wells were treated identically, but U1 snRNP was omitted during immobilization. (Right panel) Involvement of the 5′ end of U1 snRNA in the enhancement of RNA binding by SF2/ASF was determined; reactions were done in triplicate, as above, but before addition of the labeled RNA, the wells were treated with RNase H and either an oligonucleotide complementary to the 5′ end of U1 snRNA (α-U1 5′ oligo) or an arbitrary control oligonucleotide. (B) Binding of U1 snRNP to immobilized RNA was examined. (Left panel) U1 snRNP (0.04 μM) and SF2/ASF proteins (0.5 μM) were incubated with streptavidin-coated beads that had or had not been previously bound by biotinylated C175G RNA; bound U1 snRNPs were eluted and detected after SDS-PAGE by Western blotting with anti-Sm antibodies. (Right panel) U1 snRNP was incubated with α-U1 5′ or control oligonucleotides and RNase H before addition to the binding mixtures; U1 snRNP was detected by both anti-Sm and anti-U1-A antibodies; the U1 snRNP proteins detected are labeled; the light additional bands are produced by cross-reactivity with the SF2/ASF proteins.

FIG. 8

Effects of high-affinity binding sites for hnRNP A1 on splicing and U1 snRNP binding. (A) Derivatives of AdML WW containing 10-nucleotide hnRNP A1 target sites from the fibroblast growth factor receptor 2 gene at position 40 or 132 or both positions were incubated in splicing reaction mixtures supplemented where shown (shaded) with SF2/ASF (1.2 μM) or hnRNP A1 (5.5 μM) or both. The reactions contained AMT-psoralen and were irradiated after incubation for approximately 2, 5, 15, or 60 min (left to right in each block of four lanes). Samples were analyzed by electrophoresis on 5 and 8% denaturing polyacrylamide gels. The portions of the 5% gel that resolved the cross-linked adducts are shown. The asterisks above the diagrams of the pre-mRNA show the sites of insertion of the target sites. (B) Effects of hnRNP A1 on splicing efficiencies at the upstream (u/s) and downstream (d/s) 5′SS. The signals from u/s and d/s mRNA in the 60-min reactions (lanes 4, 12, 20, 28, etc., in panel A) were measured and corrected for label incorporation, and they are shown both separately, as percentages of the total RNA (pre-mRNA and splicing intermediates and products) in the reaction and as the ratios of the two. (C) Abundance of the cross-linked adducts of AdML WW and Ad 40/132. The intensities of the cross-linked U1 snRNA bands at 5 min in panel A (lanes 2, 10, 50, and 58) are shown as percentages of the pre-mRNA intensities and as a ratio for the two sites.

FIG. 9

Models for the molecular mechanisms by which hnRNP A1 and SR proteins affect 5′SS selection. (A) The top panel shows hypothetical binding sites of various affinities for two proteins on a pre-mRNA containing two alternative 5′SSs. The distributions take account of the fact that the proteins have marked sequence preferences and yet appear to be able to occupy all available sites on the RNA at concentrations used in alternative splicing assays in vitro. hnRNP A1 is shown to bind in a more regular fashion because of its cooperativity. The affinity of interaction at each site is indicated by a range from + to +++++. The middle panel depicts a possible binding pattern in nuclear extract at a high concentration of SF2/ASF. The high-, middle-, and low-affinity sites for SF2/ASF are occupied by SF2/ASF protein (small circles), although hnRNP A1 (among other proteins) competes for binding (crescent shape) and excludes SF2/ASF from the latter's lowest-affinity sites. Thus, both splice sites are close to a sequence bound by SF2/ASF, and U1 snRNP binding is enhanced by interactions (double-headed arrow) at both sites. Double occupancy leads to use of the downstream site. Double occupancy also results even at normal concentrations of SF2/ASF if the 5′SSs have the consensus sequence, because U1 snRNP binding outcompetes hnRNP A1. The lower panel illustrates the effect of elevated hnRNP A1 concentrations. Only at the highest-affinity sites can SF2/ASF or other SR proteins bind in preference to hnRNP A1. U1 snRNP binding is reduced by competition with hnRNP A1, and so double occupancy of the 5′SSs is unlikely. Thus, splicing reflects the probability of occupancy of the individual sites. In this case, the upstream site is favored because it is close to a high-affinity site for SF2/ASF, permitting some U1 snRNP binding. The equilibrium between hnRNP A1, SF2/ASF, and U1 snRNP at the downstream site is dynamic, and, on molecules in which U1 snRNP bound initially at the upstream 5′SS, it would be expected that U1 snRNP would also bind eventually to the downstream site. By this time, the upstream site might be committed to splicing. This may account for the observation of a slower rate of nonproductive and cap-independent U1 snRNP binding to unused sites (68). (B) The introduction of two high-affinity binding sites (+++++) for hnRNP A1 resulted in an inversion of the normal effects of hnRNP A1 on alternative splicing. Although the shift to the downstream 5′SS was akin to the effect of SF2/ASF, the reductions in U1 snRNP cross-linking at both 5′SSs and the results with the individual high-affinity sites showed that the mechanism is quite different. We propose that the two sites flanking the upstream 5′ splice site allow it to be inactivated when hnRNP A1 is elevated, either because they mark out a domain for unusually stable cooperative hnRNP A1 binding (patterned hnRNP A1 proteins) or because two bound sites form a stable interacting complex that mitigates against splicing. U1 snRNP occupancy at the downstream site would be low, because of competition with hnRNP A1, but this site would be used eventually. U1 snRNP may bind also to the upstream site, but the sequestered site is unable to interact with other components. It is not known whether the cooperative binding of hnRNP A1 is propagated in both directions along the RNA or is unidirectional.