Multiple activities of the human splicing factor ASF

Abstract

The effects of human alternative splicing factor, ASF, on in vitro splicing of adenovirus E1A pre-mRNA were examined. E1A pre-mRNA is a complex substrate, and splicing in HeLa cell nuclear extracts produces six different RNAs using three alternative 5' splice sites and two 3' splice sites. Addition of excess ASF to splicing reactions produced a simplified splicing pattern, in which only one spliced product, 13S RNA, was detected. Inhibition of 12S and 9S splicing, which use 5' splice sites upstream of the 13S 5' splice site, extends previous observations that when multiple 5' splice sites compete for the same 3' splice site, ASF causes preferential selection of the proximal 5' splice site. However, inhibition of the other splices, which use a different upstream 3' splice site, represents a novel activity of ASF, as competition between 5' splice sites is not involved. The effect of ASF on 12S splicing was found to depend on its position relative to competing 5' splice sites, indicating that the ability of ASF to activate proximal 5' splice sites is position- but not sequence-dependent. Finally, addition of small amounts of ASF to ASF-lacking S100 extract was able to activate distal as well as proximal 5' splice sites in two of three pre-mRNAs tested, indicating that in these cases changes in the concentration of ASF alone can be sufficient to modulate alternative 5' splice site selection.

Figure 1

A. Structure of the six alternatively spliced E1A mRNAs produced during in vitro splicing reactions. 13S, 12S, and 9S RNAs are produced by splicing from one of 3 alternative 5′ splice sites to a common 3′ splice site. Additional RNAs are produced by splicing an intron between the 9S 5′ splice site and a 3′ splice site 216 nucleotides downstream of the 9S 5′ splice site. This produces 12.5S RNA in the absence of additional splicing, US RNA when the 13S intron is also removed, and 1 0S RNA when the 12S intron is also removed. B. Products of splicing E1A pre mRNA in the absence of additional ASF (lane 1) and in the presence of 1 μl (lane 2), 2μl (lane 3), or 4μl (lane 4) of purified ASF (see Materials and Methods for purification details).

Figure 2

Sequences of E1A 5′ splice sites. The sequences of the 13S, 12S, and 9S 5′ splice sites are shown, with the sequences of mutants in the splice site shown below the wild-type sequence. Uppercase letters indicate exon sequences, and lowercase letters indicate intron sequences. For the 13S 5′ splice site mutant, dl1500 (Montell et al., 1984), the dashed line indicates the position of a 9 nt. deletion. The 12S 5′ splice site mutation is point mutant pm975 (Montell et al., 1982). Also the probable sequence of the cryptic-1 5′ splice site is indicated by underlining in the dl1500 sequence.

Figure 3

Influence of ASF on the splicing of mutant E1A pre-mRNAs. Products of splicing the 12S 5′ splice site mutant (lanes 1 and 2), 13S 5′ splice site mutant (lanes 3 and 4), 12S, 13S double 5′ splice site mutant (lanes 5 and 6), and 13S, 12S, 9S triple 5′ splice site mutant (lanes 7 and 8) pre-mRNAs; in the absence (lanes 1, 3, 5, and 7) or presence (lanes 2, 4, 6, and 8) of excess ASF. In addition to the authentic E1A splicing products, the positions of two cryptic splicing products are indicated. These are the only products of splicing the triple mutant RNA in the presence of excess ASF (lane 8); two other bands seen in this lane are the 5′ exons for cryptic 1 and cryptic 2 splicing. In this experiment 4 μl of partially purified ASF (see Materials and Methods for details of ASF purification) were added to the reactions shown in even-numbered lanes. Asterisks indicate the positions of the 12.5S, 11S, and 10S lariat + 3′ exon intermediates, which are in different positions relative to the linear products in the gel shown here, compared to that shown in Figure 1B, because the two gels were not run under identical conditions.

Figure 4

Comparison of the effects of ASF and increasing ionic strength in splicing reactions. Wild-type E1A (lanes 1–10) or the 13S 5′ splice site mutant (lanes 11–20) pre-mRNAs were spliced in under standard splicing condition as described in Materials and Methods (lanes 1 and 11); in reactions to which 1 μl (lanes 2 and 12), 2 μl (lanes 3 and 13), 3 μl (lanes 4 and 14), or 4 μl (lanes 5 and 15) of ASF were added; or in reactions containing increasing concentrations of KCl as indicated (lanes 6–10 and 16–20). Asterisks indicate the positions of the 12.5S, US, and 10S lariat + 3′ exon intermediates (see legend to Figure 3).

Figure 5

Effect of low ASF concentrations on alternative splicing of E1A pre-mRNAs. Wild-type E1A (lanes 1–5) and the 13S 5′ splice site mutant (lanes 6–10) were processed in S100 extract in the absence of ASF (lanes 1 and 6), or in the presence of 0.5 μl (lanes 2 and 7), 1 μl (lanes 3 and 8), 2 μl (lanes 4 and 9), or 4 μl (lanes 5 and 10) of ASF.

Figure 6

Effect of low ASF concentrations on alternative splicing of SV40 early region pre-mRNA. SV40-i66 pre-mRNA was processed in S100 extract in the absence of ASF (lane 1), or in the presence of 0.1 μl (lane 2), 0.5 μl (lane 3), 1 μl (lane 4), 2 μl (lane 5), or 3 μl (lane 6) of ASF.