Fig. 6. In vitro splicing analysis determining the effect of ISS and ESS3 mutations. (A) Splicing of tat pre-mRNA in NE in the absence and presence of 32 ng/µl recombinant SF2/ASF (lanes 1 and 2) as compared with tat-derived pre-mRNAs with deleted ESS3 (tatΔ4; lanes 3 and 4), scrambled ISS (tat-sA1; lanes 5 and 6), deleted ISS (tatΔA1; lanes 9 and 10), scrambled ISS and deleted ESS3 (tat-sA1Δ4; lanes 7 and 8), and deleted ISS and ESS3 (tatΔA1Δ4; lanes 11 and 12). (B) Splicing of the chimeric PIPtat pre-mRNA (lanes 1–5), as compared with PIPtat-derived pre-mRNAs with deleted ESS3 (PIPtatΔ4; lanes 6–10), and scrambled ISS and deleted ESS3 (PIPtat-sA1Δ4; lanes 11–15). (C) Splicing of PIPtat pre-mRNA (lanes 1–5) and PIPtat with scrambled ISS (PIPtat-sA1; lanes 6–10). The source of NE was: non-depleted NE, oligo(dT)ΔNE or oligo(dT)ΔNE supplemented with U2AF alone or together with 12.5 ng/µl (+) or 37.5 ng/µl (++) (final concentration) GST–hnRNP A1 as indicated above the lanes. The asterisk indicates a degradation product that co-migrates with the exon–exon product of PIPtat. The identities of the splicing products are indicated.