Fig. 1. RNA substrates used in this study. (A) The primary transcript with indications of the tat splice sites. Boxes and thin lines denote exons and introns, respectively. The tat pre-mRNA contains SD4 and SA7. (B) The names and the schematic drawings of the various splicing substrates are shown to the left. Deletions are indicated by dotted lines and point mutations or scrambling by a cross. The 3′ exon was divided into regions 1–4 for visualization. The mutagenized regions, up- or downstream from SA7, are shown to the right. Lowercase letters indicate point mutations or scrambling, and the nucleotides are numbered relative to SA7. (C) The sequences of UVtat and UVPIP that were used for UV-crosslinking and gel shift analysis derive from tat and PIP7.A, respectively. The 3′ splice sites in tat and PIP7.A are marked SA7 and SA, respectively, and the experimentally verified alternative branch points at nucleotides –48, –26 and –16 relative to SA7 in tat and the branch point in PIP7.A are indicated with bold letters (Dyhr-Mikkelsen and Kjems, 1995; T.Ø.Tange, C.K.Damgaard and J.Kjems, in preparation). The ISS, ESE3 and ESS3 sequences are underlined, and putative hnRNP A1 binding sites that match the hnRNP A1 consensus binding site, UAGGG(U/A) (Burd and Dreyfuss, 1994), at five out of six positions are indicated by brackets above the sequence.

Fig. 3. In vitro splicing of PIP7.A (A) and tat (B) in different NES. The extracts are denoted: NE (non-depleted); mockΔNE (mock immunodepleted); oligo(dT)ΔNE [oligo(dT)-chromatography depleted]; and αMC3ΔNE (U2AF65 immunodepleted). U2AF denotes the addition of U2AF activity recovered from the oligo(dT) column. The identities of the splicing products are indicated.

Fig. 5. Gel mobility-shift assay mapping the hnRNP A1 binding sites in the intron (A) and in the exon part of UVtat (B). The RNA substrates, indicated above the autoradiograms, were incubated in 10 µl reactions containing 0, 30, 100 and 300 ng of GST–hnRNP A1 in (A) and 0, 10, 30, 100 and 300 ng of GST–hnRNP A1 in (B). The migration of the free RNA probes and the complexes is indicated. The asterisk marks the position of RNA dimers of some of the substrates.

Fig. 7. Analysing the effect of hnRNP A1 on spliceosome assembly. (A) In vitro splicing of tat pre-mRNA in NE (lane 1), NE supplemented with 32 ng/µl SF2/ASF (lane 2) or NE supplemented with both 32 ng/µl SF2/ASF and 20 ng/µl hnRNP A1 (lane 3). The identities of the splicing products are indicated. (B) Autoradiogram of SDS–polyacrylamide gel showing UV-crosslinking of proteins to UVtat. Bands corresponding to U2AF65 and GST–hnRNP A1 are indicated, and a protein size marker (in kDa) is shown to the left. The experimental conditions are the same as in (A) except that the samples were incubated for 30 min. (C) Native gel electrophoresis showing complex A3′ formation on UVtat and derivatives with scrambling/deletion of the ISS and/or the ESS3. The substrates were incubated for 25 min in non-depleted NE (lanes 1, 4, 7 and 10), oligo(dT)ΔNE supplemented with U2AF (lanes 2, 5, 8 and 11) and oligo(dT)ΔNE supplemented with U2AF and 15 ng/µl hnRNP A1 (lanes 3, 6, 9 and 12).

Fig. 9. A putative model for regulating splicing of the 3′ splice site in tat pre-mRNA. Binding of hnRNP A1 (A1) to the ISS may physically block the association of factors with the branch point sequences, e.g. SF1/mBBP and/or U2 snRNP, but without affecting U2AF65 binding to the polypyrimidine tract. SF2/ASF binds the ESE3 and stimulates binding of U2AF65 while the ESS3 has a negative effect on U2AF65 binding (Tange and Kjems, 2001). In addition, our data imply that ESS3 also affects spliceosome assembly after U2AF binding via an hnRNP A1-dependent mechanism.

Fig. 2. Monitoring the depletion efficiency of U2AF65 (A) and hnRNP A1 [(B) and (C)] by western blotting. (A) Western blot of non-depleted extract (NE; lane 1), oligo(dT)-depleted extract [oligo(dT)ΔNE; lane 6], immunodepleted extract (αMC3ΔNE; lane 7), mock immunodepleted extract (mockΔNE; lane 8), and serial dilutions of non-depleted NE used for estimating the degree of depletion (lanes 2–5), using U2AF65-specific antibody (αMC3). Ponceau staining of the filter verified that equal amounts of extracts were loaded and transferred to the filter (data not shown). (B) Western blot of the same filter as in (A) probed with hnRNP A1-specific antibody (αA1). (C) Immunodetection of hnRNP A1 in the 2.5 M KCl buffer wash of the oligo(dT) column (lanes 3–7) and the eluted U2AF activity (lane 8). The samples were loaded next to samples containing NE (lane 1) and oligo(dT)ΔNE (lane 2). Bands above or below the marked bands probably derive from unspecific binding of the antibody. A size marker (kDa) is indicated to the left, and the positions of U2AF65 and hnRNP A1 are marked.

Fig. 4. hnRNP A1 specifically inhibits tat splicing. Splicing reactions containing tat (lanes 1–5) or PIP7.A (lanes 6–10) were performed in non-depleted extract (NE; lanes 1 and 6) or in oligo(dT)-depleted extract [oligo(dT)ΔNE; lanes 2–5 and 7–10]. The depleted extract was reconstituted with U2AF activity in lanes 3–6 and 8–10, and 17.5 ng/µl (+) or 35 ng/µl (++) (final concentration) GST–hnRNP A1 was added in lanes 4 and 9, and lanes 5 and 10, respectively. The identities of the splicing products are indicated.

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.

Fig. 8. Native gel electrophoresis analysing the assembly of spliceosomes on tat pre-mRNA-derived constructs in response to hnRNP A1 (15 ng/µl) and inactivation of the ISS and ESS3 elements. The pre-mRNAs, PIPtat, PIPtat-sA1Δ4 and, as control, PIP7.A, were incubated for 20 min under the indicated conditions before analysing the complexes by native polyacrylamide gel electrophoresis. The positions of complexes H, A, B and C are indicated.