1. Exon Identity Established through Differential Antagonism between Exonic Splicing Silencer-Bound hnRNP A1 and Enhancer-Bound SR Proteins 
Jun Zhu, Akila Mayeda, Adrian R. Krainer 
Molecular Cell 
Volume 8, Issue 6, Pages (December 2001)
DOI: /S (01)

2. Figure 1 Effect of hnRNP A1 on Tat23 Pre-mRNA Splicing In Vitro
(A) Diagram of the pre-mRNA substrates, with mutations or deletions in ESS2 or ESS3.
(B) The indicated pre-mRNAs were spliced in HeLa nuclear extract alone (lanes 1, 4, and 7) or supplemented with 3 pmol (lanes 2, 5, and 8) or 6 pmol (lanes 3, 6, and 9) of recombinant hnRNP A1. The asterisk indicates a cleavage product unrelated to splicing (Krainer et al., 1990).
(C) The indicated pre-mRNAs were spliced in HeLa S100 extract complemented with 4 pmol of SF2/ASF alone (lanes 1, 4, 7, 10, and 13) or with 4 pmol of SF2/ASF in the presence of 2 pmol (lanes 2, 5, 8, 11, and 14) or 4 pmol (lanes 3, 6, 9, 12, and 15) of hnRNP A1.
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2
Affinity Depletion and Add-Back of hnRNP A1 in Nuclear Extract
(A) hnRNP A1 and related proteins were selectively depleted from extract by chromatography on immobilized telomeric ssDNA oligonucleotide, and the samples were analyzed by Western blotting with an hnRNP A1-specific monoclonal antibody. Lane 1, untreated extract; lane 2, extract depleted with telomeric ssDNA; lane 3, control extract mock depleted with a mutant immobilized ssDNA oligonucleotide.
(B) A parallel blot of the same samples was probed with mAb96, which is specific for SF2/ASF.
(C) In vitro splicing of the indicated pre-mRNAs in nuclear extract (lanes 1, 5, 9, and 13), mock-depleted extract (lanes 2, 6, 10, and 14), or hnRNP A1-depleted extract alone (lanes 3, 7, 11, and 15) or in the presence of 3.2 pmol of recombinant hnRNP A1 (lanes 4, 8, 12, and 16).
(D) In vitro splicing of tat 23 pre-mRNA in nuclear extract (lane 1), hnRNP A1-depleted nuclear extract alone (lane 2) or in the presence of 3.2 pmol or 9.6 pmol of the indicated recombinant hnRNP A/B wild-type or mutant proteins.
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 3 Silencer Effects in SF2/ASF- or SC35-Dependent Splicing
(A) In vitro splicing of tat23 pre-mRNA in S100 extract (lane 1), hnRNP A1-depleted S100 extract (S100ΔA1; lane 4), or mock-depleted S100 extract (lane 7) complemented with 4 pmol of SF2/ASF (lanes 2, 5, and 8) or SC35 (lanes 3, 6, and 9).
(B) In vitro splicing of tat23m7 pre-mRNA in nuclear extract (lane 1), S100 extract alone (lane 2), or S100 extract complemented with 2 or 4 pmol of SF2/ASF (lanes 3 and 4) or SC35 (lanes 5 and 6).
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 4 Binding of hnRNP A1 and SR Proteins to tat Exon 3
(A) Coomassie-stained SDS-gel of proteins purified from nuclear extract by RNA-affinity chromatography. Proteins bound to 23 nt wild-type (ESS3; lane 2) or mutant (ESS3m; lane 3) ESS3 RNA. M: molecular weight markers, with sizes at left.
(B) Western blotting of the same samples with mAb4B10. Lane 1, 4 μl of nuclear extract; lane 2, 16 μl of proteins eluted from wild-type ESS3-RNA beads; lane 3, 16 μl of proteins eluted from mutant ESS3m-RNA beads.
(C) Competition for RNA binding and UV crosslinking between hnRNP A1 and SF2/ASF. A uniformly labeled 48 nt 3′ fragment of tat exon 3 (20 fmol) was incubated with the indicated recombinant proteins and exposed to UV light. After RNase digestion, the crosslinked proteins were analyzed by SDS-PAGE and autoradiography. The reactions contained 12 pmol of hnRNP A1 (lanes 1 and 5), 10 pmol of SF2/ASF (lane 4) or ΔRS (lane 8), or 12 pmol of hnRNP A1 plus 5 or 10 pmol of SF2/ASF (lanes 2 and 3) or ΔRS (lanes 6 and 7).
(D) Competition between hnRNP A1 and SC35. As in (C), but the reactions contained 12 pmol of hnRNP A1 (lane 1), 12 pmol of hnRNP A1 plus 5 or 10 pmol of SC35 (lanes 2 and 3), or 10 pmol of SC35 (lane 4).
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 5 Cooperative Binding of hnRNP A1 to tat Exon 3
(A) Experimental design. The diagram shows how tat exon 3 RNAs labeled in the proximal region and unlabeled in the distal ESS region were made. The black bar represents the proximal 78 nt of the exon, uniformly labeled with [α-32P]UTP. The gray and white bars represent the unlabeled, 23 nt distal segment of the exon, with either wild-type (ESS) or mutant (ESSm) versions of ESS3.
(B) UV crosslinking of 0.5–4 pmol of recombinant hnRNA1 to wild-type (T3; lanes 1–4) or mutant (T3m; lanes 5–8) tat exon 3. After RNase digestion, the reactions were analyzed by SDS-PAGE and autoradiography.
(C) UV crosslinking of endogenous hnRNP A1 to T3 or T3m RNA in nuclear extract. After RNase digestion, labeled hnRNP A1 adducts were recovered by immunoprecipitation with mAb4B10 and analyzed by SDS-PAGE and autoradiography.
(D) UV crosslinking of endogenous hnRNP A1 to T3 (lanes 1–4) or T3m (lanes 5–8) in S100 extract alone (lanes 1 and 5), or S100 extract complemented with 4 pmol of SF2/ASF (lanes 2 and 6), ΔRS (lanes 3 and 7), or SC35 (lanes 4 and 8). Crosslinked hnRNP A1 was recovered and analyzed as in (C).
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 6
Differential Off Rates of SF2/ASF and SC35 Bound to tat Exon 3
Four picomoles of SF2/ASF or SC35 was preincubated with 225 fmol of the indicated unlabeled exon 3 RNA competitors for 10 min under splicing conditions. S100 extract and 10 fmol of labeled tat23m7 pre-mRNA were then added together to initiate splicing.
(A) Autoradiogram of a representative experiment. T3, wild-type tat exon 3 competitor; T3m, exon 3 competitor with mutated ESS3; T3u, exon 3 competitor with improved SC35 ESE motifs.
(B) Quantitation of the data from three experiments. Splicing efficiency is calculated from phosphorimage data as (mRNA × 100)/pre-mRNA.
(C) Standard splicing reactions with the T3u pre-mRNA in nuclear extract (lane 1), S100 extract alone (lane 2), or S100 extract complemented with 2 or 4 pmol of SF2/ASF (lanes 3 and 4) or SC35 (lanes 5 and 6).
Molecular Cell 2001 8, DOI: ( /S (01) )

8. Figure 7 Model of SR-Protein-Specific Splicing Silencing by hnRNP A1
The four diagrams indicate different states of the HIV tat exon 3 and the preceding intron, depending on which SR protein is present and whether ESS3 is intact. The intron is shown as a horizontal line, and the exon is shown as a light-shaded box. Wild-type and mutant ESS3 are indicated as black and dark gray boxes. SF2/ASF and SC35 are shown as free or RNA-bound molecules, and hnRNP A1 is shown as free molecules, tightly bound to ESS3, or as additional molecules cooperatively bound to RNA. Top left: hnRNP A1 binds to ESS3, but SF2/ASF bound to the upstream ESE motifs in the exon blocks further cooperative binding and propagation of hnRNP A1 toward the 5′ end; silencing is ineffective, and splicing ensues. Top right: SC35 is unable to block the propagation of hnRNP A1 upstream from ESS3; splicing silencing ensues. Bottom, left and right: mutation of the high-affinity hnRNP A1 binding site within ESS3 blocks initial binding of hnRNP A1, and either SF2/ASF or SC35 are free to bind to their respective ESE motifs; splicing ensues. Note that although we showed that hnRNP A1 binding to the upstream portion of the exon depends on the high-affinity binding site in ESS3, cooperative binding of multiple consecutive hnRNP A1 molecules has so far only been demonstrated for single-stranded homopolynucleotides.
Molecular Cell 2001 8, DOI: ( /S (01) )