Polypyrimidine tract binding protein blocks the 5' splice site-dependent assembly of U2AF and the prespliceosomal E complex

Abstract

Polypyrimidine tract binding protein (PTB) represses some alternatively spliced exons by direct occlusion of splice sites. In repressing the splicing of the c-src N1 exon, we find that PTB acts by a different mechanism. PTB does not interfere with U1 snRNP binding to the N1 5' splice site. Instead, PTB prevents formation of the prespliceosomal early (E) complex across the intervening intron by preventing the assembly of the splicing factor U2AF on the 3' splice site of exon 4. When the unregulated 5' splice site of the upstream exon 3 is present, U2AF binding is restored and splicing between exons 3 and 4 proceeds in spite of the N1 exon bound PTB. Thus, rather than directly blocking the N1 splice sites, PTB prevents the 5' splice site-dependent assembly of U2AF into the E complex. This mechanism likely occurs in many other alternative exons.

Figure 1

A Pre-spliceosomal E complex forms on the BS713 pre-mRNA in WERI-1 extract but not in HeLa extract. (A) Map of the BS713 construct. The BS713 transcript has the 5′ splice site in the upstream intron deleted and has the AdML polypyrimidine tract introduced into the 3′ splice site of the downstream intron. (B) In vitro splicing of the BS713 transcript in HeLa (H) and WERI-1 (W) nuclear extract. The RNA splicing products and intermediates are diagrammed to the right. (C) Spliceosomal complex formation on the BS713 transcript in presence of ATP. BS713 pre-mRNA was incubated in HeLa (lanes1-3) and WERI-1 (lanes 4-6) nuclear extract under splicing conditions for the indicated times and separated on a 2% native agarose gel. Positions of the H, A, B and C complexes are indicated to the right. (D) ATP independent pre-spliceosomal complex formation. BS713 pre-mRNA was incubated in HeLa (lanes 1-4) and WERI-1 (lanes 5-8) nuclear extract in the absence of ATP for the indicated times and separated on a 1.5% native agarose gel. The H and E complexes are marked.

Figure 2

Mutation of one set of PTB binding sites allows E complex formation in HeLa extract (A) BS713D construct carries mutations in the PTB binding site in the 3′ splice site of exon N1 as shown. (B) In vitro splicing of the BS713D transcript (lanes 1 and 3) in WERI-1 and HeLa extract is compared to BS713 (lane 2 and 4). (C) Formation of spliceosomal complexes on the BS714 RNA in absence (lanes 1 and 2) and presence (lanes 3 and 4) of ATP in HeLa and WERI-1 extract. Gel conditions are as described in Figure 1. Positions of the H, E, A, B, and C complexes are indicated.

Figure 3

Purification and RNA content of the H and E complexes (A) Prespliceosomal complexes assembled on the BS713-MS2 pre-mRNA in HeLa or WERI-1 nuclear extract were fractionated on 15-30% glycerol density gradients. The position of the labeled pre-mRNA was determined by scintillation counting. The positions of the E.coli 50S and 30S ribosomes used as markers are indicated. The HeLa H and WERI-1 H complexes (squares and triangles) were assembled at 4°C and have equivalent mobility that is distinct from the MS2-MBP complex (diamonds) and the E complex (crosses) assembled at 30°C. The H and E complex fractions were pooled, passed over amylose resin, and eluted in Maltose (Zhou et al., 2002a; Zhou et al., 2002b; Materials and Methods) (B) RNA was extracted from the purified H and E complexes and 3′-end labeled with ³²P-pCp. Total RNA from nuclear extract (T) was used as markers for the U snRNAs. The positions of the U snRNAs and pre-mRNA are indicated after separation on an 8% Urea-PAGE gel.

Figure 4

The U1 snRNP is base-paired to the N1 exon 5′ splice site in both HeLa and WERI-1 extract and is functional. (A) The BS713 RNA was incubated alone (lane1) or in HeLa (lanes 2-6) or WERI-1 extract (lanes 7-11), in the absence of ATP, at 30°C for 90 minutes. Extracts were mock treated (lanes 4 and 9) or pretreated with oligonucleotide U1_1-15 (lanes 5 and 10) or oligonucleotide U2_28-42 (lanes 6 and 11). AMT-psoralen was added and the reactions irradiated with 366-nm light for 10 minutes as indicated at the top. RNA from each reaction was extracted and separated on 6% urea-PAGE gel. Lane 1 shows the position of intramolecular crosslinks. The U1/pre-mRNA crosslink is indicated to the right. (B) The cross-linked site was mapped by primer extension using an oligonucleotide complimentary to nucleotides 65-86 of the intron downstream of exon N1. Dideoxy sequencing reactions using the same oligonucleotide are shown: ddA (lane 1), ddT (lane 2), ddG (lane 3), and ddC (lane 4). Primer extension was carried out on RNA extracted from reactions in which the BS713/BamHI RNA was incubated in mock-treated (lanes 6 and 8) or oligonucleotide (U1_1-15) treated extract (lanes 7 and 9). (C) The U1 snRNP in the purified complexes is active for splicing. HeLa and WERI-1 nuclear extract was preincubated in the presence (lanes 3-10) or absence (1 and 2) of DNA oligonucleotide complimentary to U1 snRNA (U1_1-15). Equivalent amounts of BS713 pre-mRNA (lanes 1-4), purified HeLa H complex (lanes 5 and 6), WERI-1 H complex (lanes 7 and 8) or WERI-1 E complex (lanes 9 and 10) were added to the treated extracts and incubation was continued for 2 hrs. RNA was extracted and separated by urea-PAGE. The splicing intermediates and products are shown at the right.

Figure 5

HeLa H complex lacks the essential splicing factor U2AF (A) The purified WERI-1 and HeLa H complexes were digested with RNase A, separated by 10 % SDS-PAGE and stained with colloidal Coomassie blue. Each band was excised and treated with trypsin. The peptides were then extracted from the gel slices and the protein identified by mass spectrometry. The identity of each band is shown to the side. Proteins specific to each of the complexes are shown in bold. The question marks indicate proteins that could not be identified. Note that some proteins comigrating with other abundant species could be missed in the mass spectrometry identification. Some of these, such as U2AF35 and SF1, were confirmed by immunoblot. (B) Proteins from the purified HeLa H complex, and the WERI-1 H and E complexes were separated by SDS-PAGE, blotted to nitrocellulose, and probed with the antibodies against the indicated proteins. Total protein from HeLa and WERI-1 extract (N.E.) was run in parallel lanes as controls.

Figure 6

Depletion of PTB from HeLa extract allows binding of U2AF to BS713-MS2 pre-mRNA. (A) Immunodepletion of PTB from HeLa extract was carried out with monoclonal anti-PTB antibody, BB7. Immunoblot analysis of the untreated (U), the mock depleted (M) and the PTB depleted (ΔP) extract used monoclonal antibodies AB5 (anti-KSRP) and BB7 (anti-PTB). (B) The H and E complexes were assembled in PTB-depleted HeLa extract. Recombinant PTB was added back to the depleted extract to 500 nM and the H′ complex was assembled in this extract by incubating the RNA at 30°C for 60 min. RNA from the purified complexes was extracted, precipitated, labeled with ³²P-pCp, and separated on an 8% urea-PAGE gel. Total RNA from nuclear extract (T) was used as markers for the U snRNAs. The positions of the U snRNAs and pre-mRNA are indicated. (C) Total protein from the purified complexes assembled in the mock-depleted (MD) and PTB depleted (ΔPTB) nuclear extracts was separated on a 10% SDS-PAGE, blotted to nitrocellulose membrane, and probed using antibodies against the proteins indicated. Total protein from nuclear extract (N.E.) was run in a parallel lane as control.

Figure 7

PTB mediated inhibition of U2AF binding is dependent on the location of the 5′ splice site. (A) Map of the BS714 pre-mRNA construct. The 5′ splice site of Exon N1 is deleted and the 5′ splice site of exon 3 is restored. (B) In vitro splicing of the BS714 transcript was carried out in HeLa (H) and WERI-1 (W) extract. The RNA splicing products and intermediates are shown to the right. (C) Formation of the splicing complexes on the BS714 RNA in HeLa (H) and WERI-1 (W) extracts in the absence (−ATP) and presence of ATP (+ATP), as described in Fig. 1. (D) The H and E complexes assembled on BS714-MS2 RNA in HeLa extract and purified on amylose resin. Total protein from the purified complexes was separated on a 10% SDS-PAGE, blotted to nitrocellulose membrane, and probed with antibodies to PTB and U2AF65 indicated. Total protein from nuclear extract (N.E.) was run in a parallel lane as control.

Figure 8

Model for PTB mediated exon silencing. PTB binds to the CU elements across N1 exon and forms a silencing complex. This complex does not affect U1 snRNP binding at the N1 exon 5′ splice site, but blocks the interaction of the U1 snRNP with U2AF, and thus prevents assembly of U2AF at the downstream 3′ splice site and formation of the E complex on this intron. The PTB complex does not interfere with the interaction of U2AF with the U1 snRNP at the 5′ splice site of exon 3, allowing splicing of exon 3 to exon 4.