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. 2008 Feb;15(2):183-91.
doi: 10.1038/nsmb.1375. Epub 2008 Jan 13.

Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome

Affiliations

Polypyrimidine tract binding protein controls the transition from exon definition to an intron defined spliceosome

Shalini Sharma et al. Nat Struct Mol Biol. 2008 Feb.

Abstract

The polypyrimidine tract binding protein (PTB) binds pre-mRNAs to alter splice-site choice. We characterized a series of spliceosomal complexes that assemble on a pre-mRNA under conditions of either PTB-mediated splicing repression or its absence. In the absence of repression, exon definition complexes that were assembled downstream of the regulated exon could progress to pre-spliceosomal A complexes and functional spliceosomes. Under PTB-mediated repression, assembly was arrested at an A-like complex that was unable to transition to spliceosomal complexes. Trans-splicing experiments indicated that, even when the U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) are properly bound to the upstream and downstream exons, the presence of PTB prevents the interaction of the two exon complexes. Proteomic analyses of these complexes provide a new description of exon definition complexes, and indicate that splicing regulators can act on the transition between the exon definition complex and an intron-defined spliceosome.

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Figures

Figure 1
Figure 1. An exon definition complex forms in both HeLa and WERI extracts
(a) Maps of the BS713, BS713ED, and BSEx4 constructs. BS713 has the 5′ splice site of exon 3 deleted and the AdML polypyrimidine tract introduced into the exon 4 3′ splice site. In BS713ED, 5′ splice site is included downstream of exon 4. BSEx4 has exon 4 and 50-nucleotide sequences from the upstream and downstream introns. (b) Formation of the ATP independent EDE complex. Native agarose gel analysis of complex formation on the Ex4 RNA in HeLa (lanes 1–3) and WERI (lanes 7–9) extracts in the absence of ATP. Heparin treatment dissociates the EDE complex in both HeLa (lanes 4–6) and WERI (lanes 10–12) extracts. Positions of the H and EDE complexes are indicated. A minor heparin resistant complex, indicated by an asterisk, is sometimes seen in the WERI extract, but this band is not consistently observed. (c) Formation of ATP dependent EDA complex. Native agarose gel analysis of complex formation on the Ex4 RNA in presence of ATP in HeLa (lanes1–3) and WERI (lanes 4–6) extract after heparin treatment. Positions of the H and EDA complexes are indicated.
Figure 2
Figure 2. The U2 snRNA is basepaired to the pre-mRNA in the EDA complex
Northern analysis of the snRNAs crosslinked to the Ex4 RNA in buffer DG (lane 1, 2, 9, and 10) or in HeLa (lanes 3–5 and lanes 11–13) or in WERI (lanes 6–8 and lanes 14–16) extract in the absence (lanes 1–8) or presence (lanes 9–16) of ATP, using probes recognizing the pre-mRNA RNA (a), U1 (b), or U2 snRNAs (c). An arrow on the right indicates the crosslinked species. Note that the crosslinked U1 and U2 snRNA bands have similar migration but do not exactly overlap. Identity of the band indicated by the asterisk in not clear.
Figure 3
Figure 3. U2 snRNP assembly via exon definition does not overcome splicing repression
In vitro splicing of BS713ED RNA (lanes 3 and 4) in HeLa and WERI extract is compared to the BS713 (lanes 1 and 2). Splicing intermediates and products are indicated.
Figure 4
Figure 4. Splicing repression after exon definition in HeLa extract is PTB dependent
(a) The BS713EDM construct carries a mutation in the N1 exon 3′ splice site as shown that eliminates PTB binding. (b) In vitro splicing of the BS713EDM transcript (lanes 1 and 3) is compared to BS713ED (lanes 2 and 4) in HeLa and WERI extracts. (c) Western analysis of the mock depleted and the PTB depleted extract using antibodies against proteins PTB, U1A, and Raver1. (d) In vitro splicing of the BS713ED (lanes 1–3) and BS713EDM (lanes 4–6) transcripts in mock depleted, PTB depleted, and PTB reconstituted HeLa extract is compared.
Figure 5
Figure 5. ATP-independent E′ complex assembles in both HeLa and WERI extracts
(a) Native agarose gel analysis of complex formation on the BS713ED RNA in absence of ATP in HeLa (lanes1–3) and WERI (lanes 4–6) extract. Positions of the H and E′ complexes are indicated. (b) 32P-pCp labeling of RNA from the purified E′ complexes. Total RNA from nuclear extract (T) was used as markers for the U snRNAs. The positions of the pre-mRNA and U snRNAs are indicated.
Figure 6
Figure 6. ATP dependent splicing complex assembly in HeLa extract is stalled at the A
complex. (a) Native agarose gel analysis of complex formation on the BS713ED RNA in presence of ATP in HeLa (lanes1–3) and WERI (lanes 4–6) extract. Positions of the H, A, A′, B, and C complexes are indicated. Note that the nature of the minor high molecular weight complex in HeLa extract is not clear. (b) Native agarose gel analysis of splicing complexes assembled on BD713ED RNA in HeLa (top panel) and WERI (bottom panel) extracts and fractionated on 15–30% glycerol density gradients. (c) 32P-pCp labeling of RNA from the purified A′ complex (A′) and from total spliceosomes (S). Total RNA from nuclear extract (T) was used as markers for the U snRNAs. The positions of the pre-mRNA and U snRNAs are indicated.
Figure 7
Figure 7. Trans splicing occurs in WERI but not HeLa extract
(a) RNA substrates used in the trans splicing assay. The 5′ RNA was transcribed from BamHI cleaved BS713 and the 3′ RNA was transcribed from BSEx4. (b) Trans-splicing analysis of uniformly 32P-labeled 5′ RNA alone (lanes 1 and 7) or in presence of cold 3′ RNA in HeLa (lanes 1–6) and WERI (lanes 7–12) extract. (c) MS2-affinity tag mediated pull down of uniformly 32P-labeled 3′ RNA in the presence or absence of the 5′ RNA in HeLa and WERI extract either in the presence or absence of ATP.
Figure 8
Figure 8. Model for PTB mediated splicing repression
(a) In WERI extract spliceosomal complexes H, E, A, B, and C form efficiently, whereas is HeLa extract assembly is stalled at an A-like complex (A′). (b) Intron bound PTB does not interfere with the binding of U1 snRNP at the N1 exon 5′ splice site or with the assembly of an exon definition complex on the downstream exon. PTB prevents the intron bound U1 and U2 snRNPs from interacting and thus prevents intron definition and spliceosome assembly.

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References

    1. Smith CW, Valcarcel J. Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci. 2000;25:381–8. - PubMed
    1. Li Q, Lee JA, Black DL. Neuronal regulation of alternative pre-mRNA splicing. Nat Rev Neurosci. 2007 - PubMed
    1. Shin C, Manley JL. Cell signalling and the control of pre-mRNA splicing. Nat Rev Mol Cell Biol. 2004;5:727–38. - PubMed
    1. Matlin AJ, Clark F, Smith CW. Understanding alternative splicing: towards a cellular code. Nature. 2005;6:386–398. - PubMed
    1. Black DL. Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem. 2003;72:291–336. - PubMed

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