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Recognition of atypical 5' splice sites by shifted base-pairing to U1 snRNA.

Roca X, Krainer AR - Nat. Struct. Mol. Biol. (2009)

Bottom Line: These atypical 5' ss are phylogenetically widespread, and many of them are conserved.Moreover, shifted base-pairing provides an explanation for the effect of a 5' ss mutation associated with pontocerebellar hypoplasia.The unexpected flexibility in 5' ss-U1 base-pairing challenges an established paradigm and has broad implications for splice-site prediction algorithms and gene-annotation efforts in genome projects.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, PO Box 100, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.

ABSTRACT
Accurate pre-mRNA splicing is crucial for gene expression. The 5' splice site (5' ss)--the highly diverse element at the 5' end of introns--is initially recognized via base-pairing to the 5' end of the U1 small nuclear RNA (snRNA). However, many natural 5' ss have a poor match to the consensus sequence, and are predicted to be weak. Using genetic suppression experiments in human cells, we demonstrate that some atypical 5' ss are actually efficiently recognized by U1, in an alternative base-pairing register that is shifted by one nucleotide. These atypical 5' ss are phylogenetically widespread, and many of them are conserved. Moreover, shifted base-pairing provides an explanation for the effect of a 5' ss mutation associated with pontocerebellar hypoplasia. The unexpected flexibility in 5' ss-U1 base-pairing challenges an established paradigm and has broad implications for splice-site prediction algorithms and gene-annotation efforts in genome projects.

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Compensatory U1 mutations that restore shifted but not canonical base-pairing rescue splicing at atypical 5' ssa, Scheme of the experimental design. SMN1/2 minigenes carrying point mutations at a heterologous atypical 5' ss in exon 7 were co-transfected with suppressor U1 snRNAs. The 5' ss nucleotides at positions +3 to +6 were individually mutated to C (+3C to +6C). The 5' end of U1 was mutated so as to rescue base-pairing in the canonical or the shifted arrangement (suppressor U1 mutations G3 to G6). Mutant +4C is shown as a representative example, for which U1 mutations G5 or G6 restore base-pairing in the canonical (C) or shifted (S) register, respectively. For the other three mutations, see Supplementary Fig. 2 online. b, RT-PCR analysis of the +3C to +6C 5' ss mutations in SMN1/2 with suppressor U1. Top labels indicate the 5' ss mutant and the suppressor U1 in either register. Atp, wild-type atypical 5' ss. The percentage and Standard Deviation of exon 7 inclusion is shown below each autoradiogram.
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Figure 3: Compensatory U1 mutations that restore shifted but not canonical base-pairing rescue splicing at atypical 5' ssa, Scheme of the experimental design. SMN1/2 minigenes carrying point mutations at a heterologous atypical 5' ss in exon 7 were co-transfected with suppressor U1 snRNAs. The 5' ss nucleotides at positions +3 to +6 were individually mutated to C (+3C to +6C). The 5' end of U1 was mutated so as to rescue base-pairing in the canonical or the shifted arrangement (suppressor U1 mutations G3 to G6). Mutant +4C is shown as a representative example, for which U1 mutations G5 or G6 restore base-pairing in the canonical (C) or shifted (S) register, respectively. For the other three mutations, see Supplementary Fig. 2 online. b, RT-PCR analysis of the +3C to +6C 5' ss mutations in SMN1/2 with suppressor U1. Top labels indicate the 5' ss mutant and the suppressor U1 in either register. Atp, wild-type atypical 5' ss. The percentage and Standard Deviation of exon 7 inclusion is shown below each autoradiogram.

Mentions: First, we tested a series of mutations that introduced a consensus nucleotide at different positions of the atypical 5' ss (Fig. 2a,b; Supplementary Fig. 2a online). All mutations, with the exception of −2A and −1G in the SMN1 context, resulted in partial or complete loss of exon 7 inclusion, further indicating that canonical base-pairing with U1 does not occur at the atypical 5' ss (Fig. 2c). The corresponding suppressor U1 snRNAs in the shifted base-pairing register partially restored exon 7 inclusion for some of these mutants: +5G and +7A in SMN1 (Fig. 2c, upper panel, lanes 8–9 and 12–13), and −1G and +5G in SMN2 (Fig. 2c, lower panel, lanes 4–5 and 8–9). For one mutant 5' ss, +3A in the SMN1 context, the suppressor U1 snRNA decreased exon 7 inclusion (Fig. 2c, upper panel, lanes 6–7), perhaps reflecting a block in a subsequent step in the splicing reaction. The −2A and +6U mutations could not be rescued by suppressor U1s in either of the two contexts. The −2A mutation resulted in very slight exon 7 skipping. The +6U mutation (as well as +6C in Fig. 3, see below) was not rescued by suppressor U1, perhaps because this mutation eliminates a strong G-C base pair essential for efficient binding of U1. Nevertheless, the rescue of exon 7 inclusion by suppressor U1s for mutants −1G, +5G and +7A is consistent with the hypothesis that atypical 5' ss are recognized via shifted base-pairing to U1.


Recognition of atypical 5' splice sites by shifted base-pairing to U1 snRNA.

Roca X, Krainer AR - Nat. Struct. Mol. Biol. (2009)

Compensatory U1 mutations that restore shifted but not canonical base-pairing rescue splicing at atypical 5' ssa, Scheme of the experimental design. SMN1/2 minigenes carrying point mutations at a heterologous atypical 5' ss in exon 7 were co-transfected with suppressor U1 snRNAs. The 5' ss nucleotides at positions +3 to +6 were individually mutated to C (+3C to +6C). The 5' end of U1 was mutated so as to rescue base-pairing in the canonical or the shifted arrangement (suppressor U1 mutations G3 to G6). Mutant +4C is shown as a representative example, for which U1 mutations G5 or G6 restore base-pairing in the canonical (C) or shifted (S) register, respectively. For the other three mutations, see Supplementary Fig. 2 online. b, RT-PCR analysis of the +3C to +6C 5' ss mutations in SMN1/2 with suppressor U1. Top labels indicate the 5' ss mutant and the suppressor U1 in either register. Atp, wild-type atypical 5' ss. The percentage and Standard Deviation of exon 7 inclusion is shown below each autoradiogram.
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Figure 3: Compensatory U1 mutations that restore shifted but not canonical base-pairing rescue splicing at atypical 5' ssa, Scheme of the experimental design. SMN1/2 minigenes carrying point mutations at a heterologous atypical 5' ss in exon 7 were co-transfected with suppressor U1 snRNAs. The 5' ss nucleotides at positions +3 to +6 were individually mutated to C (+3C to +6C). The 5' end of U1 was mutated so as to rescue base-pairing in the canonical or the shifted arrangement (suppressor U1 mutations G3 to G6). Mutant +4C is shown as a representative example, for which U1 mutations G5 or G6 restore base-pairing in the canonical (C) or shifted (S) register, respectively. For the other three mutations, see Supplementary Fig. 2 online. b, RT-PCR analysis of the +3C to +6C 5' ss mutations in SMN1/2 with suppressor U1. Top labels indicate the 5' ss mutant and the suppressor U1 in either register. Atp, wild-type atypical 5' ss. The percentage and Standard Deviation of exon 7 inclusion is shown below each autoradiogram.
Mentions: First, we tested a series of mutations that introduced a consensus nucleotide at different positions of the atypical 5' ss (Fig. 2a,b; Supplementary Fig. 2a online). All mutations, with the exception of −2A and −1G in the SMN1 context, resulted in partial or complete loss of exon 7 inclusion, further indicating that canonical base-pairing with U1 does not occur at the atypical 5' ss (Fig. 2c). The corresponding suppressor U1 snRNAs in the shifted base-pairing register partially restored exon 7 inclusion for some of these mutants: +5G and +7A in SMN1 (Fig. 2c, upper panel, lanes 8–9 and 12–13), and −1G and +5G in SMN2 (Fig. 2c, lower panel, lanes 4–5 and 8–9). For one mutant 5' ss, +3A in the SMN1 context, the suppressor U1 snRNA decreased exon 7 inclusion (Fig. 2c, upper panel, lanes 6–7), perhaps reflecting a block in a subsequent step in the splicing reaction. The −2A and +6U mutations could not be rescued by suppressor U1s in either of the two contexts. The −2A mutation resulted in very slight exon 7 skipping. The +6U mutation (as well as +6C in Fig. 3, see below) was not rescued by suppressor U1, perhaps because this mutation eliminates a strong G-C base pair essential for efficient binding of U1. Nevertheless, the rescue of exon 7 inclusion by suppressor U1s for mutants −1G, +5G and +7A is consistent with the hypothesis that atypical 5' ss are recognized via shifted base-pairing to U1.

Bottom Line: These atypical 5' ss are phylogenetically widespread, and many of them are conserved.Moreover, shifted base-pairing provides an explanation for the effect of a 5' ss mutation associated with pontocerebellar hypoplasia.The unexpected flexibility in 5' ss-U1 base-pairing challenges an established paradigm and has broad implications for splice-site prediction algorithms and gene-annotation efforts in genome projects.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, PO Box 100, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.

ABSTRACT
Accurate pre-mRNA splicing is crucial for gene expression. The 5' splice site (5' ss)--the highly diverse element at the 5' end of introns--is initially recognized via base-pairing to the 5' end of the U1 small nuclear RNA (snRNA). However, many natural 5' ss have a poor match to the consensus sequence, and are predicted to be weak. Using genetic suppression experiments in human cells, we demonstrate that some atypical 5' ss are actually efficiently recognized by U1, in an alternative base-pairing register that is shifted by one nucleotide. These atypical 5' ss are phylogenetically widespread, and many of them are conserved. Moreover, shifted base-pairing provides an explanation for the effect of a 5' ss mutation associated with pontocerebellar hypoplasia. The unexpected flexibility in 5' ss-U1 base-pairing challenges an established paradigm and has broad implications for splice-site prediction algorithms and gene-annotation efforts in genome projects.

Show MeSH
Related in: MedlinePlus