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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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Related in: MedlinePlus

U1 but not U1A7 snRNA decoys reduce splicing via the atypical 5' ssa, Schematic of the U1 (black) and U1A7 (green) snRNA decoys. The D1 and D7 decoys are short RNAs expressed from the potent U6 promoter, and comprise the first 27 nucleotides of the U6 snRNA for stability, and a sequence with perfect complementarity to the 5' end of U1 (black) or U1A7 (green) snRNAs, respectively. These decoys reduce the free levels of their cognate snRNAs in the cell, affecting the splicing of certain introns. b, The D1 but not the D7 decoy reduced SMN1/2 exon 7 inclusion in minigenes carrying the natural or an atypical 5' ss. The top labels indicate the identity of the 5' ss in exon 7 and the decoy used. The triangle depicts an increasing amount of decoy plasmid transfected with the minigene.
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Figure 4: U1 but not U1A7 snRNA decoys reduce splicing via the atypical 5' ssa, Schematic of the U1 (black) and U1A7 (green) snRNA decoys. The D1 and D7 decoys are short RNAs expressed from the potent U6 promoter, and comprise the first 27 nucleotides of the U6 snRNA for stability, and a sequence with perfect complementarity to the 5' end of U1 (black) or U1A7 (green) snRNAs, respectively. These decoys reduce the free levels of their cognate snRNAs in the cell, affecting the splicing of certain introns. b, The D1 but not the D7 decoy reduced SMN1/2 exon 7 inclusion in minigenes carrying the natural or an atypical 5' ss. The top labels indicate the identity of the 5' ss in exon 7 and the decoy used. The triangle depicts an increasing amount of decoy plasmid transfected with the minigene.

Mentions: In addition, we used U1- and U1A7-specific RNA decoys to further test which of these two transacting factors is involved in the recognition of atypical 5' ss (Fig. 4). The D1 and D7 decoys are short RNAs that carry a sequence with perfect complementarity to the 5' end of U1 or U1A7 snRNAs, respectively (Fig. 4a). The D1 decoy has the consensus 5' ss sequence, and the D7 decoy has the atypical 5' ss sequence. We determined that RNA decoys bind to their cognate snRNAs only when they have perfect complementarity to them (Supplementary Fig. 6 online), thereby reducing the free levels of these snRNPs in the cell and affecting the splicing of certain introns (data not shown). The decoy RNAs were cotransfected with SMN1/2 minigenes with the natural exon 7 5' ss or the atypical 5' ss. The D1 decoy reduced recognition of both the natural (Fig. 4b, lane 11 vs. 10) and the atypical 5' ss in exon 7 (Fig. 4b, lanes 2–5 vs. 1) in a dose-dependent manner. The D7 decoy did not substantially affect recognition of the atypical 5' ss (Fig. 4b, lanes 6–9 vs. 1), and had only a subtle effect on the natural exon 7 5' ss (Fig. 4b, lane 12 vs. 10). The results obtained with the U1/U1A7 suppressors and the decoys demonstrate that both the atypical and the natural SMN1/2 exon 7 5' ss are recognized by the same transacting factor, U1 snRNA, and not by U1A7.


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

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

U1 but not U1A7 snRNA decoys reduce splicing via the atypical 5' ssa, Schematic of the U1 (black) and U1A7 (green) snRNA decoys. The D1 and D7 decoys are short RNAs expressed from the potent U6 promoter, and comprise the first 27 nucleotides of the U6 snRNA for stability, and a sequence with perfect complementarity to the 5' end of U1 (black) or U1A7 (green) snRNAs, respectively. These decoys reduce the free levels of their cognate snRNAs in the cell, affecting the splicing of certain introns. b, The D1 but not the D7 decoy reduced SMN1/2 exon 7 inclusion in minigenes carrying the natural or an atypical 5' ss. The top labels indicate the identity of the 5' ss in exon 7 and the decoy used. The triangle depicts an increasing amount of decoy plasmid transfected with the minigene.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719486&req=5

Figure 4: U1 but not U1A7 snRNA decoys reduce splicing via the atypical 5' ssa, Schematic of the U1 (black) and U1A7 (green) snRNA decoys. The D1 and D7 decoys are short RNAs expressed from the potent U6 promoter, and comprise the first 27 nucleotides of the U6 snRNA for stability, and a sequence with perfect complementarity to the 5' end of U1 (black) or U1A7 (green) snRNAs, respectively. These decoys reduce the free levels of their cognate snRNAs in the cell, affecting the splicing of certain introns. b, The D1 but not the D7 decoy reduced SMN1/2 exon 7 inclusion in minigenes carrying the natural or an atypical 5' ss. The top labels indicate the identity of the 5' ss in exon 7 and the decoy used. The triangle depicts an increasing amount of decoy plasmid transfected with the minigene.
Mentions: In addition, we used U1- and U1A7-specific RNA decoys to further test which of these two transacting factors is involved in the recognition of atypical 5' ss (Fig. 4). The D1 and D7 decoys are short RNAs that carry a sequence with perfect complementarity to the 5' end of U1 or U1A7 snRNAs, respectively (Fig. 4a). The D1 decoy has the consensus 5' ss sequence, and the D7 decoy has the atypical 5' ss sequence. We determined that RNA decoys bind to their cognate snRNAs only when they have perfect complementarity to them (Supplementary Fig. 6 online), thereby reducing the free levels of these snRNPs in the cell and affecting the splicing of certain introns (data not shown). The decoy RNAs were cotransfected with SMN1/2 minigenes with the natural exon 7 5' ss or the atypical 5' ss. The D1 decoy reduced recognition of both the natural (Fig. 4b, lane 11 vs. 10) and the atypical 5' ss in exon 7 (Fig. 4b, lanes 2–5 vs. 1) in a dose-dependent manner. The D7 decoy did not substantially affect recognition of the atypical 5' ss (Fig. 4b, lanes 6–9 vs. 1), and had only a subtle effect on the natural exon 7 5' ss (Fig. 4b, lane 12 vs. 10). The results obtained with the U1/U1A7 suppressors and the decoys demonstrate that both the atypical and the natural SMN1/2 exon 7 5' ss are recognized by the same transacting factor, U1 snRNA, and not by U1A7.

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