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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

Shifted base-pairing between atypical 5' ss and the 5' end of U1 snRNAa, Diagram of the two base-pairing registers between the 5' ss (positions are numbered) and U1. Consensus nucleotides are shown in red in all figures (see Methods). Ψ, pseudouridine. Solid dot, 2,2,7-trimethylguanosine cap at the 5' end of U1. Box, upstream exon; line, intron. Base pairs in the canonical (C) or shifted (S) register are indicated by vertical lines. Note that the atypical 5' ss can form seven more base pairs to U1 in the shifted arrangement. b, Mutations at atypical (Atp) 5' ss that disrupt shifted but enhance canonical base-pairing abolish correct splicing. The human GTF2H1 and INPP4A minigenes are schematically represented at the top, indicating the mutations introduced at the atypical 5' ss. M, Molecular weight markers. The identity of the various spliced mRNAs, detected by radioactive RT-PCR, is schematically shown on the left of the gels and corresponds to: #1, correctly spliced mRNA; #2, retention of the downstream intron; #3, use of cryptic 5' ss in the middle exon; #4, skipping of the middle exon; #5, activation of a cryptic 5' ss in the first exon. The percentage of correct splicing is shown at the bottom. See Supplementary Fig. 1 online for details about the aberrantly spliced mRNAs. c, RT-PCR analysis of the atypical 5' ss in the SMN1/2 context (schematic at the top). Nat, natural SMN1/2 exon 7 5' ss. Numbers below the panels show the percentage and Standard Deviation (SD) of exon 7 inclusion.
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Figure 1: Shifted base-pairing between atypical 5' ss and the 5' end of U1 snRNAa, Diagram of the two base-pairing registers between the 5' ss (positions are numbered) and U1. Consensus nucleotides are shown in red in all figures (see Methods). Ψ, pseudouridine. Solid dot, 2,2,7-trimethylguanosine cap at the 5' end of U1. Box, upstream exon; line, intron. Base pairs in the canonical (C) or shifted (S) register are indicated by vertical lines. Note that the atypical 5' ss can form seven more base pairs to U1 in the shifted arrangement. b, Mutations at atypical (Atp) 5' ss that disrupt shifted but enhance canonical base-pairing abolish correct splicing. The human GTF2H1 and INPP4A minigenes are schematically represented at the top, indicating the mutations introduced at the atypical 5' ss. M, Molecular weight markers. The identity of the various spliced mRNAs, detected by radioactive RT-PCR, is schematically shown on the left of the gels and corresponds to: #1, correctly spliced mRNA; #2, retention of the downstream intron; #3, use of cryptic 5' ss in the middle exon; #4, skipping of the middle exon; #5, activation of a cryptic 5' ss in the first exon. The percentage of correct splicing is shown at the bottom. See Supplementary Fig. 1 online for details about the aberrantly spliced mRNAs. c, RT-PCR analysis of the atypical 5' ss in the SMN1/2 context (schematic at the top). Nat, natural SMN1/2 exon 7 5' ss. Numbers below the panels show the percentage and Standard Deviation (SD) of exon 7 inclusion.

Mentions: Accurate pre-mRNA splicing is critical for the correct transmission of information from gene to protein1. Splicing is catalyzed by the spliceosome, a large and dynamic complex composed of five small nuclear ribonucleoprotein particles (snRNPs) made up of snRNAs and associated polypeptides, as well as many other protein factors2. Conserved sequences that match degenerate consensus motifs at both ends of introns are essential for splicing1. As first proposed in 19803,4, and definitively demonstrated in 19865, the 5' ss is initially recognized via base-pairing to the 5' end of the U1 snRNA. The 5' ss consensus sequence for the major, or U2-type GT-AG introns in mammals, which comprise >98% of all introns6, has perfect complementarity to the 5' end of the U1 snRNA3–5,7,8, establishing up to eleven base pairs in a defined register, here referred to as the 'canonical' register (Fig. 1a; see Methods). However, the major spliceosome can accurately recognize a highly diverse set of 5' ss sequences: using SpliceRack6, a comprehensive database of splice sites, we find 2,503 unique human 5' ss sequences – considering only the classical 9-nt motif (see Methods) – that are used at least three times in the transcribed genome, in 186,630 introns.


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

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

Shifted base-pairing between atypical 5' ss and the 5' end of U1 snRNAa, Diagram of the two base-pairing registers between the 5' ss (positions are numbered) and U1. Consensus nucleotides are shown in red in all figures (see Methods). Ψ, pseudouridine. Solid dot, 2,2,7-trimethylguanosine cap at the 5' end of U1. Box, upstream exon; line, intron. Base pairs in the canonical (C) or shifted (S) register are indicated by vertical lines. Note that the atypical 5' ss can form seven more base pairs to U1 in the shifted arrangement. b, Mutations at atypical (Atp) 5' ss that disrupt shifted but enhance canonical base-pairing abolish correct splicing. The human GTF2H1 and INPP4A minigenes are schematically represented at the top, indicating the mutations introduced at the atypical 5' ss. M, Molecular weight markers. The identity of the various spliced mRNAs, detected by radioactive RT-PCR, is schematically shown on the left of the gels and corresponds to: #1, correctly spliced mRNA; #2, retention of the downstream intron; #3, use of cryptic 5' ss in the middle exon; #4, skipping of the middle exon; #5, activation of a cryptic 5' ss in the first exon. The percentage of correct splicing is shown at the bottom. See Supplementary Fig. 1 online for details about the aberrantly spliced mRNAs. c, RT-PCR analysis of the atypical 5' ss in the SMN1/2 context (schematic at the top). Nat, natural SMN1/2 exon 7 5' ss. Numbers below the panels show the percentage and Standard Deviation (SD) of exon 7 inclusion.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Shifted base-pairing between atypical 5' ss and the 5' end of U1 snRNAa, Diagram of the two base-pairing registers between the 5' ss (positions are numbered) and U1. Consensus nucleotides are shown in red in all figures (see Methods). Ψ, pseudouridine. Solid dot, 2,2,7-trimethylguanosine cap at the 5' end of U1. Box, upstream exon; line, intron. Base pairs in the canonical (C) or shifted (S) register are indicated by vertical lines. Note that the atypical 5' ss can form seven more base pairs to U1 in the shifted arrangement. b, Mutations at atypical (Atp) 5' ss that disrupt shifted but enhance canonical base-pairing abolish correct splicing. The human GTF2H1 and INPP4A minigenes are schematically represented at the top, indicating the mutations introduced at the atypical 5' ss. M, Molecular weight markers. The identity of the various spliced mRNAs, detected by radioactive RT-PCR, is schematically shown on the left of the gels and corresponds to: #1, correctly spliced mRNA; #2, retention of the downstream intron; #3, use of cryptic 5' ss in the middle exon; #4, skipping of the middle exon; #5, activation of a cryptic 5' ss in the first exon. The percentage of correct splicing is shown at the bottom. See Supplementary Fig. 1 online for details about the aberrantly spliced mRNAs. c, RT-PCR analysis of the atypical 5' ss in the SMN1/2 context (schematic at the top). Nat, natural SMN1/2 exon 7 5' ss. Numbers below the panels show the percentage and Standard Deviation (SD) of exon 7 inclusion.
Mentions: Accurate pre-mRNA splicing is critical for the correct transmission of information from gene to protein1. Splicing is catalyzed by the spliceosome, a large and dynamic complex composed of five small nuclear ribonucleoprotein particles (snRNPs) made up of snRNAs and associated polypeptides, as well as many other protein factors2. Conserved sequences that match degenerate consensus motifs at both ends of introns are essential for splicing1. As first proposed in 19803,4, and definitively demonstrated in 19865, the 5' ss is initially recognized via base-pairing to the 5' end of the U1 snRNA. The 5' ss consensus sequence for the major, or U2-type GT-AG introns in mammals, which comprise >98% of all introns6, has perfect complementarity to the 5' end of the U1 snRNA3–5,7,8, establishing up to eleven base pairs in a defined register, here referred to as the 'canonical' register (Fig. 1a; see Methods). However, the major spliceosome can accurately recognize a highly diverse set of 5' ss sequences: using SpliceRack6, a comprehensive database of splice sites, we find 2,503 unique human 5' ss sequences – considering only the classical 9-nt motif (see Methods) – that are used at least three times in the transcribed genome, in 186,630 introns.

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