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Modulation of alternative splicing by long-range RNA structures in Drosophila.

Raker VA, Mironov AA, Gelfand MS, Pervouchine DD - Nucleic Acids Res. (2009)

Bottom Line: Splice site usage can be modulated by secondary structures, but it is unclear if this type of modulation is commonly used or occurs to a significant degree with secondary structures forming over long distances.Mechanistically, the RNA structures masked splice sites, brought together distant splice sites and/or looped out introns.Thus, base-pairing interactions within introns, even those occurring over long distances, are more frequent modulators of alternative splicing than is currently assumed.

View Article: PubMed Central - PubMed

Affiliation: Center for Genomic Regulation (CRG), Dr. Aiguader, 88, 08003 Barcelona, Spain. veronica.raker@crg.es

ABSTRACT
Accurate and efficient recognition of splice sites during pre-mRNA splicing is essential for proper transcriptome expression. Splice site usage can be modulated by secondary structures, but it is unclear if this type of modulation is commonly used or occurs to a significant degree with secondary structures forming over long distances. Using phlyogenetic comparisons of intronic sequences among 12 Drosophila genomes, we elucidated a group of 202 highly conserved pairs of sequences, each at least nine nucleotides long, capable of forming stable stem structures. This set was highly enriched in alternatively spliced introns and introns with weak acceptor sites and long introns, and most occurred over long distances (>150 nucleotides). Experimentally, we analyzed the splicing of several of these introns using mini-genes in Drosophila S2 cells. Wild-type splicing patterns were changed by mutations that opened the stem structure, and restored by compensatory mutations that re-established the base-pairing potential, demonstrating that these secondary structures were indeed implicated in the splice site choice. Mechanistically, the RNA structures masked splice sites, brought together distant splice sites and/or looped out introns. Thus, base-pairing interactions within introns, even those occurring over long distances, are more frequent modulators of alternative splicing than is currently assumed.

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Statistical properties of the box-containing intron set. (A) Percentage of alternative introns (alternative) and introns containing putative polyadenylation events (polyA) in the set of introns with predicted secondary structures (dark bars), as compared those in the population of all Drosophila introns (light bars). Error bars indicate standard errors; n = 202 (see Materials and Methods). (B) Percentage of alternatively-spliced introns with predicted secondary structure (dark bars) are compared to all alternatively-spliced introns (light bars) in the categories (left to right): introns with alternative donor sites, introns with alternative acceptor sites, introns containing skipped exons, retained introns and introns with both alternative acceptor sites and internal polyadenylation signals; n = 102. (C) Distribution of box positions relative to splice sites. Light grey bars indicate the position of the 5′-box relative to the donor site, while dark grey bars indicate the position of the 3′-box relative to the acceptor site. (D) and (E) Log distributions of intron lengths for the set of introns with predicted secondary structures (dark bars) as compared to that for the population of all introns (light bars) in D, and for the set of alternative introns with predicted secondary structures (dark bars) as compared to that for the population of alternative introns (light bars) in E.
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Figure 1: Statistical properties of the box-containing intron set. (A) Percentage of alternative introns (alternative) and introns containing putative polyadenylation events (polyA) in the set of introns with predicted secondary structures (dark bars), as compared those in the population of all Drosophila introns (light bars). Error bars indicate standard errors; n = 202 (see Materials and Methods). (B) Percentage of alternatively-spliced introns with predicted secondary structure (dark bars) are compared to all alternatively-spliced introns (light bars) in the categories (left to right): introns with alternative donor sites, introns with alternative acceptor sites, introns containing skipped exons, retained introns and introns with both alternative acceptor sites and internal polyadenylation signals; n = 102. (C) Distribution of box positions relative to splice sites. Light grey bars indicate the position of the 5′-box relative to the donor site, while dark grey bars indicate the position of the 3′-box relative to the acceptor site. (D) and (E) Log distributions of intron lengths for the set of introns with predicted secondary structures (dark bars) as compared to that for the population of all introns (light bars) in D, and for the set of alternative introns with predicted secondary structures (dark bars) as compared to that for the population of alternative introns (light bars) in E.

Mentions: Analysis of the set of introns that contain the complementary boxes revealed several characteristics that set this group apart statistically. There was a significant enrichment in alternatively spliced introns, as compared to the general population (of 50%, compared to 17% overall; n = 202, P = 1 × 10–36) (Figure 1A). Alternatively spliced introns are better conserved overall than are constitutively spliced introns (23), as is reflected by the enrichment in alternatively spliced introns (of 30%, P = 2 × 10–7) observed in the control set when splice sites were rewired (alternative to alternative and constitutive to constitutive). Nonetheless, the enrichment within the set of introns that contain complementary boxes is still significant compared to that in the rewired control (50% versus 30%, P = 1 × 10–12). No significant difference in equilibrium free energies was found between structures located in alternative and constitutive introns (P = 0.16).Figure 1.


Modulation of alternative splicing by long-range RNA structures in Drosophila.

Raker VA, Mironov AA, Gelfand MS, Pervouchine DD - Nucleic Acids Res. (2009)

Statistical properties of the box-containing intron set. (A) Percentage of alternative introns (alternative) and introns containing putative polyadenylation events (polyA) in the set of introns with predicted secondary structures (dark bars), as compared those in the population of all Drosophila introns (light bars). Error bars indicate standard errors; n = 202 (see Materials and Methods). (B) Percentage of alternatively-spliced introns with predicted secondary structure (dark bars) are compared to all alternatively-spliced introns (light bars) in the categories (left to right): introns with alternative donor sites, introns with alternative acceptor sites, introns containing skipped exons, retained introns and introns with both alternative acceptor sites and internal polyadenylation signals; n = 102. (C) Distribution of box positions relative to splice sites. Light grey bars indicate the position of the 5′-box relative to the donor site, while dark grey bars indicate the position of the 3′-box relative to the acceptor site. (D) and (E) Log distributions of intron lengths for the set of introns with predicted secondary structures (dark bars) as compared to that for the population of all introns (light bars) in D, and for the set of alternative introns with predicted secondary structures (dark bars) as compared to that for the population of alternative introns (light bars) in E.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2724269&req=5

Figure 1: Statistical properties of the box-containing intron set. (A) Percentage of alternative introns (alternative) and introns containing putative polyadenylation events (polyA) in the set of introns with predicted secondary structures (dark bars), as compared those in the population of all Drosophila introns (light bars). Error bars indicate standard errors; n = 202 (see Materials and Methods). (B) Percentage of alternatively-spliced introns with predicted secondary structure (dark bars) are compared to all alternatively-spliced introns (light bars) in the categories (left to right): introns with alternative donor sites, introns with alternative acceptor sites, introns containing skipped exons, retained introns and introns with both alternative acceptor sites and internal polyadenylation signals; n = 102. (C) Distribution of box positions relative to splice sites. Light grey bars indicate the position of the 5′-box relative to the donor site, while dark grey bars indicate the position of the 3′-box relative to the acceptor site. (D) and (E) Log distributions of intron lengths for the set of introns with predicted secondary structures (dark bars) as compared to that for the population of all introns (light bars) in D, and for the set of alternative introns with predicted secondary structures (dark bars) as compared to that for the population of alternative introns (light bars) in E.
Mentions: Analysis of the set of introns that contain the complementary boxes revealed several characteristics that set this group apart statistically. There was a significant enrichment in alternatively spliced introns, as compared to the general population (of 50%, compared to 17% overall; n = 202, P = 1 × 10–36) (Figure 1A). Alternatively spliced introns are better conserved overall than are constitutively spliced introns (23), as is reflected by the enrichment in alternatively spliced introns (of 30%, P = 2 × 10–7) observed in the control set when splice sites were rewired (alternative to alternative and constitutive to constitutive). Nonetheless, the enrichment within the set of introns that contain complementary boxes is still significant compared to that in the rewired control (50% versus 30%, P = 1 × 10–12). No significant difference in equilibrium free energies was found between structures located in alternative and constitutive introns (P = 0.16).Figure 1.

Bottom Line: Splice site usage can be modulated by secondary structures, but it is unclear if this type of modulation is commonly used or occurs to a significant degree with secondary structures forming over long distances.Mechanistically, the RNA structures masked splice sites, brought together distant splice sites and/or looped out introns.Thus, base-pairing interactions within introns, even those occurring over long distances, are more frequent modulators of alternative splicing than is currently assumed.

View Article: PubMed Central - PubMed

Affiliation: Center for Genomic Regulation (CRG), Dr. Aiguader, 88, 08003 Barcelona, Spain. veronica.raker@crg.es

ABSTRACT
Accurate and efficient recognition of splice sites during pre-mRNA splicing is essential for proper transcriptome expression. Splice site usage can be modulated by secondary structures, but it is unclear if this type of modulation is commonly used or occurs to a significant degree with secondary structures forming over long distances. Using phlyogenetic comparisons of intronic sequences among 12 Drosophila genomes, we elucidated a group of 202 highly conserved pairs of sequences, each at least nine nucleotides long, capable of forming stable stem structures. This set was highly enriched in alternatively spliced introns and introns with weak acceptor sites and long introns, and most occurred over long distances (>150 nucleotides). Experimentally, we analyzed the splicing of several of these introns using mini-genes in Drosophila S2 cells. Wild-type splicing patterns were changed by mutations that opened the stem structure, and restored by compensatory mutations that re-established the base-pairing potential, demonstrating that these secondary structures were indeed implicated in the splice site choice. Mechanistically, the RNA structures masked splice sites, brought together distant splice sites and/or looped out introns. Thus, base-pairing interactions within introns, even those occurring over long distances, are more frequent modulators of alternative splicing than is currently assumed.

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