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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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A stem structure regulates alternative usage of acceptor splice sites in the Nmnat (CG13645) mini-gene. (A) Top panel: Representation of the Nmnat mini-gene (chromosome 3R:20771699–20772905). Exon 5 is an internal terminal exon that is cleaved when included (the poly-adenylation/3′-processing signals are indicated), while exon 6 is an internal exon. Box 2 is located upstream of the poly(A) signal in exon 5. Primers used in the PCR amplifications are indicated. Bottom panel: Multiple sequence alignment of the sequence between exons 4 and 6. The complementary boxes are almost 100% conserved, with only one change of GU to AU in the base pairs. (B) Splicing products from the mini-gene were amplified with a reverse primer to the vector to amplify the isoforms formed by splicing to the distal acceptor (D) or to the proximal acceptor (P) that had not been cleaved. The results of three independent splicing assays are represented graphically in the bottom panel for distal acceptor usage. Samples were normalized prior to loading against an independent PCR performed in parallel with a reverse primer to exon 4, to visualize the constitutively-spliced product of exon 3–exon 4 (data not shown). (C) As in (B), except that a reverse primer in exon 5 was used to amplify splice products to proximal acceptor (P) or with intron 4 retention (IR). Proximal acceptor usage is depicted graphically at the bottom. (D) Endogenous mRNA was amplified with reverse primers in exon 6 or exon 5. (E) Predicted base pairing for the wild-type, box 1, box 2 and box 1/2 mutants (point mutations are shown in boldface), and their estimated equilibrium free energies. Since the sequence was completely exchanged during mutagenesis, no base-pairing is predicted to occur for the single box mutations (box 1 and box 2).
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Figure 4: A stem structure regulates alternative usage of acceptor splice sites in the Nmnat (CG13645) mini-gene. (A) Top panel: Representation of the Nmnat mini-gene (chromosome 3R:20771699–20772905). Exon 5 is an internal terminal exon that is cleaved when included (the poly-adenylation/3′-processing signals are indicated), while exon 6 is an internal exon. Box 2 is located upstream of the poly(A) signal in exon 5. Primers used in the PCR amplifications are indicated. Bottom panel: Multiple sequence alignment of the sequence between exons 4 and 6. The complementary boxes are almost 100% conserved, with only one change of GU to AU in the base pairs. (B) Splicing products from the mini-gene were amplified with a reverse primer to the vector to amplify the isoforms formed by splicing to the distal acceptor (D) or to the proximal acceptor (P) that had not been cleaved. The results of three independent splicing assays are represented graphically in the bottom panel for distal acceptor usage. Samples were normalized prior to loading against an independent PCR performed in parallel with a reverse primer to exon 4, to visualize the constitutively-spliced product of exon 3–exon 4 (data not shown). (C) As in (B), except that a reverse primer in exon 5 was used to amplify splice products to proximal acceptor (P) or with intron 4 retention (IR). Proximal acceptor usage is depicted graphically at the bottom. (D) Endogenous mRNA was amplified with reverse primers in exon 6 or exon 5. (E) Predicted base pairing for the wild-type, box 1, box 2 and box 1/2 mutants (point mutations are shown in boldface), and their estimated equilibrium free energies. Since the sequence was completely exchanged during mutagenesis, no base-pairing is predicted to occur for the single box mutations (box 1 and box 2).

Mentions: Within the nicotinamide mononucleotide adenylytransferase (Nmnat) pre-mRNA, the boxes surround the proximal of two alternative acceptor sites within the alternatively spliced intron 4 (Figure 4A). Splicing to the proximal acceptor site introduces an alternative terminal exon with a polyadenylation site, while splicing to the distal acceptor site introduces an internal exon, resulting in distinct C-termini of the Nmnat protein isoforms. Splicing of this intron was first analyzed for usage of the distal acceptor site (Figure 4B). Completely exchanging the sequence of either box 1 or box 2 to eliminate complementarity (Figure 4E) drastically reduced the level of splicing to the distal acceptor (Figure 4B). Re-establishing a stem structure with the novel sequence (box 1/2; Figure 4B) reversed this effect, demonstrating the role of the stem structure in modulating the distal acceptor site usage. In contrast, analysis of the use of the proximal acceptor site (by using a primer specific for exon 5) revealed that, although this site is used in the wild-type mini-gene, its usage increased with both the box 1 and the box 2 mutations (by about 1.5-fold) and again decreased with the novel stem formation (Figure 4C). Mechanistically, the actions of the stem structure could be explained in a dual manner. First, since the proximal acceptor site is the stronger of the two sites (P = 0.04), looping it out with the stem structure could make it less competitive. Second, the distal acceptor site is more than 400 nt downstream of the proximal one, making the intervening intron much longer than the average intron in Drosophila. Forming a stem by the two complementary sequences, which are separated by about 350 nt, could physically bring this distal site to the proximity of the donor site and thereby promote its usage.


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

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

A stem structure regulates alternative usage of acceptor splice sites in the Nmnat (CG13645) mini-gene. (A) Top panel: Representation of the Nmnat mini-gene (chromosome 3R:20771699–20772905). Exon 5 is an internal terminal exon that is cleaved when included (the poly-adenylation/3′-processing signals are indicated), while exon 6 is an internal exon. Box 2 is located upstream of the poly(A) signal in exon 5. Primers used in the PCR amplifications are indicated. Bottom panel: Multiple sequence alignment of the sequence between exons 4 and 6. The complementary boxes are almost 100% conserved, with only one change of GU to AU in the base pairs. (B) Splicing products from the mini-gene were amplified with a reverse primer to the vector to amplify the isoforms formed by splicing to the distal acceptor (D) or to the proximal acceptor (P) that had not been cleaved. The results of three independent splicing assays are represented graphically in the bottom panel for distal acceptor usage. Samples were normalized prior to loading against an independent PCR performed in parallel with a reverse primer to exon 4, to visualize the constitutively-spliced product of exon 3–exon 4 (data not shown). (C) As in (B), except that a reverse primer in exon 5 was used to amplify splice products to proximal acceptor (P) or with intron 4 retention (IR). Proximal acceptor usage is depicted graphically at the bottom. (D) Endogenous mRNA was amplified with reverse primers in exon 6 or exon 5. (E) Predicted base pairing for the wild-type, box 1, box 2 and box 1/2 mutants (point mutations are shown in boldface), and their estimated equilibrium free energies. Since the sequence was completely exchanged during mutagenesis, no base-pairing is predicted to occur for the single box mutations (box 1 and box 2).
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Related In: Results  -  Collection

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Figure 4: A stem structure regulates alternative usage of acceptor splice sites in the Nmnat (CG13645) mini-gene. (A) Top panel: Representation of the Nmnat mini-gene (chromosome 3R:20771699–20772905). Exon 5 is an internal terminal exon that is cleaved when included (the poly-adenylation/3′-processing signals are indicated), while exon 6 is an internal exon. Box 2 is located upstream of the poly(A) signal in exon 5. Primers used in the PCR amplifications are indicated. Bottom panel: Multiple sequence alignment of the sequence between exons 4 and 6. The complementary boxes are almost 100% conserved, with only one change of GU to AU in the base pairs. (B) Splicing products from the mini-gene were amplified with a reverse primer to the vector to amplify the isoforms formed by splicing to the distal acceptor (D) or to the proximal acceptor (P) that had not been cleaved. The results of three independent splicing assays are represented graphically in the bottom panel for distal acceptor usage. Samples were normalized prior to loading against an independent PCR performed in parallel with a reverse primer to exon 4, to visualize the constitutively-spliced product of exon 3–exon 4 (data not shown). (C) As in (B), except that a reverse primer in exon 5 was used to amplify splice products to proximal acceptor (P) or with intron 4 retention (IR). Proximal acceptor usage is depicted graphically at the bottom. (D) Endogenous mRNA was amplified with reverse primers in exon 6 or exon 5. (E) Predicted base pairing for the wild-type, box 1, box 2 and box 1/2 mutants (point mutations are shown in boldface), and their estimated equilibrium free energies. Since the sequence was completely exchanged during mutagenesis, no base-pairing is predicted to occur for the single box mutations (box 1 and box 2).
Mentions: Within the nicotinamide mononucleotide adenylytransferase (Nmnat) pre-mRNA, the boxes surround the proximal of two alternative acceptor sites within the alternatively spliced intron 4 (Figure 4A). Splicing to the proximal acceptor site introduces an alternative terminal exon with a polyadenylation site, while splicing to the distal acceptor site introduces an internal exon, resulting in distinct C-termini of the Nmnat protein isoforms. Splicing of this intron was first analyzed for usage of the distal acceptor site (Figure 4B). Completely exchanging the sequence of either box 1 or box 2 to eliminate complementarity (Figure 4E) drastically reduced the level of splicing to the distal acceptor (Figure 4B). Re-establishing a stem structure with the novel sequence (box 1/2; Figure 4B) reversed this effect, demonstrating the role of the stem structure in modulating the distal acceptor site usage. In contrast, analysis of the use of the proximal acceptor site (by using a primer specific for exon 5) revealed that, although this site is used in the wild-type mini-gene, its usage increased with both the box 1 and the box 2 mutations (by about 1.5-fold) and again decreased with the novel stem formation (Figure 4C). Mechanistically, the actions of the stem structure could be explained in a dual manner. First, since the proximal acceptor site is the stronger of the two sites (P = 0.04), looping it out with the stem structure could make it less competitive. Second, the distal acceptor site is more than 400 nt downstream of the proximal one, making the intervening intron much longer than the average intron in Drosophila. Forming a stem by the two complementary sequences, which are separated by about 350 nt, could physically bring this distal site to the proximity of the donor site and thereby promote its usage.

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.

Show MeSH