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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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Alternative splicing of Atrophin (CG6964, also called Grunge and Gug) is regulated by a conserved secondary structure element. (A) Schematic representation of the Atrophin mini-gene, which encompasses exons 8–11 (chromosome 3L:8462561–8463475). A non-annotated proximal acceptor site was determined to be located in the box 2 sequence. The multiple sequence alignment of the intronic regions containing the boxes is shown in the bottom panel. The complementary boxes and the sequence downstream of box 2 are 100% conserved. Splicing to the proximal acceptor site is predicted to add 66 nucleotides to the exon, and the predicted amino acid insertion is shown below the sequence. The legend for (B) and (C) is the same as in Figure 1, except that P is the proximal acceptor site, and D, the distal acceptor site.
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Figure 3: Alternative splicing of Atrophin (CG6964, also called Grunge and Gug) is regulated by a conserved secondary structure element. (A) Schematic representation of the Atrophin mini-gene, which encompasses exons 8–11 (chromosome 3L:8462561–8463475). A non-annotated proximal acceptor site was determined to be located in the box 2 sequence. The multiple sequence alignment of the intronic regions containing the boxes is shown in the bottom panel. The complementary boxes and the sequence downstream of box 2 are 100% conserved. Splicing to the proximal acceptor site is predicted to add 66 nucleotides to the exon, and the predicted amino acid insertion is shown below the sequence. The legend for (B) and (C) is the same as in Figure 1, except that P is the proximal acceptor site, and D, the distal acceptor site.

Mentions: Atrophin encodes a transcriptional co-repressor with histone deacetylase activity (27). Splicing analysis of the mini-gene products revealed the presence of an unannotated acceptor site within box 2, proximal to the annotated one (Figure 3A). RT–PCR analysis of the endogenous mRNA revealed that this proximal acceptor site is indeed used (Figure 3B), and correspondingly, the novel exonic region is completely conserved phylogenetically (Figure 3A). While the proximal acceptor is predicted to be stronger than the distal one (P = 0.09), both acceptor sites are used, with an approximate ratio of 1:1 for the mini-gene and endogenous mRNAs (Figure 3B). However, mutation of either box within the mini-gene construct resulted in splicing mainly to the proximal acceptor site located in box 2 (Figure 3). Re-establishing a stem with a novel sequence, and with a similar stability as the wild-type stem (Figure 3C), led to a switch in the splicing pattern to approximately that of the wild-type (Figure 3B). Thus, the stem structure suppresses the proximal acceptor site, thereby equalizing two splice sites of distinct strengths by incorporating the stronger site into a stem structure.


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

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

Alternative splicing of Atrophin (CG6964, also called Grunge and Gug) is regulated by a conserved secondary structure element. (A) Schematic representation of the Atrophin mini-gene, which encompasses exons 8–11 (chromosome 3L:8462561–8463475). A non-annotated proximal acceptor site was determined to be located in the box 2 sequence. The multiple sequence alignment of the intronic regions containing the boxes is shown in the bottom panel. The complementary boxes and the sequence downstream of box 2 are 100% conserved. Splicing to the proximal acceptor site is predicted to add 66 nucleotides to the exon, and the predicted amino acid insertion is shown below the sequence. The legend for (B) and (C) is the same as in Figure 1, except that P is the proximal acceptor site, and D, the distal acceptor site.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724269&req=5

Figure 3: Alternative splicing of Atrophin (CG6964, also called Grunge and Gug) is regulated by a conserved secondary structure element. (A) Schematic representation of the Atrophin mini-gene, which encompasses exons 8–11 (chromosome 3L:8462561–8463475). A non-annotated proximal acceptor site was determined to be located in the box 2 sequence. The multiple sequence alignment of the intronic regions containing the boxes is shown in the bottom panel. The complementary boxes and the sequence downstream of box 2 are 100% conserved. Splicing to the proximal acceptor site is predicted to add 66 nucleotides to the exon, and the predicted amino acid insertion is shown below the sequence. The legend for (B) and (C) is the same as in Figure 1, except that P is the proximal acceptor site, and D, the distal acceptor site.
Mentions: Atrophin encodes a transcriptional co-repressor with histone deacetylase activity (27). Splicing analysis of the mini-gene products revealed the presence of an unannotated acceptor site within box 2, proximal to the annotated one (Figure 3A). RT–PCR analysis of the endogenous mRNA revealed that this proximal acceptor site is indeed used (Figure 3B), and correspondingly, the novel exonic region is completely conserved phylogenetically (Figure 3A). While the proximal acceptor is predicted to be stronger than the distal one (P = 0.09), both acceptor sites are used, with an approximate ratio of 1:1 for the mini-gene and endogenous mRNAs (Figure 3B). However, mutation of either box within the mini-gene construct resulted in splicing mainly to the proximal acceptor site located in box 2 (Figure 3). Re-establishing a stem with a novel sequence, and with a similar stability as the wild-type stem (Figure 3C), led to a switch in the splicing pattern to approximately that of the wild-type (Figure 3B). Thus, the stem structure suppresses the proximal acceptor site, thereby equalizing two splice sites of distinct strengths by incorporating the stronger site into a stem structure.

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