Identification and experimental validation of splicing regulatory elements in Drosophila melanogaster reveals functionally conserved splicing enhancers in metazoans

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
RNA (Impact Factor: 4.94). 08/2011; 17(10):1884-94. DOI: 10.1261/rna.2696311
Source: PubMed


RNA sequence elements involved in the regulation of pre-mRNA splicing have previously been identified in vertebrate genomes by computational methods. Here, we apply such approaches to predict splicing regulatory elements in Drosophila melanogaster and compare them with elements previously found in the human, mouse, and pufferfish genomes. We identified 99 putative exonic splicing enhancers (ESEs) and 231 putative intronic splicing enhancers (ISEs) enriched near weak 5' and 3' splice sites of constitutively spliced introns, distinguishing between those found near short and long introns. We found that a significant proportion (58%) of fly enhancer sequences were previously reported in at least one of the vertebrates. Furthermore, 20% of putative fly ESEs were previously identified as ESEs in human, mouse, and pufferfish; while only two fly ISEs, CTCTCT and TTATAA, were identified as ISEs in all three vertebrate species. Several putative enhancer sequences are similar to characterized binding-site motifs for Drosophila and mammalian splicing regulators. To provide additional evidence for the function of putative ISEs, we separately identified 298 intronic hexamers significantly enriched within sequences phylogenetically conserved among 15 insect species. We found that 73 putative ISEs were among those enriched in conserved regions of the D. melanogaster genome. The functions of nine enhancer sequences were verified in a heterologous splicing reporter, demonstrating that these sequences are sufficient to enhance splicing in vivo. Taken together, these data identify a set of predicted positive-acting splicing regulatory motifs in the Drosophila genome and reveal regulatory sequences that are present in distant metazoan genomes.

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    • "This program is based on an approach for modelling the sequences of short motifs such as those involved in RNA splicing which accounts for non-adjacent as well as adjacent dependencies between positions. Although MaxEntScan scores are derived from human splice sites, this approach was recently used successfully to predict the D. melanogaster splice site strength according with the observation that Drosophila splice-site motifs are highly similar to human, and many spliceosomal components involved in splice-site recognition are highly conserved [51], [52], [53]. Results are reported in Figure S3–B. "
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    ABSTRACT: In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control.
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    • "However, it is obvious that additional cis sequence information is required in defining exons and influencing their inclusion in mRNAs. Such auxiliary sequences, which stimulate splicing are found in both exons (exonic splicing enhancers, ESE), and introns (intronic splicing enhancers, ISE; Aznarez et al., 2008; Lomelin et al., 2010; Brooks et al., 2011). Moreover, sequences that inhibit splicing have also been characterized, as exonic and intronic splicing silencers (ESS and ISS; Yu et al., 2008; Zhang et al., 2008; Wen et al., 2010). "
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