RNA editing and alternative splicing of the insect nAChR subunit alpha6 transcript: evolutionary conservation, divergence and regulation
© Jin et al; licensee BioMed Central Ltd. 2007
Received: 08 December 2006
Accepted: 27 June 2007
Published: 27 June 2007
RNA editing and alternative splicing play an important role in expanding protein diversity and this is well illustrated in studies of nicotinic acetylcholine receptors (nAChRs).
Here, we compare the RNA editing and alternative splicing of the nAChR alpha6 subunit genes from different insects spanning ~300 million years of evolution– Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Tribolium castaneum and Apis mellifera. The conserved and species-specific A-to-I RNA editing occurred across all species except A. gambiae, which displayed extraordinarily short flanking intronic sequences. Interestingly, some A-to-I editing sites were a genomically encoded G in other species. A combination of the experimental data and computational analysis of orthologous alpha6 genes from different species indicated that RNA editing and alternative splicing predated at least the radiation of insect orders spanning ~300 million years of evolution; however, they might have been lost in some species during subsequent evolution. The occurrence of alternative splicing was found to be regulated in distinct modes and, in some cases, even correlated with RNA editing.
On the basis of comparative analysis of orthologous nAChR alpha6 genes from different insects spanning ~300 million years of evolution, we have documented the existence, evolutionary conservation and divergence, and also regulation of RNA editing and alternative splicing. Phylogenetic analysis of RNA editing and alternative splicing, which can create a multitude of functionally distinct protein isoforms, might have a crucial role in the evolution of complex organisms beyond nucleotide and protein sequences.
RNA editing is a process that results in the synthesis of proteins that are not directly encoded in the genome. One type of RNA editing involves the modification of individual adenosine bases to inosine in RNA by ADAR enzymes (adenosine deaminases acting on RNA) [1, 2]. Because inosine acts as guanosine during translation, A-to-I conversion in coding sequences leads to amino acid changes and often entails changes in protein function [2–4]. A-to-I RNA editing is common in animals and is associated with various neurological functions [3, 4]. Caenorhabditis elegans, Drosophila melanogaster and Mus musculus mutants lacking ADAR enzymes display predominantly distinct neurological phenotypes [5–8]. In addition to amino acid changes, the editing and subsequent destabilization of the RNA duplex present in the 5' or 3'-untranslated regions (UTRs) could alter the stability, transport or translation of the mRNA [2, 9]. Moreover, RNA editing may influence alternative splicing decisions .
Alternative splicing is a major contributor to transcriptomic and proteomic complexity, disease, and development. Alternative splicing may affect the protein sequence in two ways: (i) by deleting or inserting a sequence and creating long and short isoforms, or (ii) by substituting one segment of the amino acid sequence for another . An indication for the first pathway is that truncated isoforms often act as dominant-negative regulators of the full-length isoform's activities [12, 13]. In contrast, the second mode is capable of creating, from mutually exclusive alternative sequences, a multitude of functionally distinct protein isoforms and thus might have a crucial role in the evolution of complex organisms . As both RNA editing and alternative splicing can lead to the inclusion of alternative amino acid sequences into proteins, functionally distinct isoforms are likely to be generated . Therefore, editing and alternative splicing provide a powerful posttranscriptional means for fine-tuning of gene expression at the cellular and organismal levels.
Nicotinic acetylcholine receptors (nAChRs) mediate the fast actions of the neurotransmitter acetylcholine (ACh) in both vertebrates and invertebrates . An extraordinary feature of the insect nAChR genes is that they can potentially create many different mRNAs by RNA editing and alternative splicing. More than 30,000 alpha6 nAChR isoforms are theoretically possible through RNA editing and alternative splicing, without considering any linkage between these events . The alternatively spliced exons are organized into two clusters. The exon 3 and 8 clusters contain 2 and 3 alternative versions, respectively . Seven adenosines could be modified in D. melanogaster alpha6, four of which are also edited in the alpha6 ortholog in the tobacco budworm Heliothis virescens. However, although these RNA A-to-I editing sites are conserved between D. melanogaster and H. virescens, they are not shared with the equivalent nAChR subunit of Anopheles, which is considered to be an example of convergent evolution . It is possible that different alpha6 isoforms may interact with distinct sets of receptor guidance cues. RNA editing and alternative splicing of the nAChR alpha6 pre-mRNA may therefore be central to the mechanisms specifying transmitter affinity, channel conductance and ion selectivity.
The recently sequenced genomes of 12 Drosophila species , the mosquito A. gambiae , the silkworm B. mori , the honeybee A. mellifera , and T. castaneum  have renewed interest in molecular and functional diversity in the insect nAChR alpha6 gene. Recent analysis reveals bees and wasps (Hymenoptera) are at the base of the radiation of Holometabolous insects [22, 23]. Here, we compare the RNA editing and alternative splicing of the nAChR alpha6 gene from these insects spanning ~300 million years of evolution. These sequence comparisons provide insight into the evolution of the nAChR alpha6 gene and indicate that many isoforms have arisen by RNA editing and alternative splicing events. These findings also suggest that expressing a diverse nAChR alpha6 repertoire is more important than the actual sequence of each isoform. In this article, we describe the A-to-I RNA editing and alternative splicing found in insect nAChR alpha6 genes, as well as their evolutionary conservation and divergence, and regulation. In addition, we provide an example of a strong correlation between RNA editing and alternative splicing.
Comparison of the nAChR subunit alpha6 genes from different insect species
To obtain insight into the functional diversity, the regulation of expression, and the evolution of nAChR alpha6, we have compared the sequence of the nAChR alpha6 genes in Drosophila to other species. The organisms analyzed consisted of 13 Dipteran species, including 12 Drosophila species (D. melanogaster, D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae, D. pseudoobscura, D. persimilis, D. willistoni, D. mojavensis, D. virilis and D. grimshawi) and one mosquito (A. gambiae), the Lepidopteran B. mori (silkworm), the Coleopteran T. castaneum (red flour beetle) and the Hymenopteran A. mellifera (honeybee). The sequences from these species allowed us to analyze the evolution of the nAChR alpha6 gene over at least 300 million years and across phylogenetic orders. The overall organization of the nAChR alpha6 genes of these insect species is quite similar, but there are a few subtle differences. The nAChR alpha6 genes possess two versions for exon 3 in most species, while no such tandem duplication of coding exon 3 can be found in the A. mellifera genome. Although nAChR alpha6 genes have three versions for exon 8 in most species, only two alternatives for the equivalent exon are observed in the A. gambiae and B. mori genomes.
We next analyzed the evolutionary relationship of the alternative exons within the Drosophila species. All three exon 8 variants had orthologs in each species. All three exon 8 orthologs were very highly conserved in these species, and were identical at the amino acid level. Surprisingly, the orthologs to the alternative exon 8b were identical even at the nucleotide level. We determined a similar hierarchy of nucleotide conservation of the alternative exons 8 of nAChR subunit alpha6 genes within the Drosophila species, namely exon 8b > exon 8a > exon 8c.
Conservation and divergence of alternative splicing
To elucidate how the alternative splicing patterns of exon 3 were developmentally regulated, we designed the specific primers based on three alternative variant cDNAs (Figure 3A). Consequently, the expression of splice type I and II appeared to be tightly regulated in a similar manner, exhibiting an abundant level in the embryo, and decreasing slightly during the pupal stage to the adult level. In contrast, the expression of splice type III showed a distinct mode during development, exhibiting a very low level in the embryo, and increasing rapidly during the pupal stage to the adult level (Figure 3B). The expression analysis using RT-PCR was consistent with the results derived from RT-PCR clones (Figure 3C). Thus, the alternative splicing patterns of exon 3 in silkworm were different from the other insect species.
Evolutionary conservation and divergence of nAChR alpha6 RNA editing
Some A-to-I editing sites were a genomically encoded G in other species
The nAChR subunit alpha6 genes were subject to RNA editing in D. melanogaster, B. mori, T. castaneum and A. mellifera. Interestingly, there were several examples in which some A-to-I editing sites were a genomically encoded G in some species. For example, the alpha6 site 13 was edited in B. mori and A. mellifera, while the site 13 in the alpha6 ortholog α7–2 in the tobacco budworm H. virescens (Lepidoptera) was a genomically encoded G (Figure 5A). The site 3 was edited in A. mellifera, while the homologous sites in A. gambiae, H. virescens, B. mori and T. castaneum were a genomically encoded G (Figure 5A). Similarly, the alpha6 site 2 was edited in Drosophila ; however, the alpha6 homologous sites in other species were a genomically encoded G (Figure 5A). Although we did not know how general this phenomenon was, this led us to consider the possibility that RNA editing might act as an evolutionary intermediate form between single nucleotide polymorphism (SNP) sites, maintaining partial conservation at the protein and functional level despite sequence divergence at the DNA level.
Correlation between RNA editing and alternative splicing
The vast majority of tandemly duplicated exons (99.4%) are likely to be involved in mutually exclusive alternative splicing events . The alternative splicing patterns of the duplicated exon 3 were conserved in the chosen insect species, except the silkworm. It remained to be determined whether the alternative splicing pattern of mutually exclusive exons was changed because the signal ensuring the splicing of pairs of alternative exons was disturbed in the silkworm. RT-PCR and direct sequencing of the cDNAs derived from adult transcripts showed that two A-to-G substitutions occurred in exon 4 of the silkworm nAChR subunit alpha6 gene (Figure 3D). However, these two A-to-G substitutions were undetectable in embryonic transcripts. Interestingly, sequence analysis of 80 cDNA clones derived from embryonic, larvae, pupae and adult transcripts, respectively, revealed two A-to-G substitutions in exon 4 of the silkworm in splice type III, but not in splice types I and II. PCR amplification of silkworm genomic DNA and direct sequencing revealed that adenosine was the nucleotide in the genomic DNA at both positions, confirming that A-to-I editing occurred in this case. These results suggest that specific editing in exon 4 and splice type III of exon 3 might be closely related.
To determine whether coordinated RNA editing and alternative splicing were conserved in other insects, we subsequently analyzed the nAChR alpha6 genes from D. melanogaster, T. castaneum and A. mellifera. In contrast to the silkworm, sequence analysis indicated that no A-to-G substitutions took place in exon 4 in these species. These results suggest that RNA-editing sites in exon 4 are species-specific in B. mori, which correlates with the species-specific alternative splicing pattern of the duplicated exon 3. To discern whether alternative splicing also correlates with RNA editing in other distant exons in the silkworm, we analyzed the A-to-I RNA editing sites in exon 5 of the nAChR subunit alpha6 gene. However, no evidence indicated any relation between alternative splicing patterns of exon 3 and editing in distant exon 5, suggesting that the alternative splicing patterns of exon 3 was not regulated by RNA editing in distant exon 5.
RNA editing conserved between the orders Diptera and Lepidoptera for one nAChR gene was previously considered an example of convergent evolution . However, our phylogenetic analysis of RNA editing in orthologous nAChR alpha6 genes from different species revealed a divergent evolution from a common ancestor. Moreover, this implies that divergent evolution from a common ancestor would have been accompanied by editing loss or gain in paralogous genes. We suggest that the data presented here comprise a credible phylogeny of RNA editing for a gene, graphically illustrating descent with modification (Figure 7A). RNA editing in insect nAChR subunit alpha6 genes predates at least the radiation of the Coleopteranand Hymenopteran orders, beginning with sites 5 and 10. New editing sites were probably generated and ancestral editing sites were lost in subsequent evolution through global intronic variation (Figure 7A). Our evidence suggests that Anopheles lost editing in the nAChR alpha6 gene during the evolution of the Diptera; such a loss might be consistent with the phylogenic evolution of the introns. The nAChR alpha6 genes possess tandem duplication of coding exons in their genomic sequences in insect species, which represents alternative spliced exons. Dating exon duplications through a combination of the available experimental data on alternative splicing in orthologous genes from different species and computational analysis indicated that the exon 3 and 8 duplications predated at least the radiation of insect orders spanning ~300 million years of evolution. Our results disproved the previous hypothesis that a duplication event gave rise to exon 8b and 8c before the divergence of an ancestor of Drosophila and Anopheles, whereas after the divergence a further duplication gave rise to an extra exon (exon 8a) in the Drosophila lineage . However, our evidence suggests the possibility that divergent evolution from a common ancestor was accompanied by exon loss and generation of paralogous genes (Figure 7B). In the nAChR alpha6 gene, our evidence suggests that recent loss of copies of duplicated exons has occurred (e.g. exon 8a in A. gambiae). Moreover, divergent evolution from a common ancestor would have been accompanied by change in the alternative splicing pattern of mutually exclusive exons in paralogous genes (e.g. exon 3 in B. mori)
RNA editing and SNP sites
The nAChR subunits alpha6 are subject to RNA editing in insect species. Interestingly, there were several examples where some A-to-I editing sites were a genomically encoded G in closely- related species. Although we do not know how general this interesting phenomenon is, this led us to consider the possibility that mRNA editing might act to maintain similarity at the protein and functional level despite sequence divergence at the DNA level. In plant mitochondria, mRNA editing might act to maintain similarity at the protein level . Some genetic restoration events in plants and animals are proposed to be the result of a template-directed process that makes use of an ancestral RNA-sequence cache [27, 28]. Therefore, the edited RNA-sequence might be taken as a template to synthesize DNA, thus causing changes at the DNA level. In addition, the most prevalent changes of substitutional RNA editing in the nucleus of higher eukaryotes are hydrolytic deaminations where a genomically encoded C or A is converted to U and I, respectively  Interesting, A/G and C/T(U) substitution were much more prevalent than other forms of SNP. Given these relations between RNA editing and SNP, it is pertinent to ask whether RNA editing might be an evolutionary predecessor to genomically fixed SNPs.
Regulation of mutually exclusive alternative splicing
RNA editing and alternative splicing play an important role in expanding protein diversity and are commonly employed to enlarge the proteome. Since both processes may require conserved exonic and intronic elements, RNA editing may influence alternative splicing decisions or vice versa. There are a few examples of an association between alternative splicing and editing [30–34]. As RNA editing usually occurs close to exon/intron boundaries, this is likely to be a general phenomenon and suggests an important and novel role for RNA editing . Moreover, the ADAR2 protein regulates its own synthesis by creating an alternative splice site that leads to an out-of-frame product [30, 34]. The auto-editing of ADAR2 intron 4 by the ADAR2 adenosine deaminase is tightly coupled to splicing, as the modification of the dinucleotide AA to AI creates a new 3' splice site . The editing site and the affected splice site are usually in close proximity to one another, and so RNA editing affected alternative splicing by creating or deleting splice sites [30–34]. Only one example indicates that the editing efficiency of a Drosophila gene correlates with a distant splice site selection where alternative splicing occurs downstream of editing. In contrast, no correlation is seen when editing occurs downstream of alternative splicing . However, the result remains to be determined without considering the fact that RNA editing and alternative splicing are not regulated by similar developmental patterns.
In this study, RNA editing could affect the alternative splicing pattern by another mechanism because no new splice sites were generated or deleted. The vast majority of tandemly duplicated exons (99.4%) are likely to be involved in mutually exclusive alternative splicing events , therefore, mechanisms must exist to ensure that the splicing of pairs of alternative exons is strictly mutually exclusive, involved in competing base-pairing interactions , the steric hindrance of snRNP binding  and the dual spliceosome mechanisms . If the signal involved in these mechanisms was disturbed, the alternative splicing pattern of mutually exclusive exons might be changed. Taken together, a model can be proposed to explain how editing and alternative splicing of pre-mRNA is coordinated. RNA editing in exon 4 might disrupt a splicing enhancer signal within exon 4, which can prevent the exon 3 cluster from splicing together. The disrupted splice site is now more efficient at splicing out the shorter intron, leading to the longer product. It is not exactly clear how an enhancer within this exon would alter the choice of a distant acceptor site, but there are previous studies showing that longer introns tend to be flanked by stronger splice sites . To test whether A-to-I editing disrupts exon splicing enhancer (ESE) elements, we analyzed edited and unedited exon sequences with an ESE-finder program . A-to-I editing in two sites was predicted by the ESE-finder to destroy the SF2/ASF (GGAACGA) and SRp40 (CGTCAAG) ESE motifs, respectively. Taken together, our results suggest that ESE disruption is the underlying mechanism of A-to-I editing that results in the change of the alternative splicing pattern. Conversely, if RNA editing does not occur, for example, in the silkworm embryo and in D. melanogaster and T. castaneum, mutually exclusive alternative splicing of the duplicated exons has arisen in the majority of transcripts. Alternatively, A-to-I editing might disrupt the other splice signals within exon 4 in the silkworm nAChR subunit alpha6, which controls mutually exclusive splicing of the duplicated exons. One protein has recently been identified that prevents all of the duplicated exon variants from being spliced together, which demonstrates that the duplicated exon variants are in fact capable of being spliced together but protein factors exist that repress this reaction . A-to-I editing possibly relieves the repression on the upstream alternative duplicated exons, and as a result, the duplicated exon variants might be spliced together.
We have documented the existence, evolutionary conservation, and regulation of RNA editing and alternative splicing in nAChR alpha6 from five insects spanning ~300 million years of evolution– D. melanogaster, A. gambiae, B. mori, T. castaneum and A. mellifera. A combination of the experimental data and computational analysis of orthologous alpha6 genes from different species indicated that RNA editing and alternative splicing predated at least the radiation of insect orders spanning ~300 million years of evolution; however, they might be lost in some species during subsequent evolution. The occurrence of alternative splicing was found to be developmentally regulated, and even correlated with RNA editing in some cases. Interestingly, some A-to-I editing sites represented a genomically encoded G in other species. Phylogenetic analysis of RNA editing and alternative splicing, which are capable of creating the multitude of functionally distinct protein isoforms, might have a crucial role in the evolution of complex organisms beyond nucleotide and protein sequences.
Total RNA was isolated from different developmental stages of D. melanogaster, B. mori (Qingsong X Haoyue), A. mellifera (ligustica Spinola) and T. castaneum (the red flour beetle) using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer's protocol. Genomic DNA was isolated using the Universal Genomic DNA Extraction Kit (TaKaRa). RNA was stored at -80°C and genomic DNA was stored at 4°C. Plasmid DNA was purified using Qiagen plasmid isolation kit.
Gene assemblies and analysis
The sequences of the nAChR subunit alpha6 genes from D. melanogaster and A. gambiae have been previously described [16, 17]. The sequences of the nAChR subunit alpha6 genes for the other Drosophila species(D. simulans, D. sechellia, D. yakuba, D. erecta, D. ananassae, D. pseudoobscura, D. persimilis, D. willistoni, D. mojavensis, D. virilis and D. grimshawi), the silkworm B. mori, the honeybee A. mellifera and the red flour beetle T. castaneum were assembled from individual raw sequence reads available from the NCBI trace archives. Vertebrate alpha6 orthologs in Homo sapiens (human),Danio rerio (zebrafish), Mus musculus (mouse),Gallus gallus (chicken) and Takifugu rubripes (pufferfish) were identified by BLAST searches using the sequence of the most closely related organisms. The intron 7 sequence of the silkworm nAChR subunit alpha6 gene was determined using PCR and sequencing.
Generation of full-length cDNA
Primers used for the RT-PCR and PCR analysis
Analysis of gene expression by RT-PCR
Silkworm total RNA was reverse transcribed using SuperScript II RT and the resulting single-stranded cDNA product was treated with DNase at 37°C for 30 min. PCR amplification was carried out using cDNA from 10 ng of total RNA template in each reaction. The gene-specific primers for PCR were designed according to the nAChR alpha6 genomic sequence. Each splice product was amplified separately from bulk cDNA using a single spliceform-specific primer and a shared primer. Primer1, 2, 3, 4, 5 refer to BmDa-5-4, BmDa-5-8, BmDa-5-9, BmDa-5-10, and BmDa-3-1, respectively (Table 1). Amplification conditions were 35 cycles of 94°C for 30 s, 55-65°C for 30 s and 72°C for 30 s, followed by one cycle of 72°C for 10 min. Silkworm glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) transcripts were amplified as an external control.
Analysis of alternative splicing forms
Total RNA was reverse transcribed using SuperScript II RT and the resulting single-stranded cDNA product was treated with DNase at 37°C for 30 min. The gene specific primers for PCR were designed according to the nAChR alpha6 genomic sequence (Table 1). PCR amplification was carried out using cDNA from 10 ng of total RNA template in each reaction. The products of RT-PCR were purified and cloned into the pGEM-T Easy vector (Promega, USA) and transformed with a JM109 competent cell. Recombinant clones were identified by restriction enzymes and PCR. Sequencing of selected clones was done using automatic DNA sequencer. cDNA sequences were determined by amplifying portions of the gene and directly sequencing the PCR product.
Analysis of RNA editing
Analysis of RNA editing was performed using total RNA as the template for RT-PCR. RT-PCR was performed with the primer pairs mentioned above for nAChR alpha6 genes in different species. RT-PCR amplicons were either directly sequenced after gel purification or subcloned and individual cDNA-bearing plasmid clones subjected to sequencing. Primers for the nAChR subunit alpha6 exon 5 were used to amplify genomic DNA from the same tissues used for RNA isolation. The genomic PCR amplification product was subjected to direct sequencing to demonstrate that genomic products give a pure A signal at editing sites, ruling out a polymorphism. For subcloning, RT-PCR splice products were cloned into the pGEM-T Easy vector. Relative A-to-G abundance was determined by sequencing individual clones with plasmids containing appropriately sized inserts.
nAChR subunit alpha6 sequences
The GenBank accession numbers of the nAChR alpha6 subunit genes are as follows: alpha6 cDNAs of B. mori variants are EF127797, EF127798, EF127799; alpha6 cDNAs of T. castaneum variants are from EF127806 to EF127810; alpha6 cDNAs of A. mellifera variants are from EF127800 to EF127805.
nicotinic acetylcholine receptor
adenosine deaminase acting on RNA
reverse transcription-polymerase chain reaction
expressed sequence tag
single nucleotide polymorphism
editing site complementary sequence
exon splicing enhancer
This work was partly supported by research grants from the National Natural Science Foundation of China (90508007 and 30277056), and 863 Program (2006AA10A119) and the Program for New Century Excellent Talents in University (NCET-04-0531).
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