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RNA editing and alternative splicing of the insect nAChR subunit alpha6 transcript: evolutionary conservation, divergence and regulation.

Jin Y, Tian N, Cao J, Liang J, Yang Z, Lv J - BMC Evol. Biol. (2007)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biochemistry, College of Life Sciences, Zhejiang University (Zijingang Campus), Hangzhou, Zhejiang, PR of China. jinyf@zju.edu.cn

ABSTRACT

Background: 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).

Results: 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.

Conclusion: 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.

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Comparison of the editing levels among four orders. The editing sites (in red) and their editing levels (A/G signal) of sites 3–6 and 9–13 among the nAChR alpha6 orthologs from D. melanogaster (Dme), B. mori (Bmo), T. castaneum (Tca) and A. mellifera (Ame) are shown. RNA editing of the nAChR subunit alpha6 genes from D. melanogaster have been previously described [16]. Some RNA editing sites were not evidently detected as a mixed sequence signal G and A, but revealed by sequencing of cDNA clones.
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Figure 6: Comparison of the editing levels among four orders. The editing sites (in red) and their editing levels (A/G signal) of sites 3–6 and 9–13 among the nAChR alpha6 orthologs from D. melanogaster (Dme), B. mori (Bmo), T. castaneum (Tca) and A. mellifera (Ame) are shown. RNA editing of the nAChR subunit alpha6 genes from D. melanogaster have been previously described [16]. Some RNA editing sites were not evidently detected as a mixed sequence signal G and A, but revealed by sequencing of cDNA clones.

Mentions: We have subsequently analyzed the nAChR alpha6 genes from T. castaneum and A. mellifera. We demonstrated that editing also occurred in both the alpha6 homolog of T. castaneum and A. mellifera, with a quite different pattern of editing (Figure 5, 6). These sites were originally discovered through sequence analysis of cDNAs that were subsequently compared with genomic DNA. In each case, A was observed in the genomic sequence with G at the corresponding position in numerous cDNAs. A-to-I RNA editing occurred across the species except A. gambiae with its extraordinarily short flanking intronic sequences (Figure 5, 6). There are up to 11 A-to-I RNA editing sites in A. mellifera. During the preparation of this paper, six A-to-I RNA editing sites were found in A. mellifera [24], which were identical with 6 of the 11 RNA editing sites in our experiment. The editing sites 5 and 10 in nAChR alpha6 were conserved among the four orders of insect, represented by D. melanogaster, B. mori, T. castaneum and A. mellifera (Figure 5). The RT-PCR clone data revealed that editing was detectable at site 1 in B. mori and A. mellifera, as in Drosophila, albeit at a very low level. Site 4 was edited at low levels in D. melanogaster, but almost completely edited in B. mori, and undetectable in T. castaneum and A. mellifera. Interestingly, site 3 was specifically edited in A. mellifera but it constituted a genomically encoded G in other species. Sites 7 and 8 were markedly edited in A. mellifera, while editing was undetectable in other species. In contrast, site 6 was edited in D. melanogaster, B. mori and T. castaneum, but undetectable in A. mellifera (Figure 5). Overall, editing sites and levels of the alpha6 homolog differed among species, possessing conserved and species-specific editing sites in each species. However, this was the most highly conserved RNA editing event yet reported in invertebrates.


RNA editing and alternative splicing of the insect nAChR subunit alpha6 transcript: evolutionary conservation, divergence and regulation.

Jin Y, Tian N, Cao J, Liang J, Yang Z, Lv J - BMC Evol. Biol. (2007)

Comparison of the editing levels among four orders. The editing sites (in red) and their editing levels (A/G signal) of sites 3–6 and 9–13 among the nAChR alpha6 orthologs from D. melanogaster (Dme), B. mori (Bmo), T. castaneum (Tca) and A. mellifera (Ame) are shown. RNA editing of the nAChR subunit alpha6 genes from D. melanogaster have been previously described [16]. Some RNA editing sites were not evidently detected as a mixed sequence signal G and A, but revealed by sequencing of cDNA clones.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919356&req=5

Figure 6: Comparison of the editing levels among four orders. The editing sites (in red) and their editing levels (A/G signal) of sites 3–6 and 9–13 among the nAChR alpha6 orthologs from D. melanogaster (Dme), B. mori (Bmo), T. castaneum (Tca) and A. mellifera (Ame) are shown. RNA editing of the nAChR subunit alpha6 genes from D. melanogaster have been previously described [16]. Some RNA editing sites were not evidently detected as a mixed sequence signal G and A, but revealed by sequencing of cDNA clones.
Mentions: We have subsequently analyzed the nAChR alpha6 genes from T. castaneum and A. mellifera. We demonstrated that editing also occurred in both the alpha6 homolog of T. castaneum and A. mellifera, with a quite different pattern of editing (Figure 5, 6). These sites were originally discovered through sequence analysis of cDNAs that were subsequently compared with genomic DNA. In each case, A was observed in the genomic sequence with G at the corresponding position in numerous cDNAs. A-to-I RNA editing occurred across the species except A. gambiae with its extraordinarily short flanking intronic sequences (Figure 5, 6). There are up to 11 A-to-I RNA editing sites in A. mellifera. During the preparation of this paper, six A-to-I RNA editing sites were found in A. mellifera [24], which were identical with 6 of the 11 RNA editing sites in our experiment. The editing sites 5 and 10 in nAChR alpha6 were conserved among the four orders of insect, represented by D. melanogaster, B. mori, T. castaneum and A. mellifera (Figure 5). The RT-PCR clone data revealed that editing was detectable at site 1 in B. mori and A. mellifera, as in Drosophila, albeit at a very low level. Site 4 was edited at low levels in D. melanogaster, but almost completely edited in B. mori, and undetectable in T. castaneum and A. mellifera. Interestingly, site 3 was specifically edited in A. mellifera but it constituted a genomically encoded G in other species. Sites 7 and 8 were markedly edited in A. mellifera, while editing was undetectable in other species. In contrast, site 6 was edited in D. melanogaster, B. mori and T. castaneum, but undetectable in A. mellifera (Figure 5). Overall, editing sites and levels of the alpha6 homolog differed among species, possessing conserved and species-specific editing sites in each species. However, this was the most highly conserved RNA editing event yet reported in invertebrates.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biochemistry, College of Life Sciences, Zhejiang University (Zijingang Campus), Hangzhou, Zhejiang, PR of China. jinyf@zju.edu.cn

ABSTRACT

Background: 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).

Results: 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.

Conclusion: 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.

Show MeSH