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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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Species-specific alternative splicing patterns. (A) Analysis of species-specific alternative splicing patterns using an RT-PCR-based strategy. 1: D. melanogaster; 2: A. mellifera; 3:B. mori; 4: T. castaneum. Primers used for amplification of splice products were DmDa-5-1 and DmDa-3-1 for D. melanogaster, AmDa-5-3 and AmDa-3-1 for A. mellifera, BmDa-5-4 and BmDa-3-1 for B. mori, TcDa-5-1 and TcDa-3-1 for T. castaneum, respectively (Table 1). The migration positions of PCR products corresponding to transcripts with one or two alternative duplicated exon variants are indicated. Because the sequence between the specific primers in T. castaneum is smaller than in other species, its band is smaller. (B) Comparison of the boundary sequences of sites in exon 2 and alternative exons 3 among nAChR alpha6 orthologs of D. melanogaster (Dme), B. mori (Bmo), T. castaneum (Tca) and A. mellifera (Ame). Direct sequencing of these RT-PCR products (A) confirmed that these duplicated exons are alternatively spliced. Different nucleotides in the alternative exons 3a and 3b (in box) showed a mixed signal.
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Figure 2: Species-specific alternative splicing patterns. (A) Analysis of species-specific alternative splicing patterns using an RT-PCR-based strategy. 1: D. melanogaster; 2: A. mellifera; 3:B. mori; 4: T. castaneum. Primers used for amplification of splice products were DmDa-5-1 and DmDa-3-1 for D. melanogaster, AmDa-5-3 and AmDa-3-1 for A. mellifera, BmDa-5-4 and BmDa-3-1 for B. mori, TcDa-5-1 and TcDa-3-1 for T. castaneum, respectively (Table 1). The migration positions of PCR products corresponding to transcripts with one or two alternative duplicated exon variants are indicated. Because the sequence between the specific primers in T. castaneum is smaller than in other species, its band is smaller. (B) Comparison of the boundary sequences of sites in exon 2 and alternative exons 3 among nAChR alpha6 orthologs of D. melanogaster (Dme), B. mori (Bmo), T. castaneum (Tca) and A. mellifera (Ame). Direct sequencing of these RT-PCR products (A) confirmed that these duplicated exons are alternatively spliced. Different nucleotides in the alternative exons 3a and 3b (in box) showed a mixed signal.

Mentions: We were interested in understanding the alternative splicing of the nAChR alpha6 transcripts and in particular whether this is regulated. We first analyzed how the alternative exons 3 were regulated. The vast majority of tandemly duplicated exons (99.4%) are likely to be involved in mutually exclusive alternative splicing events [14]. The Reverse Transcription Polymerase Chain Reaction (RT-PCR) showed a very clear band in D. melanogaster and T. castaneum adult cDNA, as in A. mellifera with constitutive exon 3 (Figure 2A). Direct sequencing of these amplification products confirmed that these duplicated exons were alternatively spliced (Figure 2B). Sequence analysis of 30 cDNAs also showed that no duplicated exons were spliced together. These results indicated that the vast majority of exons were likely to be involved in mutually exclusive alternative splicing, which was consistent with EST and cDNA data analysis, although a duplicate of exon 3 was even found in cDNA [16].


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)

Species-specific alternative splicing patterns. (A) Analysis of species-specific alternative splicing patterns using an RT-PCR-based strategy. 1: D. melanogaster; 2: A. mellifera; 3:B. mori; 4: T. castaneum. Primers used for amplification of splice products were DmDa-5-1 and DmDa-3-1 for D. melanogaster, AmDa-5-3 and AmDa-3-1 for A. mellifera, BmDa-5-4 and BmDa-3-1 for B. mori, TcDa-5-1 and TcDa-3-1 for T. castaneum, respectively (Table 1). The migration positions of PCR products corresponding to transcripts with one or two alternative duplicated exon variants are indicated. Because the sequence between the specific primers in T. castaneum is smaller than in other species, its band is smaller. (B) Comparison of the boundary sequences of sites in exon 2 and alternative exons 3 among nAChR alpha6 orthologs of D. melanogaster (Dme), B. mori (Bmo), T. castaneum (Tca) and A. mellifera (Ame). Direct sequencing of these RT-PCR products (A) confirmed that these duplicated exons are alternatively spliced. Different nucleotides in the alternative exons 3a and 3b (in box) showed a mixed signal.
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Figure 2: Species-specific alternative splicing patterns. (A) Analysis of species-specific alternative splicing patterns using an RT-PCR-based strategy. 1: D. melanogaster; 2: A. mellifera; 3:B. mori; 4: T. castaneum. Primers used for amplification of splice products were DmDa-5-1 and DmDa-3-1 for D. melanogaster, AmDa-5-3 and AmDa-3-1 for A. mellifera, BmDa-5-4 and BmDa-3-1 for B. mori, TcDa-5-1 and TcDa-3-1 for T. castaneum, respectively (Table 1). The migration positions of PCR products corresponding to transcripts with one or two alternative duplicated exon variants are indicated. Because the sequence between the specific primers in T. castaneum is smaller than in other species, its band is smaller. (B) Comparison of the boundary sequences of sites in exon 2 and alternative exons 3 among nAChR alpha6 orthologs of D. melanogaster (Dme), B. mori (Bmo), T. castaneum (Tca) and A. mellifera (Ame). Direct sequencing of these RT-PCR products (A) confirmed that these duplicated exons are alternatively spliced. Different nucleotides in the alternative exons 3a and 3b (in box) showed a mixed signal.
Mentions: We were interested in understanding the alternative splicing of the nAChR alpha6 transcripts and in particular whether this is regulated. We first analyzed how the alternative exons 3 were regulated. The vast majority of tandemly duplicated exons (99.4%) are likely to be involved in mutually exclusive alternative splicing events [14]. The Reverse Transcription Polymerase Chain Reaction (RT-PCR) showed a very clear band in D. melanogaster and T. castaneum adult cDNA, as in A. mellifera with constitutive exon 3 (Figure 2A). Direct sequencing of these amplification products confirmed that these duplicated exons were alternatively spliced (Figure 2B). Sequence analysis of 30 cDNAs also showed that no duplicated exons were spliced together. These results indicated that the vast majority of exons were likely to be involved in mutually exclusive alternative splicing, which was consistent with EST and cDNA data analysis, although a duplicate of exon 3 was even found in cDNA [16].

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