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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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Editing of B. mori nAChR alpha6 splice forms (A) The structure and alternative splicing patterns of the alpha6 gene in the regions surrounding the alternative exons 3. Three main variants, depending on the alternative exon 3 versions: type I (alternative exon 3a), type II (alternative exon 3b), type III (alternative exons 3a and 3b). Boxes represent exons and the line represents introns. The black boxes represent constitutive exons and the open boxes represent exons that are duplicated in tandem. "*" represents the editing sites. The migration positions of PCR products corresponding to transcripts with one or two alternative duplicated exon variants are indicated with arrows. 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). (B) Analysis of alternative splicing in silkworm embryo, larvae, pupae, and adult using RT-PCR. 1, 2, 3, 4 indicated splice forms amplified using forward primer (primer1, or 2, or 3, or 4), and reverse primer (primer5), respectively. (C) Frequency of splice forms in silkworm embryo, larvae, pupae, and adult. RNA was isolated from each developmental stage and used for RT-PCR. The RT-PCR products were cloned and analyzed. 20 cDNA clones were sequenced for every stage. (D) Comparison of the editing levels (A/G signal) of sites in exon 4 among distinct splice forms. cDNA 1, 2, 3, 4 indicated splice forms in Figure 3B.
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Figure 3: Editing of B. mori nAChR alpha6 splice forms (A) The structure and alternative splicing patterns of the alpha6 gene in the regions surrounding the alternative exons 3. Three main variants, depending on the alternative exon 3 versions: type I (alternative exon 3a), type II (alternative exon 3b), type III (alternative exons 3a and 3b). Boxes represent exons and the line represents introns. The black boxes represent constitutive exons and the open boxes represent exons that are duplicated in tandem. "*" represents the editing sites. The migration positions of PCR products corresponding to transcripts with one or two alternative duplicated exon variants are indicated with arrows. 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). (B) Analysis of alternative splicing in silkworm embryo, larvae, pupae, and adult using RT-PCR. 1, 2, 3, 4 indicated splice forms amplified using forward primer (primer1, or 2, or 3, or 4), and reverse primer (primer5), respectively. (C) Frequency of splice forms in silkworm embryo, larvae, pupae, and adult. RNA was isolated from each developmental stage and used for RT-PCR. The RT-PCR products were cloned and analyzed. 20 cDNA clones were sequenced for every stage. (D) Comparison of the editing levels (A/G signal) of sites in exon 4 among distinct splice forms. cDNA 1, 2, 3, 4 indicated splice forms in Figure 3B.

Mentions: The RT-PCR showed a distinct pattern in B. mori (Figure 2A), not seen in D. melanogaster and T. castaneum. Total silkworm RNA harvested from silkworms at various stages of development was used as a template for RT-PCR with primers surrounding the exon 3 (Figure 3A). At the embryo stage, only one band could be amplified, and direct sequencing indicated a mixed sequence signal of two nucleotides, confirming the existence of alternative splicing of exon 3. The existence of alternative splicing in the embryonic transcripts was further confirmed by independent sequencing of cloned cDNAs of RT-PCR products. This band, which resulted from type I and type II alternative splicing, decreased slightly during development, exhibiting a very low level in the pupal and adult stages (Figure 3B). However, another slightly larger band appeared during development, displaying a very low level in the larvae but increasing rapidly during the stages from pupa to adult (Figure 3B). Sequencing of cloned cDNAs of this product indicated the existence of splice type III alternative splicing, which included both alternative exons, and retained the opening frame of the spliced RNA.


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)

Editing of B. mori nAChR alpha6 splice forms (A) The structure and alternative splicing patterns of the alpha6 gene in the regions surrounding the alternative exons 3. Three main variants, depending on the alternative exon 3 versions: type I (alternative exon 3a), type II (alternative exon 3b), type III (alternative exons 3a and 3b). Boxes represent exons and the line represents introns. The black boxes represent constitutive exons and the open boxes represent exons that are duplicated in tandem. "*" represents the editing sites. The migration positions of PCR products corresponding to transcripts with one or two alternative duplicated exon variants are indicated with arrows. 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). (B) Analysis of alternative splicing in silkworm embryo, larvae, pupae, and adult using RT-PCR. 1, 2, 3, 4 indicated splice forms amplified using forward primer (primer1, or 2, or 3, or 4), and reverse primer (primer5), respectively. (C) Frequency of splice forms in silkworm embryo, larvae, pupae, and adult. RNA was isolated from each developmental stage and used for RT-PCR. The RT-PCR products were cloned and analyzed. 20 cDNA clones were sequenced for every stage. (D) Comparison of the editing levels (A/G signal) of sites in exon 4 among distinct splice forms. cDNA 1, 2, 3, 4 indicated splice forms in Figure 3B.
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Figure 3: Editing of B. mori nAChR alpha6 splice forms (A) The structure and alternative splicing patterns of the alpha6 gene in the regions surrounding the alternative exons 3. Three main variants, depending on the alternative exon 3 versions: type I (alternative exon 3a), type II (alternative exon 3b), type III (alternative exons 3a and 3b). Boxes represent exons and the line represents introns. The black boxes represent constitutive exons and the open boxes represent exons that are duplicated in tandem. "*" represents the editing sites. The migration positions of PCR products corresponding to transcripts with one or two alternative duplicated exon variants are indicated with arrows. 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). (B) Analysis of alternative splicing in silkworm embryo, larvae, pupae, and adult using RT-PCR. 1, 2, 3, 4 indicated splice forms amplified using forward primer (primer1, or 2, or 3, or 4), and reverse primer (primer5), respectively. (C) Frequency of splice forms in silkworm embryo, larvae, pupae, and adult. RNA was isolated from each developmental stage and used for RT-PCR. The RT-PCR products were cloned and analyzed. 20 cDNA clones were sequenced for every stage. (D) Comparison of the editing levels (A/G signal) of sites in exon 4 among distinct splice forms. cDNA 1, 2, 3, 4 indicated splice forms in Figure 3B.
Mentions: The RT-PCR showed a distinct pattern in B. mori (Figure 2A), not seen in D. melanogaster and T. castaneum. Total silkworm RNA harvested from silkworms at various stages of development was used as a template for RT-PCR with primers surrounding the exon 3 (Figure 3A). At the embryo stage, only one band could be amplified, and direct sequencing indicated a mixed sequence signal of two nucleotides, confirming the existence of alternative splicing of exon 3. The existence of alternative splicing in the embryonic transcripts was further confirmed by independent sequencing of cloned cDNAs of RT-PCR products. This band, which resulted from type I and type II alternative splicing, decreased slightly during development, exhibiting a very low level in the pupal and adult stages (Figure 3B). However, another slightly larger band appeared during development, displaying a very low level in the larvae but increasing rapidly during the stages from pupa to adult (Figure 3B). Sequencing of cloned cDNAs of this product indicated the existence of splice type III alternative splicing, which included both alternative exons, and retained the opening frame of the spliced RNA.

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