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Cross-species EST alignments reveal novel and conserved alternative splicing events in legumes.

Wang BB, O'Toole M, Brendel V, Young ND - BMC Plant Biol. (2008)

Bottom Line: Intron retention is the most common form of AS in all four plant species (~50%), with slightly lower frequency in legumes compared to Arabidopsis and rice.The results also indicate that the frequency of AS in plants is comparable to that observed in mammals.Finally, our results highlight the importance of normalizing EST levels when estimating the frequency of alternative splicing.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Pathology, University of Minnesota, St, Paul, MN 55108, USA. wangx741@umn.edu

ABSTRACT

Background: Although originally thought to be less frequent in plants than in animals, alternative splicing (AS) is now known to be widespread in plants. Here we report the characteristics of AS in legumes, one of the largest and most important plant families, based on EST alignments to the genome sequences of Medicago truncatula (Mt) and Lotus japonicus (Lj).

Results: Based on cognate EST alignments alone, the observed frequency of alternatively spliced genes is lower in Mt (approximately 10%, 1,107 genes) and Lj (approximately 3%, 92 genes) than in Arabidopsis and rice (both around 20%). However, AS frequencies are comparable in all four species if EST levels are normalized. Intron retention is the most common form of AS in all four plant species (~50%), with slightly lower frequency in legumes compared to Arabidopsis and rice. This differs notably from vertebrates, where exon skipping is most common. To uncover additional AS events, we aligned ESTs from other legume species against the Mt genome sequence. In this way, 248 additional Mt genes were predicted to be alternatively spliced. We also identified 22 AS events completely conserved in two or more plant species.

Conclusion: This study extends the range of plant taxa shown to have high levels of AS, confirms the importance of intron retention in plants, and demonstrates the utility of using ESTs from related species in order to identify novel and conserved AS events. The results also indicate that the frequency of AS in plants is comparable to that observed in mammals. Finally, our results highlight the importance of normalizing EST levels when estimating the frequency of alternative splicing.

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Completely conserved ExonS event in plant enoyl-CoA hydratase/isomerase genes. A: same-species and cross-species EST alignments in Mt gene locus AC145499_47. Filled boxes and arrows indicate exons, and lines indicate introns. Green open or filled boxes indicate exons skipped or retained in certain ESTs. The top black scale indicates coordinates for the gene locus on BAC (AC145499). The blue bar represents the IMGAG annotated gene model, with the green triangle representing the protein translation start codon and the red triangle representing the stop codon. Red bars represent individual same species EST alignments. Purple bars represent Lj ESTs, dark yellow bars represent soybean ESTs, and gray bars represent ESTs from other legume species. B. Multiple sequence alignments of the mutual exclusive exons. E3 indicates the Exon 3 and E4 indicates the Exon 4. At2E3 refers to the exon in the second copy of At gene (At4g13360). Amino acids encoded by Mt sequences are list at the top of sequence alignment. Degenerate positions (change in nucleotide will not change amino acids) which are conserved in all exons are highlighted in colors. C. EST alignment in the second copy of At gene (At4g13360). Only exon E3 exists in this gene and no ExonS can be detected. D, E. EST alignment in At and Os genes where the ExonS pattern is completely conserved.
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Figure 4: Completely conserved ExonS event in plant enoyl-CoA hydratase/isomerase genes. A: same-species and cross-species EST alignments in Mt gene locus AC145499_47. Filled boxes and arrows indicate exons, and lines indicate introns. Green open or filled boxes indicate exons skipped or retained in certain ESTs. The top black scale indicates coordinates for the gene locus on BAC (AC145499). The blue bar represents the IMGAG annotated gene model, with the green triangle representing the protein translation start codon and the red triangle representing the stop codon. Red bars represent individual same species EST alignments. Purple bars represent Lj ESTs, dark yellow bars represent soybean ESTs, and gray bars represent ESTs from other legume species. B. Multiple sequence alignments of the mutual exclusive exons. E3 indicates the Exon 3 and E4 indicates the Exon 4. At2E3 refers to the exon in the second copy of At gene (At4g13360). Amino acids encoded by Mt sequences are list at the top of sequence alignment. Degenerate positions (change in nucleotide will not change amino acids) which are conserved in all exons are highlighted in colors. C. EST alignment in the second copy of At gene (At4g13360). Only exon E3 exists in this gene and no ExonS can be detected. D, E. EST alignment in At and Os genes where the ExonS pattern is completely conserved.

Mentions: One example of a completely conserved ExonS event occurs in an enoyl-CoA hydratase/isomerase gene (Mt: AC145449_47). As shown in Figure 4A, the IMGAG-annotated gene structure for AC145449_47 contains 11 exons, each with strong EST support. Exon3 (65 nt) and Exon4 (53 nt) are mutually exclusive. In one isoform, Exon3 is retained and Exon4 is skipped (Mt: 7206545, 90656179; Lj: 45578881; Lupine: 27458685). In another isoform, Exon4 is retained with Exon3 skipped (Mt: 7567285, 11904359, 13596489, 33106093; Lj: 7719575). The two mRNA isoforms therefore encode two proteins (418 aa and 414 aa) differing slightly in their predicted Enoyl-CoA hydratase domain (ECH, pfam00378). No isoform contains both exons, while it is possible to skip both (Mt: 83667352). Two genes in At (At4g13360 and At3g24360), one gene in Os (LOC_Os06g39344) and one in Lj (LjTC_2465, AP006370.1: 88858–94512) are the closest homologs to AC145449_47. Exactly the same AS pattern was observed in all the homologous genes except for At4g13360, where the 65-nt exon (Exon3) was retained constitutively and no trace of the 53-nt exon can be found in the corresponding region (Figure 4C–E). Sequence comparison revealed several nucleotide bases in degenerate codons conserved in all four species (Figure 4B). These bases may contribute to the recognition of (or skipping) the exon.


Cross-species EST alignments reveal novel and conserved alternative splicing events in legumes.

Wang BB, O'Toole M, Brendel V, Young ND - BMC Plant Biol. (2008)

Completely conserved ExonS event in plant enoyl-CoA hydratase/isomerase genes. A: same-species and cross-species EST alignments in Mt gene locus AC145499_47. Filled boxes and arrows indicate exons, and lines indicate introns. Green open or filled boxes indicate exons skipped or retained in certain ESTs. The top black scale indicates coordinates for the gene locus on BAC (AC145499). The blue bar represents the IMGAG annotated gene model, with the green triangle representing the protein translation start codon and the red triangle representing the stop codon. Red bars represent individual same species EST alignments. Purple bars represent Lj ESTs, dark yellow bars represent soybean ESTs, and gray bars represent ESTs from other legume species. B. Multiple sequence alignments of the mutual exclusive exons. E3 indicates the Exon 3 and E4 indicates the Exon 4. At2E3 refers to the exon in the second copy of At gene (At4g13360). Amino acids encoded by Mt sequences are list at the top of sequence alignment. Degenerate positions (change in nucleotide will not change amino acids) which are conserved in all exons are highlighted in colors. C. EST alignment in the second copy of At gene (At4g13360). Only exon E3 exists in this gene and no ExonS can be detected. D, E. EST alignment in At and Os genes where the ExonS pattern is completely conserved.
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Related In: Results  -  Collection

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Figure 4: Completely conserved ExonS event in plant enoyl-CoA hydratase/isomerase genes. A: same-species and cross-species EST alignments in Mt gene locus AC145499_47. Filled boxes and arrows indicate exons, and lines indicate introns. Green open or filled boxes indicate exons skipped or retained in certain ESTs. The top black scale indicates coordinates for the gene locus on BAC (AC145499). The blue bar represents the IMGAG annotated gene model, with the green triangle representing the protein translation start codon and the red triangle representing the stop codon. Red bars represent individual same species EST alignments. Purple bars represent Lj ESTs, dark yellow bars represent soybean ESTs, and gray bars represent ESTs from other legume species. B. Multiple sequence alignments of the mutual exclusive exons. E3 indicates the Exon 3 and E4 indicates the Exon 4. At2E3 refers to the exon in the second copy of At gene (At4g13360). Amino acids encoded by Mt sequences are list at the top of sequence alignment. Degenerate positions (change in nucleotide will not change amino acids) which are conserved in all exons are highlighted in colors. C. EST alignment in the second copy of At gene (At4g13360). Only exon E3 exists in this gene and no ExonS can be detected. D, E. EST alignment in At and Os genes where the ExonS pattern is completely conserved.
Mentions: One example of a completely conserved ExonS event occurs in an enoyl-CoA hydratase/isomerase gene (Mt: AC145449_47). As shown in Figure 4A, the IMGAG-annotated gene structure for AC145449_47 contains 11 exons, each with strong EST support. Exon3 (65 nt) and Exon4 (53 nt) are mutually exclusive. In one isoform, Exon3 is retained and Exon4 is skipped (Mt: 7206545, 90656179; Lj: 45578881; Lupine: 27458685). In another isoform, Exon4 is retained with Exon3 skipped (Mt: 7567285, 11904359, 13596489, 33106093; Lj: 7719575). The two mRNA isoforms therefore encode two proteins (418 aa and 414 aa) differing slightly in their predicted Enoyl-CoA hydratase domain (ECH, pfam00378). No isoform contains both exons, while it is possible to skip both (Mt: 83667352). Two genes in At (At4g13360 and At3g24360), one gene in Os (LOC_Os06g39344) and one in Lj (LjTC_2465, AP006370.1: 88858–94512) are the closest homologs to AC145449_47. Exactly the same AS pattern was observed in all the homologous genes except for At4g13360, where the 65-nt exon (Exon3) was retained constitutively and no trace of the 53-nt exon can be found in the corresponding region (Figure 4C–E). Sequence comparison revealed several nucleotide bases in degenerate codons conserved in all four species (Figure 4B). These bases may contribute to the recognition of (or skipping) the exon.

Bottom Line: Intron retention is the most common form of AS in all four plant species (~50%), with slightly lower frequency in legumes compared to Arabidopsis and rice.The results also indicate that the frequency of AS in plants is comparable to that observed in mammals.Finally, our results highlight the importance of normalizing EST levels when estimating the frequency of alternative splicing.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Pathology, University of Minnesota, St, Paul, MN 55108, USA. wangx741@umn.edu

ABSTRACT

Background: Although originally thought to be less frequent in plants than in animals, alternative splicing (AS) is now known to be widespread in plants. Here we report the characteristics of AS in legumes, one of the largest and most important plant families, based on EST alignments to the genome sequences of Medicago truncatula (Mt) and Lotus japonicus (Lj).

Results: Based on cognate EST alignments alone, the observed frequency of alternatively spliced genes is lower in Mt (approximately 10%, 1,107 genes) and Lj (approximately 3%, 92 genes) than in Arabidopsis and rice (both around 20%). However, AS frequencies are comparable in all four species if EST levels are normalized. Intron retention is the most common form of AS in all four plant species (~50%), with slightly lower frequency in legumes compared to Arabidopsis and rice. This differs notably from vertebrates, where exon skipping is most common. To uncover additional AS events, we aligned ESTs from other legume species against the Mt genome sequence. In this way, 248 additional Mt genes were predicted to be alternatively spliced. We also identified 22 AS events completely conserved in two or more plant species.

Conclusion: This study extends the range of plant taxa shown to have high levels of AS, confirms the importance of intron retention in plants, and demonstrates the utility of using ESTs from related species in order to identify novel and conserved AS events. The results also indicate that the frequency of AS in plants is comparable to that observed in mammals. Finally, our results highlight the importance of normalizing EST levels when estimating the frequency of alternative splicing.

Show MeSH