Cross-kingdom patterns of alternative splicing and splice recognition

gb-2008-9-3-r50 GBJ Research Cross-kingdom patterns of alternative splicing and splice recognition McGuire M Abigail amcguire@broad.mit.edu Pearson D Matthew mdp@broad.mit.edu Neafsey E Daniel neafsey@broad.mit.edu Galagan E James jgalag@mit.edu

The Broad Institute of MIT and Harvard, Cambridge Center, Cambridge, MA 02142, USA

Genome Biology 1465-6906 2008 9 3 R50 http://genomebiology.com/2008/9/3/R50 18321378 10.1186/gb-2008-9-3-r50 15 10 2007 28 1 2008 5 3 2008 05 03 2008 2008 Manson McGuire et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cross-kingdom alternative splicing

A comprehensive survey of alternate splicing across 42 eukaryotes so as to gain insight into how spliceosomal introns are recognized.

Abstract

Background

Variations in transcript splicing can reveal how eukaryotes recognize intronic splice sites. Retained introns (RIs) commonly appear when the intron definition (ID) mechanism of splice site recognition inconsistently identifies intron-exon boundaries, and cassette exons (CEs) are often caused by variable recognition of splice junctions by the exon definition (ED) mechanism. We have performed a comprehensive survey of alternative splicing across 42 eukaryotes to gain insight into how spliceosomal introns are recognized.

Results

All eukaryotes we studied exhibit RIs, which appear more frequently than previously thought. CEs are also present in all kingdoms and most of the organisms in our analysis. We observe that the ratio of CEs to RIs varies substantially among kingdoms, while the ratio of competing 3' acceptor and competing 5' donor sites remains nearly constant. In addition, we find the ratio of CEs to RIs in each organism correlates with the length of its introns. In all 14 fungi we examined, as well as in most of the 9 protists, RIs far outnumber CEs. This differs from the trend seen in 13 multicellular animals, where CEs occur much more frequently than RIs. The six plants we analyzed exhibit intermediate proportions of CEs and RIs.

Conclusion

Our results suggest that most extant eukaryotes are capable of recognizing splice sites via both ID and ED, although ED is most common in multicellular animals and ID predominates in fungi and most protists.

Bioinformatics Genome studies

Background

Intron splicing occurs in all domains of life, but the splicing methods employed and the frequencies of splicing vary among organisms. Bacteria and archaea lack the spliceosomal pathway and splice infrequently via self-splicing introns. Among unicellular eukaryotes, there is substantial range in splicing frequency 12. Many early-branching eukaryotes, including the protists Giardia, Cryptosporidia, Trypanosoma, Entamoeba, and Trichomonas, have few or no introns. Only 5% of genes are spliced in Saccharomyces cerevisiae 3, a yeast, while the average number of introns per gene among other fungi is generally low (with a few noteworthy exceptions). Their average intron density ranges from just over one in Schizosaccharomyces pombe to approximately five in Cryptococcus neoformans 4. Protists have similarly low rates of splicing. In contrast, multicellular animals often have large numbers of introns (over seven per gene in vertebrates), while plants have intermediate numbers of introns (approximately four per gene in Oryza sativa and Arabidopsis thaliana).

The number of introns and recognized splice sites may vary between individual mRNA transcripts of a single gene, giving rise to the phenomena of splice variation and alternative splicing. In this paper we use 'splice variation' to describe any difference in intron processing, reserving the term 'alternative splicing' for splice variation that is regulated and functionally significant. Observed splice variation is a combination of programmed alternative splicing events and splicing errors. Functional alternative splicing may result from various causes, including ontogenic changes and environmental stimuli. As the number of genes in an organism is not well correlated with its complexity, alternative splicing may provide an additional layer of regulation that permits greater complexity in higher organisms 5. Multicellular organisms may generate different splice forms of the same gene in different tissues, or even within different cells in the same tissue 56. More recently, it has also been demonstrated that alternative splicing can vary between individuals in a heritable manner 7.

Splice variants can be divided into four broad categories: retained introns (RIs), cassette exons (CEs), competing 5' splice sites, and competing 3' splice sites. CEs are the predominant form of splice variation in multicellular eukaryotes 8910, whereas RIs are more frequent in multicellular plants such as A. thaliana and O. sativa 111213, as well as the fungus Cryptococcus and in yeast 14151617.

The profile of splice variants in a given organism is likely influenced by the mechanisms it uses to identify and process splice sites. In eukaryotes, it has been proposed that the spliceosome recognizes splice sites in pairs, either across the intron (intron definition (ID)) or across the exon (exon definition (ED)) 9. In ID, splice sites on either side of an intron are recognized as a unit, while in ED, splice sites on either side of an exon are recognized as a unit. Experiments in both yeast and Drosophila have shown that when splice sites are presumably recognized by ID, mutating a single splice site disrupts splicing of the intron adjacent to the mutation. This leads to an RI, but has no effect on the splicing of nearby introns 1718 (Figure 1). In contrast, when splice sites are presumably recognized by ED, mutating a single splice site affects not only the splicing of the intron adjacent to the mutation, but also the intron on the other side of the exon adjacent to the mutation. This causes cassette exons to be skipped 1920. Therefore, it is believed that with ID, splicing errors are more likely to result in RIs, while with ED, splicing errors are more likely to result in CEs. ID and ED are not mutually exclusive; in Drosophila melanogaster, ID and ED have been shown to operate within a single mRNA 21.

Figure 1

Effects of splicing errors under the intron definition (ID) and exon definition (ED) models

Effects of splicing errors under the intron definition (ID) and exon definition (ED) models. Arrowheads connected by horizontal bars illustrate the paired recognition of splice sites. (a) When splice sites are recognized in pairs across introns by ID, an error at a single splice site (marked 'x') prevents the removal of an intron, leading to a RI. Under ID, two adjacent splice sites must be mis-spliced, and the splicing machinery must operate over a greater distance, to generate a CE. (b) In the ED model, splice sites are recognized in pairs across exons. An error at a single splice site results in a CE. Obtaining an RI via ED requires coordinated mis-recognition of two adjacent splice sites over a longer distance. Observed RIs can be parsimoniously explained by ID-mediated splicing, while observed CEs likely indicate splicing via ED.

The method used to recognize splice sites has been associated with restrictions on exon and intron length. Recognition of splice sites with ED appears to constrain exon length 2022, while recognition with ID limits intron length 1823. Fox-Walsh et al. 24 suggest that splice site recognition across the intron in D. melanogaster ceases at lengths greater than around 200-250 bp. A review of previous studies suggests that phylogenetic trends in exon and intron length may be correlated with the relative occurrence of RIs and CEs and the use of ID or ED for splice junction recognition 1618202324. However, previous results have been limited in their phylogenetic scope.

In this paper, we report a comprehensive survey of splice variants in 42 eukaryotic organisms. Our survey covers a wide phylogenetic range, including 13 multicellular animals, 6 plants, 14 fungi, and 9 protists. We observe variation across major phylogenetic groups in the representation of RIs and CEs among splice variants that is consistent with variation in the mode of splice site recognition (ID or ED) used by these groups. We infer that groups with a high ratio of RIs to CEs (fungi and protists) operate predominantly by ID, while groups with a low ratio of RIs to CEs (multicellular animals) operate predominantly by ED. In organisms with evidence of both RIs and CEs (thus, employing both ID and ED), CEs are shorter than constitutive exons (exons that show no evidence of splice variation), and RIs are shorter than constitutive introns, suggesting that splice mechanisms are closely tied to gene structure.

Results

To assess splice variation in eukaryotes, we selected 42 organisms with genome assemblies and large numbers of publicly available expressed sequence tags (ESTs; Table 1), spanning the plants, fungi, protists and multicellular animals. We aligned ESTs to genome assemblies and constructed transcript fragments, examined all loci where the EST data indicated two or more overlapping non-compatible transcripts, and labeled every instance of splice variation. Table 2 shows the numbers of ESTs for each organism, as well as the numbers of transcripts and loci constructed. Table 3 lists the splice variants we found. A complete list of the locations of predicted sites of splice variation, as well as control introns and exons that show no splice variation despite high EST coverage, is available on the Broad Institute's ftp site 25.

Table 1

Genomes and ESTs used in the analysis

Genome name

Kingdom

Reference

ESTs

Danio rerio

Animal

Assembly Zv6, Sanger Institute [66]

Genbank 06/15/07

Takifugu rubripes

Animal

[67]

Genbank 06/22/07

Branchistoma floridae

Animal

JGI [60] (v1.0)

Genbank 06/22/07

Ciona savignyi

Animal

Broad Institute [58] (ci1.0)

Genbank 06/15/07

Ciona intestinalis

Animal

[68]

Genbank 06/15/07

Nematostella vectensis

Animal

[69]

Genbank 06/22/07

Strongylocentrotus purpuratus

Animal

[70]

Genbank 06/27/07

Drosophila melanogaster

Animal

[71]

Genbank 06/15/07

Aedes aegypti

Animal

[72]

Vectorbase [61,73]

Anopheles gambiae.

Animal

[74]

Vectorbase [61,73]

Apis mellifera

Animal

[75]

Genbank 06/18/07

Caenorhabditis elegans

Animal

[76]

Genbank 02/1/07

Schistosoma mansoni

Animal

TIGR/JCVI [59]

Genbank 06/22/07

Rhizopus oryzae

Fungi

Broad Institute [58]

Genbank 06/15/07 + Broad Institute [58]

Cryptococcus neoformans JEC21

Fungi

[15]

Genbank 06/19/07

Ustilago maydis

Fungi

[77]

Genbank 06/15/07

Schizosaccharomyces pombe

Fungi

[78]

Genbank 01/14/08

Saccharomyces cerevisiae

Fungi

[79]

Genbank 01/14/08

Neurospora crassa

Fungi

[80]

Genbank 06/15/07

Magnaporthe grisea 70-15

Fungi

[81]

Genbank 06/15/07

Stagnospora nodorum

Fungi

Broad Institute [58]

Genbank 06/15/07

Aspergillus flavus NRRL3357

Fungi

TIGR/JCVI [59], GenBank AAIH00000000

Genbank 06/15/07

Aspergillus nidulans

Fungi

[82]

Genbank 06/15/07

Histoplasma capsulatum

Fungi

Broad Institute [58]

Genbank 06/15/07

Coccidioides posadasii

Fungi

TIGR/JCVI [59]

Genbank 06/15/07

Coccidioides immitis RS

Fungi

Broad Institute [58]

Genbank 06/15/07

Sclerotinia sclerotiorum 1980

Fungi

Broad Institute [58]

Genbank 06/15/07 + Broad Institute [58]

Dictyostelium discoideum

Protist

[83]

Genbank 06/15/07

Populus trichocarpa

Plant

[84]

Genbank 06/18/07

Arabidopsis thaliana

Plant

[85]

Genbank 06/16/07

Oryza sativa

Plant

[86]

Genbank 06/15/07

Physcomitrella patens

Plant

JGI [60] (v1.1)

Genbank 06/28/07

Chlamydomonas reinhardtii

Plant

JGI [60] (v3.0)

Genbank 06/18/07

Ostreococcus lucimarinus

Plant

[87]

JGI [60]

Phytophthora infestans

Protist

Broad Institute [58] (C Nusbaum, personal communication)

Genbank 06/15/07

Phytophthora sojae

Protist

[88]

Genbank 06/15/07

Plasmodium yoelii

Protist

[89]

Genbank 06/15/07

Plasmodium falciparum 3D7

Protist

[90]

Genbank 06/19/07

Paramecium tetraurelia

Protist

[91]

Genbank 06/15/07

Tetrahymena thermophila

Protist

[92]

Genbank 06/19/07

Entamoeba hystolitica

Protist

[4]

Genbank 06/18/07

Phaeodactylum tricornutum

Protist

JGI [60]

Genbank 06/28/07

Table 2

Numbers of ESTs, transcripts, and loci

Kingdom

Total ESTs*

Spliced. Filtered ESTs^†

Transcripts^‡

Loci^§

D. rerio

Animal

1350,105

556,175

76,066

33,338

T. rubripes

Animal

26,069

14,197

5,890

4,988

B. floridae

Animal

277,538

57,883

15,784

12,054

C. savignyi

Animal

84,302

24,196

7,027

8,382

C. intestinalis

Animal

686,396

334,137

31,794

15,108

N. vectensis

Animal

16,619

6,206

4,609

4,294

S. purpuratus

Animal

141,833

24,770

23,256

20,773

D. melanogaster

Animal

532,557

242,235

25,241

14,965

A. aegypti

Animal

303,409

120,523

15,878

11,389

A. gambiae

Animal

216,617

95,607

13,511

9,835

A. mellifera

Animal

78085

32,860

9,581

7,800

C. elegans

Animal

346,064

219,812

28,438

21,304

S. mansoni

Animal

158,841

55,392

14,494

10,137

R. oryzae

Fungi

25,393

12,238

3,263

3,052

C. neoformans

Fungi

59,041

46,693

8,173

6,361

U. maydis

Fungi

39,308

11,236

1,289

1,109

S. pombe

Fungi

5,574

843

274

269

S. cerevisiae

Fungi

32,653

2,251

259

245

N. crassa

Fungi

28,089

7,577

1,571

1,495

M. grisea

Fungi

53,102

14,563

3,229

2,795

S. nodorum

Fungi

15,973

5,925

1,637

1,537

A. flavus

Fungi

20,371

8,495

3,004

2,772

A. nidulans

Fungi

16,848

5,499

2,240

2,090

H. capsulatum

Fungi

26,389

2,334

850

950

C. posadasii

Fungi

54,217

30,604

6,769

5,296

C. immitis

Fungi

65,754

32,162

6,484

5,133

S. sclerotiorum

Fungi

65,884

30,203

4,314

3,704

D. discoideum

Protist

155,032

46,116

4,687

4,246

P. trichocarpa

Plant

89,943

38,299

11,462

9,377

A. thaliana

Plant

1,276,692

350,380

35,856

23,412

O. sativa

Plant

977,774

374,397

43,265

25,610

P. patens

Plant

194,822

106,309

21,962

15,402

C. reinhardtii

Plant

167,641

72,903

11,353

8,514

O. lucimarinus

Plant

19,200

1,043

328

304

P. infestans

Protist

94,091

17,381

4,762

4,104

P. sojae

Protist

28,357

7,418

2,125

2,012

P. yoelii

Protist

13,925

2,863

1,019

931

P. falciparum

Protist

21,349

3,928

1,417

1,219

P. tetraurelia

Protist

86,070

44,772

11,423

10,258

T. thermophila

Protist

56,543

21,073

6,035

5,540

E. histolytica

Protist

20,404

599

174

166

P. tricornutum

Protist

89,139

14,576

3,325

2,937

*Number of raw ESTs before filtering. ^†Number of ESTs aligned after applying our set of filters, containing at least one splice site (see Materials and methods). ^‡Number of 'transcripts' constructed from the ESTs. ^§Number of 'loci' (overlapping clusters of transcripts) (genes with 1+ splice site).

Table 3

Types of splice variants observed

Retained introns

Kingdom

No. of splice variants

Cassette exons

Discarding unspliced ESTs

Keeping unspliced ESTs*

Competing 5' sites

Competing 3' sites

CERI+CE MathType@MTEF@5@5@+=feaafiart1ev1aqatCvAUfeBSjuyZL2yd9gzLbvyNv2Caerbhv2BYDwAHbqedmvETj2BSbqee0evGueE0jxyaibaiKI8=vI8viVeY=Nipec8Eeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaqcfa4aaSaaaeaacaWGdbGaamyraaqaaiaadkfacaWGjbGaey4kaSIaam4qaiaadweaaaaaaa@35ED@ (CE fraction)

D. rerio

Animal

6,137

2,475

1,176

2,324

931

1,555

0.68

T. rubripes

Animal

201

0.53

B. floridae

Animal

812

516

123

121

149

0.95

C. savignyi

Animal

181

0.84

C. intestinalis

Animal

2,779

834

907

2,084

394

644

0.48

N. vectensis

Animal

108

0.87

S. purpuratus

Animal

284

147

0.92

D. melanogaster

Animal

2,163

453

712

2,343

442

556

0.39

A. aegypti

Animal

983

280

284

606

186

233

0.50

A. gambiae

Animal

882

207

283

635

190

202

0.42

A. mellifera

Animal

561

153

124

338

135

149

0.55

C. elegans

Animal

1,589

361

452

828

285

491

0.44

S. mansoni

Animal

1,236

235

599

974

143

259

0.28

R. oryzae

Fungi

C. neoformans

Fungi

1,091

768

1,117

101

204

0.02

U. maydis

Fungi

105

0.21

S. pombe

Fungi

S. cerevisiae

Fungi

N. crassa

Fungi

0.20

M. grisea

Fungi

151

194

0.05

S. nodorum

Fungi

0.09

A. flavus

Fungi

162

124

302

0.02

A. nidulans

Fungi

100

216

0.01

H. capsulatum

Fungi

0.03

C. posadasii

Fungi

950

648

1,259

120

167

0.02

C. immitis

Fungi

861

542

1,035

131

180

0.01

S. sclerotiorum

Fungi

323

210

419

0.04

D. discoideum

Protist

107

0.15

P. trichocarpa

Plant

664

144

215

392

103

202

0.40

A. thaliana

Plant

3,255

251

1,260

2,609

547

1,197

0.17

O. sativa

Plant

3,893

450

1,339

3,076

706

1,398

0.25

P. patens

Plant

2,068

249

534

1,156

558

727

0.32

C. reinhardtii

Plant

490

211

520

133

0.22

O. lucimarinus

Plant

P. infestans

Protist

406

262

564

0.02

P. sojae

Protist

0.03

P. yoelii

Protist

0.04

P. falciparum

Protist

0.21

P. tetraurelia

Protist

535

407

786

T. thermophila

Protist

282

202

423

0.03

E. histolytica

Protist

P. tricornutum

Protist

215

126

643

*For comparison, we include the total number of RIs predicted when unspliced ESTs are not discarded. Discarding unspliced ESTs primarily affects the number of predicted RIs. Complete results for all forms of alternative splicing, when unspliced ESTs are not discarded, are included in Additional data file 5.

All eukaryotes exhibit splice variation

We found that splice variation is present in all organisms we analyzed. Every eukaryote we studied exhibited RIs, and almost every organism exhibited competing 5' splice sites, competing 3' splice sites, and CEs. Several organisms showed zero or very few CEs or competing splice sites due to having only a small EST library or a small overall number of predicted splice variants (for example, Histoplasma capsulatum, Rhizopus oryzae, Entamoeba histolytica), or a small number of introns (for example, S. cerevisiae). We also found no CEs in Paramecium tetraurelia, despite a large EST library and numerous predicted splice variants. However, P. tetraurelia is unusual in that it has extraordinarily short introns (25 bp on average). As CEs are usually associated with longer introns and shorter exons, it is possible that this organism's gene structure renders CEs impossible.

Figure 2 illustrates the relative proportions of the four different kinds of splice variants in each organism, along with previously published data for human for comparison 8. Our results for Caenorhabditis elegans, D. melanogaster, A. thaliana, and O. sativa confirmed those of previous studies 101213.

Figure 2

Frequencies of different forms of splice variation, arranged by phylogenetic group

Frequencies of different forms of splice variation, arranged by phylogenetic group. The two bar charts show the relative frequencies of each type of splice variation. The ratio of CEs to RIs is shown in the chart on the left, while the one on the right displays competing 5' and competing 3' splice sites. Note that the CE/RI ratio shows wide variation among kingdoms while the ratio of competing 5' to competing 3' splice sites is remarkably consistent. A high-level overview of the phylogenetic tree is shown on the far left, and the organisms' names are colored according to their phylogenetic grouping. To see all four forms of splice variation on a single bar plot, see Additional data file 1. The data for H. sapiens was taken from a previous study [8].

The ratio of competing 3' splice sites to competing 5' splice sites was fairly constant, with more competing 3' splice sites than competing 5' splice sites in almost every case (Table 3). This is consistent with results of previous studies of splice variation, including the nine organisms in the Altextron database 10 and several other organisms 8912. When we combine the data from all the organisms in our analysis, there are 1.7 times more competing 3' splice sites than competing 5' splice sites. Interestingly, Zavolan et al. 26 found that competing 3' splice sites are more likely to preserve the reading frame than competing 5' splice sites.

In contrast to the uniform ratio of competing splice sites, the ratio of CEs to RIs varies widely between organisms. We found the ratio CERI+CE MathType@MTEF@5@5@+=feaafiart1ev1aqatCvAUfeBSjuyZL2yd9gzLbvyNv2Caerbhv2BYDwAHbqedmvETj2BSbqee0evGueE0jxyaibaiKI8=vI8viVeY=Nipec8Eeeu0xXdbba9frFj0xb9qqpG0dXdb9aspeI8k8fiI+fsY=rqGqVepae9pg0db9vqaiVgFr0xfr=xfr=xc9adbaqaaeGaciGaaiaabeqaaeqabiWaaaGcbaqcfa4aaSaaaeaacaWGdbGaamyraaqaaiaadkfacaWGjbGaey4kaSIaam4qaiaadweaaaaaaa@35ED@ (which we will refer to as the CE fraction) to be a useful metric for summarizing the pattern of these splice variants. The CE fraction is listed in Table 3 and illustrated in Figure 2 for each organism.

CE and RI prevalence vary by kingdom and by intron length

Major eukaryotic groups (animals, plants, fungi, and protists) exhibit very divergent CE fractions (Figure 2). We found that RIs are the dominant form of splice variation in fungi and most protists, while CEs are the dominant form in multicellular animals. Plants have intermediate proportions of CEs and RIs. The difference in the proportions of RIs and CEs between the group of animals and the group consisting of all fungi and protists is highly statistically significant (p < 1e-10 by Fisher's exact test).

The 13 multicellular animals in our analysis have 1.3 times more CEs than RIs, which corresponds to an overall average CE fraction of 55%. CE fractions for these organisms range from 28% for the flatworm Schistosoma mansoni to 95% for the chordate Branchiostoma floridae. The four insects we studied have an average CE fraction of 44%. Moreover, variation in the CE fraction within chordates appears to be associated with genome size, serving as a partial control for phylogenetic effects. Takifugu rubripes has a more compact genome than the other two chordates in our analysis (Danio rerio and B. floridae), and has a correspondingly lower CE fraction (53%) than they do (D. rerio has a CE fraction of 68%; B. floridae, 95%).

In contrast, among the unicellular fungi and protists, we see very few CEs and an overwhelming preference for RIs. Overall, fungi and protists have 37 times more RIs than CEs (an average CE fraction of 3%). This preference for intron retention is consistent with previous reports on baker's yeast and fission yeast 1727, although our kingdom-wide sampling indicates RI predominance is not limited to the highly derived yeasts. RIs also dominate in C. neoformans 15, a member of a group of intron-rich fungi, indicating that RI dominance in fungi is not coupled with intron density.

Plants in turn appear intermediate between animals and fungi in their relative amounts of CEs and RIs. We examined four multicellular plants: A. thaliana, O. sativa (rice), Populus trichocarpa (cottonwood), and Physcomitrella patens (a moss), as well as two unicellular green algae (Chlamydomonas. reinhardtii and Ostreococcus lucimarinus). Overall, we found 3.1 times more RIs than CEs, with an average CE fraction of 24%, consistent with previous studies in A. thaliana and O. sativa 111213. The unicellular algae C. reinhardtii has a CE fraction of 22%, which is closer to the values seen in multicellular plants than other unicellular organisms. However, it has a large genome size for a unicellular organism (118 Mb). In contrast, O. lucimarinus is a much simpler unicellular green algae with smaller genome size (13 Mb), minimal cellular organization and no CEs, a genome structure that is more like those of unicellular fungi and protists.

The observed variation in CE fraction closely parallels variation in intron length. Animals and plants have more long introns (introns greater than 200 bp) than do protists and fungi (Table 4). In Figure 3 we plot the fraction of constitutive introns greater than 200 bp versus the CE fraction, and demonstrate a direct correlation between the presence of long introns and high incidence of CEs (y = 0.84x + 0.00; R²= 0.73). This correlation also holds within each kingdom (fungi, protists, plants, and animals), providing a phylogenetic control. As discussed below, this correlation is consistent with the hypothesis that splice site recognition differs within these groups.

Table 4

Intron and exon lengths for controls and splice variants

Kingdom

Intron density*

Genome assembly size (Mb)^†

Average intron length^‡

Average RI length^§¶

Average intron length next to CE^¥

No. of CEs with unambiguous boundaries^#

% introns >200 bp**

Average exon length^¶††

Average internal exon length^¶‡‡

Average CE length^§§

D. rerio

Animal

7.2

1,547

2,940

130

3,485

903

71%

180

132

113

T. rubripes

Animal

8.1

393

582

126

641

45%

140

119

103

B. floridae

Animal

5.7

923

1,283

115

1,424

135

91%

151

124

111

C. savignyi

Animal

9.1

164

710

595

82%

148

130

123

C. intestinalis

Animal

7.4

117

457

152

544

327

76%

181

143

122

N. vectensis

Animal

4.1

357

823

167

820

79%

165

110

S. purpuratus

Animal

907

1,819

176

2,085

99%

174

141

133

D. melanogaster

Animal

3.9

120

829

1,632

218

25%

279

246

167

A. aegypti

Animal

3.1

1,384

4,614

106