Volume 12 Supplement 9
Evidence for intron length conservation in a set of mammalian genes associated with embryonic development
© Seoighe and Korir; licensee BioMed Central Ltd. 2011
Published: 5 October 2011
We carried out an analysis of intron length conservation across a diverse group of nineteen mammalian species. Motivated by recent research suggesting a role for time delays associated with intron transcription in gene expression oscillations required for early embryonic patterning, we searched for examples of genes that showed the most extreme conservation of total intron content in mammals.
Gene sets annotated as being involved in pattern specification in the early embryo or containing the homeobox DNA-binding domain, were significantly enriched among genes with highly conserved intron content. We used ancestral sequences reconstructed with probabilistic models that account for insertion and deletion mutations to distinguish insertion and deletion events on lineages leading to human and mouse from their last common ancestor. Using a randomization procedure, we show that genes containing the homeobox domain show less change in intron content than expected, given the number of insertion and deletion events within their introns.
Our results suggest selection for gene expression precision or the existence of additional development-associated genes for which transcriptional delay is functionally significant.
One of the salient features of eukaryotic genomes is the pervasive presence of introns, comprising up to 95% of transcribed primary protein-coding sequences in mammals . The functions, origins and evolutionary trajectory of introns have long been of great interest in genomics and genome evolution. Although some theories of the spread of introns postulate that they can accumulate passively as a consequence of insufficient purifying selection to remove them in organisms with relatively low effective population sizes [2, 3], introns have been shown to play a number of functional roles . They give rise to the possibility of alternative splicing, contributing to the diversity of biomolecules, and they can also have a substantial impact on levels of gene expression [5–7]. Introns contain most of the sequence features required for splicing (5’ and 3’ splice sites, branch point sites (BPS), poly-pyrimidine tracts and various intronic splicing elements). Many introns also contain functional non-coding RNAs , which can play critical roles in fine-tuning gene expression . Lastly, introns have been proposed to control the timing of gene expression by delaying transcription .
Negative feedback loops with a time delay can result in oscillating patterns of gene expression, which can be exploited by living organisms as biological time-keeping devices. This appears to be particularly important in development . The hairy and enhancer of split 7 (Hes7) gene is involved in the control of somite formation through oscillatory patterns of gene expression . Recently, Takashima et al. investigated in vivo the impact of removing the introns from the mouse Hes7 gene. They found that expression of the mutant Hes7 gene occurred approximately 19 minutes earlier than for the wild-type and that this reduction in gene expression delay resulted in abolition of oscillations and segmentation defects. Given that the delay associated with transcribing introns appears to play a crucial role in the functioning of this gene, the length of the introns may be evolving under purifying selective pressure. Moreover, further examples of genes with highly conserved intron content across species may reveal more genes for which transcriptional delay is an important aspect of gene regulation. We investigated the conservation of the intron content of Hes7 across a diverse set of nineteen mammals and carried out a search for other genes for which total intron length shows evidence of evolutionary conservation. In such cases introns are likely to play an important functional role and, in a subset, time delays associated with transcribing introns may be a significant aspect of gene regulation.
Results and discussion
Conservation of Hes7 intron length Intron content of orthologues of human Hes7 (OrthoDB ID EOG45TDC4). The tarsier orthologue was omitted. This gene had no annotated introns but the annotation appeared to be incomplete.
Little brown bat
Grey short-tailed opossum
To determine whether intron content, and thus potentially transcription time, of Hes7 was exceptionally conserved compared to other genes and to discover additional examples of genes for which there is evidence of intron content conservation we compared intron content (defined as the sum of the lengths of all introns in the canonical transcript) between human gene models from Ensembl and sets of orthologous genes, obtained from OrthoDB . Because the pattern of insertion and deletion may differ between very large and smaller introns we restricted to genes with introns of similar lengths to Hes7 (specifically we considered genes for which the sum of the intron lengths in the canonical transcript was between one and five kilobases). Restricting also to orthologous groups represented in at least half of the species, only 11 other orthologous groups (from a total of 1875 satisfying these criteria) had intron content within 10% of the human gene in as high a proportion of the orthologues as the Hes7 group. Remarkably, of the corresponding 12 human genes (including Hes7), five are annotated with the gene ontology (GO) term GO:0007389 (pattern specification process) from the biological process component of GO, defined as “any developmental process that results in the creation of defined areas or spaces within an organism to which cells respond and eventually are instructed to differentiate”. Using the DAVID functional annotation tool [16, 17], this is the most statistically enriched GO term (p = 9.4 × 10–5) and remains significant following correction for multiplicity of testing using the Benjamini-Hochberg method (FDR = 0.03). Six of the genes were annotated with the Swissprot and Protein Information Resource (SP-PIR) keywords term ‘developmental protein’ (FDR = 0.003).
Gene set enrichment analysis Gene set enrichment analysis of genes in size class 1 showing evidence of intron content conservation in mammals. Top twenty most significantly enriched terms are shown.
2 × 10–10
8 x 10–8
Homeobox, conserved site
9 × 10–10
2 x 10–7
2 × 10–9
4 x 10–7
5 x 10–9
6 x 10–7
Regulation of transcription, DNA-dependent
7 x 10–9
1 x 10–5
1 x 10–8
8 x 10–7
Regulation of RNA metabolic process
1 x 10–8
8 x 10–6
Positive regulation of transcription from RNA polymerase II promoter
1 x 10–8
6 x 10–6
Positive regulation of biosynthetic process
3 x 10–8
1 x 10–5
Positive regulation of cellular biosynthetic process
3 x 10–8
1 x 10–5
Transcription regulator activity
7 x 10–8
2 x 10–5
Positive regulation of gene expression
8 x 10–8
2 x 10–5
Positive regulation of transcription, DNA-dependent
8 x 10–8
2 x 10–5
Positive regulation of RNA metabolic process
8 x 10–8
2 x 10–5
Positive regulation of macromolecule biosynthetic process
9 x 10–8
2 x 10–5
Positive regulation of macromolecule metabolic process
1 x 10–7
2 x 10–5
Positive regulation of nitrogen compound metabolic process
1 x 10–7
2 x 10–5
Sequence-specific DNA binding
1 x 10–7
2 x 10–5
2 x 10–7
8 x 10–6
Positive regulation of transcription
2 x 10–7
3 x 10–5
Intronic insertions and deletions along lineages leading to human and mouse
To investigate evolution of intron length through insertion and deletion in greater detail we considered changes in intron length along the human and mouse lineages following divergence from their last common ancestor. Genomic sequences at ancestral nodes of the mammalian phylogeny were obtained from Ensembl. These sequences were inferred using a probabilistic method that has been shown to have high accuracy for the inference of insertion and deletion events. We mapped insertion and deletion events to introns.
We also identified the complete set of genes in size class one that were annotated with a homeobox domain by Interpro . To investigate whether the change in intron content since the common ancestor of human and mouse was less than expected, given the inferred numbers of indels we used a randomization procedure (described in Data and methods). We applied this procedure to the set of homeobox domain proteins and found that the change in intron content of these genes along both the human and mouse lineages was far less than expected, given the number of indels inferred to have occurred in each lineage and under the null assumption that the ratio of insertions and deletions and size distribution of these events did not differ for these genes compared to the rest of genes in the size class. In the case of human, the mean absolute change in intron content was 450 bp per gene, whereas the median of this value in the randomized data was 846 bp per gene. The randomized values were less than or equal to the observed value in 87 of 10,000 randomizations (one-tailed p = 0.009). In mouse the mean absolute change in intron length was just 9 bp per gene. The median of this quantity across randomizations was 398 bp per gene. The values in the random data were lower than or equal to the observed data in 33 of 10,000 randomizations (one-tailed p = 0.003).
Contribution of conserved sequence elements to intron length conservation
To explain our observations, conservation acting on functional elements would, perhaps, have to be balanced with selection for rapid induction, as rapidly induced genes have been shown to have short introns . If the conservation of intron content is a consequence of balance between conservation of intronic functional elements and selection either for efficiency  or for rapid induction, this balance appears to be particularly significant for development-associated genes. For these genes, expression precision at the critical developmental junctures in which patterns are established in the developing embryo may be particularly crucial. An intriguing role for introns that has recently gained experimental support is in the establishment of oscillating patterns of gene expression through the control of time delays in expression . These authors attributed a 19 minute delay to the presence of introns. However, the introns of Hes7 are relatively short (< 2 kb) and RNA polymerase II processes nucleotides at a rate in the order of 2 kb per minute [22, 23]. This may suggest either the existence of other functional elements within the intron that caused the delay in transcription or specific properties of the intron that result in an exceptionally slow transcription rate.
Some theories of intron evolution have focussed on the energy cost associated with introns. Reduced intron lengths in highly expressed genes were proposed to result from selection for efficiency . In support of this model, greater selective efficiency associated with larger effective population sizes of unicellular versus multicellular organisms, is associated with shorter introns [2, 3]. Alternatively longer introns in highly and ubiquitously expressed genes may be associated with greater regulatory complexity and the preservation of regulatory elements in introns . Selection to reduce energetic costs of transcription and for rapid induction of some genes as well as selection to preserve regulatory elements in highly regulated genes may all be important factors for intron evolution. However, comparison of genes with very conserved intron contents carried out here, suggests that there may be a substantial number of genes for which the size of the primary transcript is an important and conserved feature. These genes are enriched for key developmental processes, particularly the establishment of early embryonic patterns. Since time delays in gene induction have been shown to be important for some such genes, we propose that selection to conserve transcription time is an important factor in the evolution of intron lengths, particularly in development-associated genes. Our analysis involved a combination of a heuristic examination of intron content across a panel of mammalian species and a statistical randomization approach, which does not take into account all of the factors that may affect the fixation probabilities of insertions and deletions in introns. A better understanding of the evolution of intron sizes through insertion and deletion requires the development of evolutionary models of intron length evolution, though this is challenging because of the diversity of insertion and deletion events and the difficulty in modelling the constraints imposed by functional elements within the introns on the occurrence and size distribution of these events. If an appropriate model of neutral evolution by insertion and deletion can be derived, such a model could be used to identify and quantify purifying selection acting on intron lengths and, perhaps, to discover examples of positive selection acting on changes in intron length over a phylogeny or specific branches of a phylogenetic tree.
Data and methods
Complete sets of gene models were downloaded from Ensembl release 62  via BioMart for 19 mammalian species. These were Homo sapiens, Bos taurus, Canis familiaris, Ornithorhynchus anatinus, Equus caballus, Erinaceus europaeus, Gorilla gorilla, Loxodonta africana, Monodelphis domestica, Myotis lucifugus, Microcebus murinus, Mus musculus, Oryctolagus cuniculus, Sus scrofa, Spermophilus tridecemlineatus, Tarsius syrichta, Tursiops truncatus, Vicugna pacos and Felis catus. Species were selected to sample a broad range of mammalian evolutionary history. A phylogenetic tree of these taxa, obtained from the interactive tree of life [25, 26] is provided for illustrative purposes (Fig. 1). For each Ensembl gene in each species we considered all annotated exons and calculated the total intronic content of the gene as the sum of the gaps between non-overlapping successive exons in the canonical transcript associated with the gene. Genes were divided into four classes, depending on the total intron content of the gene (1 – 5 kb, 5 – 20 kb, 20 – 50 kb and 50 – 100 kb). Orthologous groups of mammalian proteins were downloaded from OrthoDB . We extracted the protein identifiers from OrthoDB corresponding to each of the mammalian species included in the study. Where more than one protein from a species was included in the same orthologous group, we selected one paralogue at random. For each orthologous group, with a representative in at least half of the mammalian species considered we determined the number of species in which the intron content, as defined above, was within 10% of the length of the intron content of the human gene. This was done separately for genes in different intron content classes. Functional analysis of genes with conserved intron content was carried out using DAVID [16, 17]. For each size class, the genes for which the intron content showed evidence of conservation was used as the foreground set and the complete set of genes in the size class was used as the background set.
Genomic multiple sequence ancestor alignments, inferred using the Ensembl Enredo-Pecan-Ortheus pipeline , were downloaded from the comparative genomics section of the Ensembl FTP site. Insertions and deletions were inferred for ancestral sequences included in these alignments using a branch transducer method that has been shown to achieve high accuracy . This allowed insertions and deletions to be placed on branches of the mammalian phylogeny. For reconstructed insertion and deletion events we focussed on the branches leading from the common ancestor of the euarchontoglires (which includes rodents and primates) to humans and mouse. All insertion and deletion events occurring within introns (at least 20 bp from exon-intron) boundaries were identified, based on Ensembl gene models. In this case, intronic indels were defined as indels that occur within the boundaries of the gene but not within 20 bp of any annotated exon associated with the gene.
Randomization study of intron length evolution along the human lineage
For each intron size class we used the complete set of insertion and deletion events within the introns to define the null expectation of events in that intron size class. To test for evidence of purifying selection acting on the intron content of a gene or a set of genes we considered all of the insertion and deletion events in the gene(s) under consideration and for each event sampled an insertion or deletion from the total set of events in the introns of genes in that class. Given a set of insertions and deletions randomly sampled in this way we calculated the change in intron content implied by this set of insertions and deletions (sum of insertion lengths minus the sum of the lengths of the deletions), separately along the human and mouse lineages. This was repeated 10,000 times and in each case the implied change in intron content in the randomized data was compared to the change in intron content, given the actual insertions and deletions inferred to have occurred. The number of times the absolute value of the change in intron content in the randomized data was less than or equal to the absolute value of the change in intron content in the observed data was used to calculate a p-value, with a separate p-value calculated for the human and mouse lineages. These p-values indicate the probability of observing as little or less variation in the intron content of a gene or set of genes, given the number of insertion and deletion events that have occurred and under the assumption that these indel events have the same distribution as events in other genes in the same intron size class.
CS is funded by Science Foundation Ireland (grant number 07/SK/M1211b). PK is funded by the National University of Ireland Galway.
This article has been published as part of BMC Bioinformatics Volume 12 Supplement 9, 2011: Proceedings of the Ninth Annual Research in Computational Molecular Biology (RECOMB) Satellite Workshop on Comparative Genomics. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/12?issue=S9.
- Mattick J, Gagen M: The evolution of controlled multitasked gene networks: The role of introns and other noncoding RNAs in the development of complex organisms. Molecular Biology and Evolution 2001, 18(9):1611–1630. 10.1093/oxfordjournals.molbev.a003951PubMedView ArticleGoogle Scholar
- Lynch M: Intron evolution as a population-genetic process. Proceedings of the National Academy of Sciences of the United States of America 2002, 99(9):6118–6123. 10.1073/pnas.092595699PubMedPubMed CentralView ArticleGoogle Scholar
- Lynch M: The origins of eukaryotic gene structure. Molecular Biology and Evolution 2006, 23(2):450–468.PubMedView ArticleGoogle Scholar
- Fedorova L, Fedorov A: Introns in gene evolution. Genetica 2003, 118(2–3):123–131.PubMedView ArticleGoogle Scholar
- Tange T, Nott A, Moore M: The ever-increasing complexities of the exon junction complex. Current Opinion in Cell Biology 2004, 16(3):279–284. 10.1016/j.ceb.2004.03.012PubMedView ArticleGoogle Scholar
- Nott A, Meislin S, Moore M: A quantitative analysis of intron effects on mammalian gene expression. RNA 2003, 9: 607–617. 10.1261/rna.5250403PubMedPubMed CentralView ArticleGoogle Scholar
- Lu S, Cullen B: Analysis of the stimulatory effect of splicing on mRNA production and utilization in mammalian cells. RNA 2003, 9: 618–630. 10.1261/rna.5260303PubMedPubMed CentralView ArticleGoogle Scholar
- Rearick D, Prakash A, McSweeny A, Shepard S, Fedorova L, Fedorov A: Critical association of ncRNA with introns. Nucleic Acids Research 2011, 39(6):2357–2366. 10.1093/nar/gkq1080PubMedPubMed CentralView ArticleGoogle Scholar
- Swinburne I, Silver P: Intron delays and transcriptional timing during development. Developmental cell 2008, 14(3):324–330. 10.1016/j.devcel.2008.02.002PubMedPubMed CentralView ArticleGoogle Scholar
- Aulehla APO: Oscillating signaling pathways during embryonic development. Current Opinion in Cell Biology 2008, 20(6):632–637. 10.1016/j.ceb.2008.09.002PubMedView ArticleGoogle Scholar
- Takashima Y, Ohtsuka T, González A, Miyachi H, Kageyama R: Intronic delay is essential for oscillatory expression in the segmentation clock. Proceedings of the National Academy of Sciences of the United States of America 2011, 108(8):3300–3305. 10.1073/pnas.1014418108PubMedPubMed CentralView ArticleGoogle Scholar
- Bessho Y, Sakata R, Komatsu S, Shiota K, Yamada S, Kageyama R: Dynamic expression and essential functions of Hes7 in somite segmentation. Genes and Development 2001, 15(20):2642–2647. 10.1101/gad.930601PubMedPubMed CentralView ArticleGoogle Scholar
- Oates A, Ho R: HairyE/(spl)-related (Her) genes are central components of the segmentation oscillator and display redundancy with the Delta/Notch signaling pathway in the formation of anterior segmental boundaries in the zebrafish. Development 2002, 129(12):2929–2946.PubMedGoogle Scholar
- Brend T, Holley S: Expression of the oscillating gene her1 is directly regulated by hairy/enhancer of split, T-box, and suppressor of hairless proteins in the zebrafish segmentation clock. Developmental Dynamics 2009, 238(11):2745–2759. 10.1002/dvdy.22100PubMedPubMed CentralView ArticleGoogle Scholar
- Waterhouse R, Zdobnov E, Tegenfeldt F, Li J, Kriventseva E: OrthoDB: The hierarchical catalog of eukaryotic orthologs in 2011. Nucleic Acids Research 2011, 39(SUPPL. 1):D283-D288.PubMedPubMed CentralView ArticleGoogle Scholar
- Huang D, Sherman B, Lempicki R: Bioinformatics enrichment tools: Paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Research 2009, 37: 1–13. 10.1093/nar/gkn923PubMed CentralView ArticleGoogle Scholar
- Huang D, Sherman B, Lempicki R: Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nature Protocols 2009, 4: 44–57.View ArticleGoogle Scholar
- Zdobnov EAR, Apweiler R: InterProScan – An integration platform for the signature-recognition methods in InterPro. Bioinformatics 2001, 17(9):847–848. 10.1093/bioinformatics/17.9.847PubMedView ArticleGoogle Scholar
- Vinogradov A: “Genome design” model: Evidence from conserved intronic sequence in human-mouse comparison. Genome Research 2006, 16(3):347–354. 10.1101/gr.4318206PubMedPubMed CentralView ArticleGoogle Scholar
- Elkon R, Zlotorynski E, Zeller K, Agami R: Major role for mRNA stability in shaping the kinetics of gene induction. BMC Genomics 2010, 11: 259. 10.1186/1471-2164-11-259PubMedPubMed CentralView ArticleGoogle Scholar
- Castillo-Davis C, Mekhedov S, Hartl D, Koonin E, Kondrashov F: Selection for short introns in highly expressed genes. Nature Genetics 2002, 31(4):415–418.PubMedGoogle Scholar
- Larson D, Zenklusen D, Wu B, Chao J, Singer R: Real-time observation of transcription initiation and elongation on an endogenous yeast gene. Science 2011, 332(6028):475–478. 10.1126/science.1202142PubMedPubMed CentralView ArticleGoogle Scholar
- Boireau S, Maiuri P, Basyuk E, De La Mata M, Knezevich A, Pradet-Balade B, Bäcker V, Kornblihtt A, Marcello A, Bertrand E: The transcriptional cycle of HIV-1 in real-time and live cells. Journal of Cell Biology 2007, 179(2):291–304. 10.1083/jcb.200706018PubMedPubMed CentralView ArticleGoogle Scholar
- Flicek P, Amode M, Barrell D, Beal K, Brent S, Chen Y, Clapham P, Coates G, Fairley S, Fitzgerald S, Gordon L, Hendrix M, Hourlier T, Johnson N, Kähäri A, Keefe D, Keenan S, Kinsella R, Kokocinski F, Kulesha E, Larsson P, Longden I, McLaren W, Overduin B, Pritchard B, Riat H, Rios D, Ritchie G, Ruffier M, Schuster M, Sobral D, Spudich G, Tang Y, Trevanion S, Vandrovcova J, Vilella A, White S, Wilder S, Zadissa A, Zamora J, Aken B, Birney E, Cunningham F, Dunham I, Durbin R, Fernández-Suarez X, Herrero J, Hubbard T, Parker A, Proctor G, Vogel J, Searle S: Ensembl 2011. Nucleic Acids Research 2011, 39(SUPPL. 1):D800-D806.PubMedPubMed CentralView ArticleGoogle Scholar
- Letunic I, Bork P: Interactive Tree Of Life (iTOL): An online tool for phylogenetic tree display and annotation. Bioinformatics 2007, 23: 127–128. 10.1093/bioinformatics/btl529PubMedView ArticleGoogle Scholar
- Letunic I, Bork P: Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Research, in press.
- Hubbard T, Aken B, Ayling S, Ballester B, Beal K, Bragin E, Brent S, Chen Y, Clapham P, Clarke L, Coates G, Fairley S, Fitzgerald S, Fernandez-Banet J, Gordon L, Graf S, Haider S, Hammond M, Holland R, Howe K, Jenkinson A, Johnson N, Kahari A, Keefe D, Keenan S, Kinsella R, Kokocinski F, Kulesha E, Lawson D, Longden I, Megy K, Meidl P, Overduin B, Parker A, Pritchard B, Rios D, Schuster M, Slater G, Smedley D, Spooner W, Spudich G, Trevanion S, Vilella A, Vogel J, White S, Wilder S, Zadissa A, Birney E, Cunningham F, Curwen V, Durbin R, Fernandez-Suarez X, Herrero J, Kasprzyk A, Proctor G, Smith J, Searle S, Flicek P: Ensembl 2009. Nucleic Acids Research 2009, 37(SUPPL. 1):D690-D697.PubMedPubMed CentralView ArticleGoogle Scholar
- Paten B, Herrero J, Fitzgerald S, Beal K, Flicek P, Holmes I, Birney E: Genome-wide nucleotide-level mammalian ancestor reconstruction. Genome Research 2008, 18(11):1829–1843. 10.1101/gr.076521.108PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to 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.