UC Davis UC Davis Previously Published Works Title Longer first introns are a general property of eukaryotic gene structure. Permalink https://escholarship.org/uc/item/9j42z8fm Journal PloS one, 3(8) ISSN 1932-6203 Authors Bradnam, Keith R Korf, Ian Publication Date 2008-08-29 DOI 10.1371/journal.pone.0003093 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Longer First Introns Are a General Property of Eukaryotic Gene Structure Keith R. Bradnam*, Ian Korf Genome Center, University of California Davis, Davis, California, United States of America Abstract While many properties of eukaryotic gene structure are well characterized, differences in the form and function of introns that occur at different positions within a transcript are less well understood. In particular, the dynamics of intron length variation with respect to intron position has received relatively little attention. This study analyzes all available data on intron lengths in GenBank and finds a significant trend of increased length in first introns throughout a wide range of species. This trend was found to be even stronger when using high-confidence gene annotation data for three model organisms (Arabidopsis thaliana, Caenorhabditis elegans, and Drosophila melanogaster) which show that the first intron in the 59 UTR is - on average - significantly longer than all downstream introns within a gene. A partial explanation for increased first intron length in A. thaliana is suggested by the increased frequency of certain motifs that are present in first introns. The phenomenon of longer first introns can potentially be used to improve gene prediction software and also to detect errors in existing gene annotations. Citation: Bradnam KR, Korf I (2008) Longer First Introns Are a General Property of Eukaryotic Gene Structure. PLoS ONE 3(8): e3093. doi:10.1371/ journal.pone.0003093 Editor: Alan Christoffels, University of Western Cape, South Africa Received June 12, 2008; Accepted August 11, 2008; Published August 29, 2008 Copyright: ß 2008 Bradnam et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work funded by NIH grant Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction that have been shown to be essential for high expression, or required for normal expression, have been found in human [19], The discovery of RNA splicing in the late 1970s paved the way mice [20], A. thaliana [21], rice [22,23] and C. elegans [24]. A for our understanding of the exon-intron structure of eukaryotic sequence motif that might contribute to enhancing expression via genes [1]. Since then, data on intron and exon sequences have IME has been predicted in A. thaliana and in rice [25]. been steadily accumulating in databases such as GenBank [2]. There are other reasons to suggest that early introns might be While there have been several studies that have used data from functionally different from those that occur later on in the gene. multiple species to look at intron position [3–5], or intron length First introns in humans (in comparison to later introns) contain [6,7], details concerning the length of introns at different positions fewer SNPs [26] and transcription factor binding sites [27], and within a gene are less well known. In particular, there is very little have a reduced insertion frequency of SINE elements [28]. information concerning differences between introns that occur in Comparisons between orthologous introns in different vertebrate coding sequences (CDSs) and introns that occur in the species have also shown that the first intron (and the 59 most untranslated regions (UTRs) of a gene. region of the first intron in particular) tends to be the most The few studies that have combined intron data from multiple conserved of all introns [15,29–31]. Comparison of introns from species have found that the first intron is - on average - longer than different Drosophila species have also shown that first introns later introns [8,9], and that introns from the 59 UTR region of a gene contain more conserved elements than later introns [14,32–34]. tend to be longer than introns from within the CDS or 39 UTR In this study, we have looked at the issue of first intron length in [10,11]. Other single-species studies have also confirmed the several different ways. Firstly, and most importantly, we used increased length of first introns in humans [12,13], Drosophila information from GenBank to look at intron length data from a melanogaster [14], mice [15], and Arabidopsis thaliana [16]. All of the much larger number of species than has been previously studied. aforementioned studies have been limited to studying low numbers Secondly, by also studying high-quality gene annotations from of introns, or low numbers of species, or both. Hence the generality three model organisms, it has been possible to systematically of increased first-intron length across all eukaryotes is still unclear. evaluate differences in intron length at different positions within a One reason for distinguishing between introns that occur at gene, ensuring that UTR introns are evaluated separately from different positions in a gene, is that introns from the 59 proximal CDS introns. By finding that a trend for increased length occurs in region of a gene (‘early’ introns) have been shown to have the vast majority of eukaryotic species, we establish that this is a important functional properties, often relating to gene expression. general feature of eukaryotic gene structure. This phenomenon The ability of an intron to enhance gene expression has been exhibits itself most strongly in the first intron of the 59 UTR termed intron-mediated enhancement (IME) [17], though not all (though the first intron of the CDS also tends to be longer than introns produce an IME effect and the sequence of any intron may downstream introns). Additionally, we find that in A. thaliana, this be less important than its position [18]. Examples of early introns increased length can partly be accounted for by the presence of an PLoS ONE | www.plosone.org 1 August 2008 | Volume 3 | Issue 8 | e3093 First Introns Are Longer IME motif that increases in frequency in early introns. Finally, we was only the 1st intron that could be distinguished from other also show that because first introns tend to be longer, this introns (data not shown). Significant differences between 1st and information can be used to detect errors in existing gene annotations. 2nd intron lengths occurred in CDSs that contained as few as 2 introns (e.g. mean lengths of 1st and 2nd introns in Aspergillus niger: Results 99 nt vs 91 nt, P,0.01), or as many as 20 introns (e.g Oryza sativa: 554 nt vs 344 nt, P,0.01). Analysis of intron lengths using GenBank data Using data from GenBank, intron lengths were calculated for all Analysis of introns using high-confidence gene available species with sufficient numbers of full-length CDSs (at annotations least 500). For each species, the average length of all first introns A major limitation to using CDS data from GenBank is that this was compared to the average length of all other introns (‘non-first’ data excludes any introns that might be located in the 59 UTR of introns). For 30 of 36 species with sufficient data, the average the gene. Therefore, many ‘first’ introns in the GenBank data may length of the first intron was seen to be significantly (Z test, P,0.05) longer than the average of the non-first introns (Figure 1). actually be the second or third intron in the gene. Another The most extreme case was observed in D. melanogaster where the problem is that GenBank contains redundancy; identical intron mean length of the first intron is nearly three times as long as the sequences may be present in multiple entries as alternative splice mean length of later introns. Combined data from all species variants and/or as sequences from different strains of the same suggests that the first intron of the CDS tends to be ,40% longer species. To counter these problems, we used intron data from sets than later introns. Significantly longer first introns were found in of high-confidence gene annotations in three model organism species from diverse phylogenetic groups (including vertebrates, species (taking just one isoform per locus). These annotations were nematodes, insects, plants, and fungi), suggesting that this all supported by cDNA evidence and revealed introns in the 59 increased length is typical for all eukaryotic species. UTR of many coding transcripts (the proportion of genes with 59 The trend for longer first introns was examined in more detail UTR introns was 23%, 15%, and 7% for D. melanogaster, A. thaliana, by partitioning the data for ‘non-first’ introns into separate classes, and C. elegans respectively). Analysis of these introns confirmed the i.e. when 1st and 2nd intron lengths are compared in CDSs that prior observation of longer first introns and also revealed that have exactly the same number of introns. In total, 118 while the first intron of the coding region remains significantly comparisons were made for species that have specific numbers longer than downstream coding introns, it is the first intron of the of introns (excluding single intron CDSs). Of these, the majority 59 UTR, if present, that is the longest intron within a transcript (78%) showed a significant difference between the lengths of the (Figure 3). Because most genes do not have 59 UTR introns, we 1st and 2nd intron (Z test: P,0.05, selected examples shown in looked for differences between first introns of the CDS in genes Figure 2).