Exon creation and establishment in human genes

Abstract

Background: A large proportion of species-specific exons are alternatively spliced. In primates, Alu elements play a crucial role in the process of exon creation but many new exons have appeared through other mechanisms. Despite many recent studies, it is still unclear which are the splicing regulatory requirements for de novo exonization and how splicing regulation changes throughout an exon's lifespan.

Results: Using comparative genomics, we have defined sets of exons with different evolutionary ages. Younger exons have weaker splice-sites and lower absolute values for the relative abundance of putative splicing regulators between exonic and adjacent intronic regions, indicating a less consolidated splicing regulation. This relative abundance is shown to increase with exon age, leading to higher exon inclusion. We show that this local difference in the density of regulators might be of biological significance, as it outperforms other measures in real exon versus pseudo-exon classification. We apply this new measure to the specific case of the exonization of anti-sense Alu elements and show that they are characterized by a general lack of exonic splicing silencers.

Conclusions: Our results suggest that specific sequence environments are required for exonization and that these can change with time. We propose a model of exon creation and establishment in human genes, in which splicing decisions depend on the relative local abundance of regulatory motifs. Using this model, we provide further explanation as to why Alu elements serve as a major substrate for exon creation in primates. Finally, we discuss the benefits of integrating such information in gene prediction.

Figure 1

EST inclusion level and symmetry. (a) EST inclusion levels for the three age groups. The x-axis shows the inclusion levels in ranges of 10, and the y-axis shows the proportion of exons from each subset falling within each range. For each exon, the EST inclusion level is defined as N_i/(N_i + N_s) × 100%, where N_i is the number of ESTs including the exon and N_s the number of ESTs skipping the exon. Only exons with N_i + N_s ≥ 10 were considered. On the left of the dashed line we plot the frequencies for exons with zero EST inclusion level. (b) Percentage of symmetric exons (length multiple of three) for each age group. (c) Percentage of symmetric exons by EST inclusion level category for each age group. Only alternative spliced exons with N_i + N_s ≥ 10 were considered.

Figure 2

Intronic densities for the main classes of repetitive elements. (a) Primate specific, (b) mammalian specific and (c) vertebrate and older. At each intronic position, the density was calculated as the proportion of cases in which the base was covered by a given type of repetitive element. We give on the x-axis the relative position from the splice junctions as negative if upstream of the acceptor site or positive if downstream of the donor site.

Figure 3

Performance comparison in real/pseudo-exon discrimination between different measures. ROC curves (vertically averaged) for exonic density, intronic density and ERA, using (a) ESEcomb, (b) SRall and (c) ESScomb as informative features. The average was calculated from 10 different subsets of the data (see text for details). (d) The corresponding AUCs. The error bars represent the standard error. FPR, false positive rate; TPR, true positive rate.

Figure 4

SRE ERA changes with age. Mean exonic relative abundance values for the three age groups (PS, MS and VO) and a set of pseudo-exons not overlapping any repeats (pseudo-INT) calculated for the three motif sets (ESEcomb, ESScomb and SRall). Exons overlapping Alu elements were excluded from the PS set. The standard error is also shown.

Figure 5

SRE functional conservation between human and mouse. SRE FCS between human and mouse of exonic regions covered by ESEcomb, SRall and ESScomb motifs for mammalian specific and vertebrate or older exons. See Materials and methods section for formula.

Figure 6

SRE exonic relative abundance and EST inclusion levels. Cumulative plot of ERA variation (y-axis) for bins of increasing maximum EST inclusion levels (x-axis) for (a) ESEcomb, (b) SRall and (c) ESScomb. The standard errors are also shown.

Figure 7

Alu's unique cis-regulatory context. Exonic and intronic densities of (a) ESEcomb, (b) SRall and (c) ESScomb motifs on primate specific exons overlapping Alu elements (PS-Alu) and on Alu pseudo-exons (pseudo-Alu). (d) Exonic relative abundance of ESEcomb, SRall and ESScomb motifs for primate specific exons overlapping Alu elements (PS-Alu) and for Alu pseudo-exons (pseudo-Alu). (e) Exonic relative abundance for the same sets of motifs in pseudo-exons overlapping other classes of repeats, namely DNA, LTR, LINE and SINE non-Alu (MIR) repeats. The error bars represent the standard error.