Department of Biology, Indiana University
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Abstract |
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Key Words: genome complexity genome evolution introns mRNA processing mRNA surveillance nonsense-mediated decay null alleles splicing
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Introduction |
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Although it is unlikely that many intron-containing alleles were immediately advantageous at the time of origin, introns are no longer passive players in genome evolution. Instead, what once may have been a simple by-product of small population size provided the raw material for the evolution of novel mechanisms for regulating gene expression and processing gene products. Indeed, because almost all of the major events in the production of mature mRNAs (including transcription initiation, elongation, polyadenylation, termination, 5' capping, and mRNA export and surveillance) are now highly coupled with exon definition and/or intron splicing (Maniatis and Reed 2002), it is likely that few if any of today's eukaryotes can survive without introns. A few examples will suffice to make this point.
First, a direct interaction between various splicing factors and elongation factors promotes transcription elongation (Ares et al. 1999; Fong and Zhou 2001). Second, for some intron-containing genes, splicing appears to be required for efficient mRNA export to the cytoplasm, with a functional coupling of these two processes being mediated by a protein complex deposited on spliced mRNAs (Luo and Reed 1999; Le Hir et al. 2001; Read and Hurt 2002). Among other things, this protects the cell from the accumulation of error-containing transcripts by ensuring that unspliced pre-mRNAs are retained in the nucleus. Third, the definition of the final exon, via the splicing signals at the 3' end of the upstream intron, appears to be essential for efficient polyadenylation of transcripts (Niwa, Rose, and Berget 1990; Niwa, MacDonald, and Berget 1992) and is also involved in transcriptional termination (Dye and Proudfoot 1999; McCracken, Lambermon, and Blencowe 2002). Fourth, the full spectrum of introns contained within a gene may mutually facilitate one anothers' removal from pre-mRNAs, with the coordinated use of splice sites for exon recognition imposing stabilizing selection for an optimal exon size (Robberson, Cote, and Berget 1990; Nesic and Maquat 1994; Berget 1995; Cooke, Hans, and Alwine 1999). Finally, as now described in detail, introns provide an additional benefit to their eukaryotic hosts by providing coordinates for the identification of premature termination codons (PTCs) contained within aberrant mRNAs.
Nonsense-Mediated Decay as a Facilitator of Intron Proliferation
Premature termination codoncontaining mRNAs arise in a variety of ways, including the direct transcription of inherited mutant alleles, transcriptional or splicing errors involving otherwise functional alleles, and (in animals) the stochastic production of somatic recombinants of immune system genes. The transcriptional error rate alone is on the order of 10-5 per nucleotide (Ninio 1991; Shaw, Bonawitz, and Reines 2002), so with 5% of random codons denoting stop and with 103 to 104 coding nucleotides comprising a typical gene, at least 0.05% to 0.5% of primary transcripts can be expected to contain a PTC. To a considerable extent, eukaryotes are protected from the accumulation of such transcripts by nonsense-mediated decay (NMD), a mRNA surveillance mechanism that leads to selective degradation of PTC-containing transcripts. Although much remains to be learned about NMD, substantial insight into the underlying molecular mechanisms has emerged for yeasts, Caenorhabditis elegans, and mammals (for reviews, see Hentze and Kulozik 1999; Gonzalez et al. 2001; Lykke-Andersen 2001; Mango 2001; Maquat and Carmichael 2001; Wilusz et al. 2001). It remains to be determined whether any protists are capable of NMD, and NMD is not known to occur in any prokaryote. However, the apparent presence of NMD in plants (Isshiki et al. 2001) suggests that NMD was probably present prior to the divergence of most of the major eukaryotic lineages.
Discriminating PTCs from correct termination codons is the major challenge for a successful NMD pathway, and fungi and animals accomplish this in quite different ways. In mammals, a series of proteins is deposited approximately 20 nucleotides 5' to every exon-exon junction at the time of splicing. These ornamented junctions then serve as markers for the true termination codon, which generally lies further downstream in the mature mRNA than the final exon-exon junction. If during translation a termination codon is detected 50 or more nucleotides upstream of this final marker, the mRNA is targeted for selective degradation (Nagy and Maquat 1998). Although intron-free mammalian genes generally appear to be NMD insensitive (Maquat and Li 2001; Brocke et al. 2002), "failsafe" sequences embedded within exons can sometimes elicit NMD in cases where there is no intron downstream of a PTC (Cheng et al. 1994; Zhang et al. 1998; Rajavel and Neufeld 2001), a situation that has parallels in yeast. Introns are extremely rare in Saccharomyces cerevisiae, which greatly reduces their utility as substrates for transcript marking. In this species, PTC recognition relies entirely on downstream sequence elements (DSEs) within coding DNA, with stop codons less than 200 bp upstream of a DSE being interpreted as premature (Ruiz-Echevarria, González, and Peltz 1998).
It is an open question whether the mammalian intron-based or the S. cerevisiae exon-based PTC-recognition pathway more closely represents the ancestral mode of NMD. However, several observations suggest that S. cerevisiae is derived with respect to NMD and other aspects of mRNA processing. First, some spliceosomal components common to both animals and the fission yeast Schizosaccharomyces pombe are absent from S. cerevisiae, and in terms of sequence variation, splicing genes in S. pombe tend to be much more similar to those in human than to those in S. cerevisiae (Aravind et al. 2000; Käufer and Potashkin 2000). Second, two proteins deployed in the exon-junction complex (EJC) in animals and also known to be present in S. pombe, Mago and Y14, appear to be absent from S. cerevisiae (Zhao et al. 2000). At the same time, empirical work demonstrates that NMD can operate on intron-free genes in S. pombe (Mendell et al. 2000), and as noted above, the presence of introns is a nonessential element for NMD in a minority of mammalian genes, and this seems also to be true for C. elegans (Pulak and Anderson 1993) and plants (Isshiki et al. 2001). Thus, it remains a formal possibility that two NMD pathways exist within eukaryotic lineages, with mammals coming to rely predominantly on the EJC pathway and S. cerevisiae on the DSE pathway. The mode of PTC detection via the EJC pathway may also vary among taxa. For example, suggestions have been made that NMD in C. elegans requires two marked exon junctions (Mango 2001), and that the first exon junction plays a role in rice (Isshiki et al. 2001), although in neither case have the destabilizing elements yet been identified.
Because NMD frequently relies on exon junctions for orientation in identifying PTCs, and because the phenomenon either predates (or coincides with) the proliferation of introns in plants, fungi, and animals, it is likely that the evolution of NMD played a role in the colonization of introns within eukaryotes. Once a reliable intron-dependent system of NMD was in place, a positive feedback in genomic evolution may have then been initiatedthe types of splicing errors that are unique to intron-containing alleles as well as the excess mutation rate for such alleles would have intensified selection for efficient NMD. This, in turn, would have relaxed the selective constraints against the further accumulation of introns and may very well have encouraged their addition and/or movement to sites that maximize the efficiency of PTC detection. The intimate spatial and temporal associations among introns, splicing, and surveillance appear to provide an optimal setting for the coevolution of transcript processing mechanisms. Thus, it is notable that (1) at least one of the elements of the EJC involved in mRNA export also functions to recruit a key factor involved in NMD (Kim, Kataoka, and Dreyfuss 2001; Le Hir et al. 2001; Lykke-Andersen, Shu, and Steitz 2001), (2) at least one of the splicing proteins also plays a role in NMD (Luo et al. 2001; Strasser and Hurt 2001), and (3) several of the proteins involved in conventional translation termination are also involved in NMD (Wang et al. 2001).
The Initial Fixation of Introns Facilitated by NMD
We first consider the probability that a newly arisen intron will become fixed in a population with an established NMD mechanism that employs exon junctions to identify PTCs. Starting with a base population fixed for an intron-free allele Ao, the new intron-containing allele (Ai) will have initial frequency 1/(2N), where N is the size of the population, assumed to be diploid and randomly mating. Although both types of alleles incur coding-region mutations that produce a PTC at rate µc per gene per generation, for intron-containing alleles, a fraction p of such mutations falls in locations of the gene that are subject to NMD (alleles in the class ai), whereas the remaining fraction (1-p) does not (alleles in the class ao) (fig. 1). Also unique to intron-containing alleles is a pathway to nonfunctionality resulting from mutations occurring in nucleotide sites critical for proper splicing (Lynch 2002). Alleles with defective splice sites in their only intron are not expected to be subject to NMD, so denoting this excess mutation rate as µi, the total rate of mutation from the Ai to the ao allele is µi + (1- p)µc.
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In addition to the molecular-genetic features , p, and µi, the effective population size N is a key determinant of the probability of fixation of a newly arisen intron under this model, as N defines the degree to which allele-frequency changes are determined by random genetic drift. k plays a negligible role in the fixation process, because it only influences the fitness of heterozygotes containing rare nonfunctional mutations incurred within the lineage of intron-containing alleles en route to fixation. Denoting the net selective advantage of an intron-containing allele subject to NMD by s = p
- µi, the fixation probability is closely approximated by the usual diffusion equation (Crow and Kimura 1970),
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The scaled probability of fixation (F = 2NuF), which is a simple function of 4Ns, is approximately equal to 1 when |4Ns| << 1, approximately equal to 4Ns when 4Ns >> 1, and asymptotically approaches zero as 4Ns
-
. If B denotes the rate of birth of new introns (per gene per unit time), then B
F can be interpreted as the rate of fixation of initial introns in previously intron-free genes. These results imply that if |s| is sufficiently small relative to 1/N, the rate of fixation of the first introns in genes will correspond to the neutral expectation B. If, however, |s| is sufficiently large relative to 1/N, intron establishment is expected to be negligible if s is negative (the advantages of NMD being outweighed by the increased mutation rate to nulls for an intron-containing allele) and to increase with increasing population size if s is positive.
Because the location of an intron dictates the selective advantage associated with NMD (through its influence on p), a biased spatial distribution for initially colonizing introns is expected under this model. Consider, for example, the situation in mammals, where NMD for a construct for one particular gene (triose phosphate isomerase) appears to only be effective with PTCs lying within a span of nucleotides 50 to 550 bases upstream of the intron (Zhang et al. 1998). Assuming equal efficiency of NMD throughout the entire 500 bp range, and letting L be the number of nucleotides in the coding region and I be the position of the intron, p = 0 if I 50, p = (I - 50)/L if50 < I < 550, and p = 500/L if I
550. Using these relationships and equation (1), along with the mutation rate µi = 10-6 and the NMD-associated selective advantage
= 10-5, the expected spatial distribution of initial colonizing introns is given in figure 2 (upper panel). For populations with sufficiently small effective sizes, the distribution is nearly uniform over the entire length of the gene, because the magnitude of random genetic drift is large relative to s. However, as N increases, a strong bias develops toward a 3' location. For this example, the distribution is always flat for introns located beyond nucleotide 550, as all of these enjoy the maximum selective advantage associated with NMD.
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Secondary Accumulation of Introns
For a species with an EJC-dependent system of NMD, the establishment of the first intron within a gene will modify the selective environment for subsequent intron colonization events because the first intron already covers a fraction of the total potential for NMD. The average selective advantage of a secondarily arising intron is necessarily less than or equal to that of the first, with the magnitude of reduction depending on the spatial configuration of the two introns (fig. 3). Because this same logic applies to all subsequently arising introns, the rate of colonization of introns is expected to be negatively associated with intron number, and once sufficient coverage for NMD has been acquired, all newly arisen introns will be weakly selected against (as a consequence of the enhanced mutation rate to nonfunctional alleles). Ultimately, this negative density dependence should result in a quasisteady state number of introns per gene, and an overdispersion of intron positions is expected to result from the selective advantage of alleles with introns with minimal overlap in their regions of NMD coverage.
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Even in the absence of detailed knowledge of the spatial requirements of NMD, these analyses lead to three qualitative predictions. If maximization of the span of protective coverage by NMD is a significant evolutionary force dictating the location of introns, then (1) the number of introns should scale linearly with the length of coding DNA in a gene, (2) the average positions of consecutive introns should be approximately evenly distributed over the length of a gene, and (3) introns should be overdispersed, i.e., exon sizes should be more uniform than expected under random insertion.
The Spatial Distribution of Introns
To gain information on the spatial distribution of introns, we surveyed the sequenced genomes of Homo sapiens, C. elegans, Drosophila melanogaster, and Arabidopsis thaliana. For all four species, there is a striking linear relationship between the average number of introns contained within a gene and the length of coding DNA (fig. 4). For H. sapiens, on average an intron is present for each 125 (SE = 3) nucleotides of coding DNA, whereas the average exon size for C. elegans is 180 (3) nucleotides, that for D. melanogaster is 325 (13) nucleotides, and that for A. thaliana is 101 (3) nucleotides. The average human and C. elegans gene size (in coding nucleotides) at the point of first intron colonization is 500 bp, whereas that for D. melanogaster and A. thaliana is nearly twofold greater,
900 bp. (We did not include intron-free genes in these analyses, as there is compelling evidence that large numbers of such annotated open reading frames are actually processed pseudogenes; Chen et al. 2002).
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Discussion |
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Our whole-genome analyses demonstrating the linear increase in the mean number of introns with gene size and the overdispersed distribution of introns strongly implicate the operation of some general form of selection for relatively uniform coverage of the coding regions of genes. However, considerable interspecific differences in the spatial geometry of intron positions also imply phylogenetic variation in the internal selective pressures and/or birth/death processes driving such patterns. Among the four species analyzed herein, humans conform most closely with a model for uniform coding-region coverage by introns. Human exons are quite diminutive with only a small minority exceeding 300 bp in length (Berget 1995). Taken at face value, the 125-bp average size of human exons might appear to be inconsistent with a model in which NMD is the sole selective force for exon size, because the few studies that have searched for an upper limit to surveillance span have found this to be in excess of 500 bp in mammalian cell lines. Given the very small number of genes that have been examined, however, most of which involve constructs with single introns, it is not yet possible to rule out the existence significant interlocus variation in the spatial requirements of NMD. Nor can a gradient in NMD efficiency with PTC-EJC distance or an enhanced efficiency of NMD with greater numbers of introns be ruled out.
These caveats aside, it must be acknowledged that additional factors associated with intron processing may contribute to stabilizing selection for small exon size in the human genome. Relatively small long-term effective population sizes enhance the vulnerability of mammalian genomes to the accumulation of introns, perhaps beyond the density necessary for efficient NMD, and also reduce the efficiency of selection against insertions within introns (Lynch 2002). As a consequence, mammalian exons are generally dwarfed by their large (often tens of kb) intervening introns. Such conditions have apparently selected for a mammalian splicing machinery that is dependent on exon (rather than intron) recognition, with significant errors arising when exon length exceeds 300 bp or so (Berget 1995; Sterner, Carlo, and Berget 1996). Thus, even if the efficiency of NMD remains high over a surveillance tract as long as 1,000 bp in mammals, the presence of exons of this length would virtually guarantee a very high incidence of splicing errors. This suggests that selection for small enough exon sizes to minimize the incidence of splicing errors combined with selection for efficient mRNA surveillance capacity may be mutually responsible for the exceptionally regular distribution of relatively small mammalian exons.
Unlike the situation in humans, the more 5'-located introns in C. elegans, D. melanogaster, and S. pombe tend to be shifted in the 5' direction relative to expectations under a model of uniform spacing. A number of selective forces might favor such locations. First, NMD will more strongly select for coverage at the 5' end of a gene if truncated transcripts of short to medium length are more harmful than those that are nearly complete. Second, 5'-located introns are likely to be more integrated with other intron-associated activities, such as gene regulation, the facilitation of transcript elongation, and the guidance of mRNA export. Third, the distribution of introns will also depend on the mechanisms of intron gain and loss if these vary over the length of a gene. For example, it has been suggested that reverse transcription followed by homologous recombination may be a common mechanism of intron loss (Fink 1986). If this is the case, however, exons at the 3' ends of genes are expected to be exceptionally long. Although the final exon of a gene does tend to be unusually long (Hawkins 1988), our data show that this is true only if the noncoding portion of the 3'-most exon is included.
The lower number of introns/coding DNA as well as the higher level of variance in exon size in invertebrates relative to mammals may be a consequence of weaker stabilizing selection for exon size in the former. Intron lengths in the two invertebrates are much smaller than those in humans, and it has been argued that splicing of genes with small introns relies on intron recognition (Berget 1995), which would reduce the need for a high density of relatively evenly spaced introns per coding DNA. Moreover, if as suggested above, there are additional selective pressures favoring 5'-located introns, a lower overall abundance of introns in invertebrates would tend to select for more 5' bias than in mammals, where the first introns are already close to the 5' end because of their higher density.
Other intron-dependent aspects of mRNA processing may have influential roles in determining the spatial distribution of introns. The essential role of spliceosomal introns in the production of alternatively spliced gene products, which are elicited by up to 30% of the genes in metazoan species (Graveley 2001), makes them an obvious candidate. Nevertheless, there are still only a few convincing demonstrations of the adaptive significance of alternative splicing, and a large number of the products of this process may just be inevitable consequences of an imperfect splicing system (Levine and Durbin 2001). Moreover, selection for alternative splicing provides no obvious explanation for the substantial differences in average intron density and dispersion that exist among species. Finally, Castillo-Davis et al. (2002) have shown that although there is a dramatic decline in average intron size with increasing level of gene expression in nematodes and mammals, intron number is independent of gene-expression level. The latter observation is consistent with the hypothesis that factors unassociated with gene regulation impose stabilizing selection for intron number. Thus, we suggest that species-specific patterns of intron locations may be driven primarily by a coevolutionary loop involving the physical limitations on both splicing and mRNA surveillance.
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Acknowledgements |
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Footnotes |
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