Department of Ecology and Evolutionary biology, University of Michigan, Ann Arbor
Correspondence: E-mail: jianzhi{at}umich.edu.
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Abstract |
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Key Words: substitution rate neutrality pseudogene Makorin rodents mice
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Introduction |
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Direct evidence for biological function of operationally defined pseudogenes is available in only a few cases. Nitric oxide synthase (NOS) is involved in intracellular signaling in the nervous system and is coexpressed with a NOS-related pseudogene in the snail Lymnaea stagnalis (Korneev, Park, and O'Shea 1999). It was experimentally shown that the pseudogene transcript plays an important role in the regulation of the NOS protein synthesis by forming a stable RNA-RNA duplex with the transcript of the functional NOS gene. The formation of the duplex significantly suppresses the translation of NOS mRNA and is possible because of antisense identity between the transcripts of the functional gene and pseudogene. In another dramatic example, targeted deletion of the pseudogene Makorin1-p1 in mice led to 80% mortality within 2 days of birth (Hirotsune et al. 2003). Affected individuals suffered from severe bone deformity and failure to thrive (Hirotsune et al. 2003). Although Makorin1-p1 (on chromosome 5) showed typical features of a truncated pseudogene derived from the functional gene Makorin1 (on chromosome 6), it was robustly transcribed. Sequence analysis of the approximately 700-bp pseudogene transcript revealed high similarity to the 5' end of Makorin1 mRNA. Hirotsune et al. (2003) experimentally showed that the Makorin1-p1 transcript regulates the stability of Makorin1 mRNA in trans. In the presence of Makorin1-p1 transcript, the half-life of Makorin1 mRNA significantly increased. Although the exact molecular mechanism involved in this expression regulation is not clear, Hirotsune et al. (2003) proposed a plausible RNA-mediated model in which transcripts of Makorin1-p1 and Makorin1 directly compete for a destabilizing factor (e.g., RNase) that binds to the highly similar 5' sequence found in both transcripts (see also Lee [2003]). The pseudogene transcript effectively titrates out the destabilizing factor and protects the coding mRNA from decay.
This gene regulation model, as well as the high sequence similarity (95.4%) between the Makorin1-p1 transcript and the 5' portion of Makorin1 mRNA, suggests that Makorin1-p1 and Makorin1 evolve in a coordinated fashion. Is it because of concerted evolution? Are there functional constraints on the transcribed and untranscribed portions of Makorin1-p1? To address these questions, we sequenced Makorin1-p1 in five additional Mus species. Our results show functional constraints on transcribed regions of Makorin1-p1, yet no evidence for concerted evolution.
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Materials and Methods |
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Sequence Analysis
The nucleotide sequences of Makorin1-p1 from Mus musculus and the above five Mus species were aligned using ClustalX (Thompson et al. 1997) followed by manual adjustment. A well-supported phylogenetic tree of the six taxa used in our analysis was recently obtained using nuclear and mitochondrial DNA sequences (Lundrigan, Jansa, and Tucker 2002). We used this tree topology in our analysis of substitution rates. Insertion and deletion substitutions were manually counted using the parsimony principle based on the species tree. Nucleotide substitution rates were estimated using MEGA version 2.0 (Kumar et al. 2001). Likelihood ratio tests were used to examine nucleotide substitution rate variation among regions of the sequenced Makorin1-p1 pseudogene. Because likelihood ratio tests may give different results under different assumptions (Zhang 1999), we implemented JC69 (Jukes and Cantor 1969), HKY85 (Hasegawa, Kishino, and Yano 1985), TN93 (Tamura and Nei 1993), and general reversible (GTR [Yang 1997]) nucleotide substitution models. JC69 is the simplest of these four models, and it assumes equal probabilities for all types of nucleotide substitutions. A more complex HKY85 model allows for unequal base frequencies and incorporates different substitution rates for transitions and transversions. TN93 generalizes the HKY85 model to allow for different rates for transitions between purines and between pirimidines. The GTR model is the most complex and includes three parameters describing nucleotide frequencies and five parameters describing relative substitution rates among nucleotides. We also used a gamma distribution that describes rate variation among sites in GTR. PAML (Yang 1997) was used for the likelihood computation. To test whether there is concerted evolution between Makorin1-p1 and Makorin1, we made a gene tree using the neighbor-joining method (Saitou and Nei 1987) and examined sister relationships among genes. Sequences used in this phylogenetic reconstruction included Makorin1-p1 sequences of all six Mus species mentioned above, Makorin1 sequences from M. musculus, M. caroli, and M. spretus, and representatives of the Makorin gene family from mouse, rat, and human. The number of synonymous nucleotide substitution per synonymous site (dS) was computed by the modified Nei-Gojobori method (Zhang, Rosenberg, and Nei 1998).
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Results |
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Although the exact molecular mechanism by which the Makorin1-p1 transcript regulates the stability of Makorin1 mRNA is unknown, the suggested model (Hirotsune et al. 2003) implies that high sequence similarity between them is necessary for the functionality of the pseudogene. That is, Makorin1-p1 must evolve in concordance with Makorin1. To examine whether gene conversion and concerted evolution is responsible for this coordinated evolution, we reconstructed a gene phylogeny using Makorin1-p1 sequences from all six species and the Makorin1 sequence from M. musculus, M. caroli, and M. spretus (fig. 4). Included in the tree were also other members of the Makorin family from the human, mouse, and rat. Only regions homologous to Makorin1-p1 region B were used for tree-making. Concerted evolution will generate phylogenetic clustering of the functional gene and pseudogene from the same species. In other words, Makorin1-p1 and Makorin1 of M. musculus, for example, should form a monophyletic group, in exclusion of Makorin1-p1 of other species. This, however, was not observed. Makorin1-p1 of M. musculus is more closely related to pseudogenes of other Mus species than to Makorin1 of M. musculus, with high bootstrap support. The same result was obtained when the partial sequence of M. pahari Makorin1-p1 was included in the phylogenetic analysis. We noted, however, that the clade of Makorin1 from M. caroli, M. musculus, and M. spretus does not show the same phylogenetic relationships as the clade of Makorin1-p1 for the same taxa. Makorin1 shows a sister relationship of M. spretus to M. musculus, but Makorin1-p1 exhibits a sister relationship of M. spretus to M. caroli. This discrepancy is most likely caused by the limited number of nucleotides (610) used in this phylogenetic analysis. At any rate, our result suggests that gene conversion is not responsible for the presumably coordinated evolution between Makorin1-p1 and Makorin1. Functional conservation and purifying selection may be the sole reason for the high sequence similarity between them. Figure 4 shows that the branch leading to Makorin1-p1 of a Mus species is significantly longer than that to Makorin1 of the same species when the rat Makorin is used as an outgroup (P < 0.001; Tajima's [1993] test). This suggests that although the transcribed region of Makorin1-p1 is under purifying selection, the selection is weaker than that on Makorin1.
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Discussion |
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Makorin1 belongs to the Makorin family of transcription factors. Putative orthologs of Makorin1 are known in human, mouse, wallaby, chicken, fruitfly, and nematode (Gray, Azama, and Whitmore 2000). Such wide phylogenetic distribution suggests its functional importance. Hirotsune et al.'s (2003) experiments showed a novel Makorin1 regulatory mechanism involving the transcript of the pseudogene Makorin1-p1. Makorin1-p1 knockout mice suffer from severe bone deformities, and most of them die in 2 days. Because Makorin1-p1 is most likely absent in rats, an interesting question is why rats do not suffer from the severe phenotypes that the knockout mice exhibit. How is Makorin1 expression regulated in rats and other species without Makorin1-p1? How and why was Makorin1-p1 co-opted into the regulatory mechanism of Makorin1? Based on experimental evidence, Hirotsune et al. (2003) proposed that Makorin1-p1 functions by titrating out a destabilizing factor that otherwise destabilizes the mRNA of Makorin1. Assuming that this hypothesis is correct, we here propose a duplication-degeneration model (fig. 5) that explains the evolutionary origin of Makorin1-p1's involvement in Makorin1's expression regulation. Before the emergence of Makorin1-p1, the production of Makorin1 mRNA was high enough to titrate out the destabilizing factor. Immediately after Makorin1-p1 was produced by duplication from Makorin1, Makorin1-p1 had the same sequence and expression level as Makorin1. The doubling of the amount of mRNA may be unnecessary or even slightly deleterious. Thus, degenerate mutations that reduce the expression level of Makorin1 could be fixed. If this happened, Makorin1-p1 became indispensable, as its transcript would be needed to titrate out the destabilizing factor. Mutations that disrupted the ORF of Makorin1-p1 could still be fixed because the transcript could still titrate out the destabilizing factor. This would explain the conservation in both expression pattern and transcript sequence of the pseudogene. This model could be tested in the future by examining the expression levels of Makorin1 and Makorin1-p1 in mice and the expression level of Makorin1 in rats. The hypothesis predicts that the amount of Makorin1 expression in rats is higher than that in mice.
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In this report, we provided evidence for evolutionary constraints on the transcribed portion of Makorin1-1p. Many examples of transcribed pseudogenes are known (e.g., Board, Coggan, and Woodcock 1992; Brandt et al. 1993; Furbass and Vanselow 1995; Bard et al. 1995), and it is possible that some of them also play physiological roles. Even retrotransposed genes, which are generally thought to become pseudogenes immediately after retrotransposition, have been shown to be able to evolve into new functional genes (Long et al. 2003). The findings of functional "pseudogenes" are relevant when pseudogenes are used for estimating rates and patterns of mutations (Li, Gojobori, and Nei 1981; Gojobori, Li, and Graur 1982; Li, Wu, and Luo 1984; Graur, Shuali, and Li 1989; Petrov and Hartl 1999). Care should be taken to ensure that such pseudogenes are indeed nonfunctional. Our approach of comparing point and indel substitution rates in transcribed and untranscribed regions of pseudogenes may be used to systematically look for functional "pseudogenes," particularly when complete genome sequences of closely related species are available, and thereby expedite the discovery of more functional elements in genomes.
Another question arising from the study of Makorin1-p1 is whether we can still call such sequences pseudogenes. The meaning of nonfunctionality in the pseudogene definition is quite ambiguous. From studies on pseudogene evolution mentioned above, functionality can be described on two levels: operational and biological. At the operational level, one can examine whether a particular gene contains frame-shifting indels, premature stop codons, and disrupted splice recognition sites. At the biological level, functionality can be described as the physiological role and fitness effect of the presence of the pseudogene. Of these two, biological function is much more difficult to examine. Decisions on whether to call a particular DNA sequence a gene or pseudogene can be made only after careful scrutiny and experimental investigations of its function. To strictly adhere to the pseudogene definition, only gene relics that lack biological function should be called pseudogenes. Once a biological function of a gene relic is found, depending on whether the functionality involves the DNA sequence or its transcript, such a relic should be called either a regulatory element or a gene, respectively. In the present case, Makorin1-p1 is probably better called an RNA gene, rather than a pseudogene.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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References |
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