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. 2012 Mar 21;29(9):2169–2176. doi: 10.1093/molbev/mss085

Rewiring of Posttranscriptional RNA Regulons: Puf4p in Fungi as an Example

Huifeng Jiang 1,2, Xiaoxian Guo 1,3, Lin Xu 1,4, Zhenglong Gu 1,*
PMCID: PMC3424410  PMID: 22438588

Abstract

It has been increasingly clear that changes in gene regulation play important roles in physiological and phenotypic evolution. Rewiring gene-regulatory networks, i.e., alteration of the gene-regulation system for different biological functions, has been demonstrated in various species. Posttranscriptional regulons have prominent roles in coordinating gene expression in a variety of eukaryotes. In this study, using Puf4p in fungi as an example, we demonstrate that posttranscriptional regulatory networks can also be rewired during evolution. Although Puf4p is highly conserved in fungi, targets of the posttranscriptional regulon are functionally diverse among known fungal species. In the Saccharomycotina subdivision, target genes of Puf4p mostly conduct function in the nucleolus; however, in the Pezizomycotina subdivision, they are enriched in the mitochondria. Furthermore, we demonstrate different regulation efficiencies of mitochondrial function by PUF proteins in different fungal clades. Our results indicate that rewiring of posttranscription regulatory networks may be an important way of generating genetic novelties in gene regulation during evolution.

Keywords: posttranscriptional regulon, evolution of gene regulation, yeast

Introduction

Understanding how gene regulation evolves is a key area in research on evolution (King and Wilson 1975; Wray et al. 2003; Carroll 2005; Ihmels et al. 2005; Frankel et al. 2010; Kaplan et al. 2011; Rozpedowska et al. 2011; Shou et al. 2011). Gene regulation can occur at various levels. The concept of the posttranscriptional regulon was recently proposed to describe the phenomenon that mRNAs encoding functionally related proteins are coordinately regulated for stability, localization, and translation (Keene 2007). Previous works on the evolution of gene regulation have been conducted mostly at the gene-transcription level (Borneman et al. 2007; Wray 2007; Emerson et al. 2010), whereas the evolution of posttranscriptional regulation has received far less attention. Illustrating how changes occur at the level of posttranscriptional regulation is crucial for gaining better understanding of how gene regulation evolves in nature (Chen and Rajewsky 2007).

Posttranscriptional gene regulation plays an important role in determining biological complexity. Posttranscriptional regulons, consisting of RNA-binding proteins (RBPs) and their target genes, are essential for the coordination of gene expression (Mata et al. 2005; Keene 2007; Woodson and Chory 2008). Although extensive functional researches have been conducted on various RBPs and divergences of RBP targets in several species have been revealed (Keene 2007; Hogan et al. 2008), the evolutionary mode of posttranscriptional regulatory networks remains largely unknown. We have shown that a tinkering evolutionary mode of the PUF3 posttranscriptional regulon occurred during fungal evolution (Jiang et al. 2010). In this study, we focus on investigating the evolutionary mode of the PUF4 posttranscriptional regulon, which plays an important role in regulating genes that are associated with ribosome biogenesis in budding yeast, Saccharomyces cerevisiae (Gerber, Herschlag and Brown 2004; Grigull et al. 2004; Foat et al. 2005).

Puf3p and Puf4p belong to a group of well-characterized posttranscriptional regulators that contain a PUM-HD-type (PUMilio homology domain, which contains eight helical repeats) RNA-binding domain to bind mRNAs on the 3’UTR (un-translated region). By binding with their target mRNAs, PUF proteins can recruit factors to transfer the target mRNAs into various subcellular locations, repress translation of mRNAs, and promote mRNA degradation (Chritton and Wickens 2010; Quenault et al. 2011). The degeneration of mRNA by PUF proteins requires the recruitment of the Ccr4p–Pop2p–Not deadenylase complex (Goldstrohm et al. 2006). The mechanism of mRNA repression is likely conserved for all PUF proteins across species (Hook et al. 2007; Kadyrova et al. 2007; Lee et al. 2010).

By altering the binding elements in genes, functions of targets for different PUF regulons vary in different species (Crittenden et al. 2002; Gerber et al. 2004, 2006; Galgano et al. 2008; Tam et al. 2010). In budding yeast, different PUF proteins recognize different elements and regulate genes in different functional categories by recruiting the RNA decay complex. For example, Puf3p and Puf4p mostly regulate genes that function in mitochondria and the nucleolus, respectively (Gerber et al. 2004). Only one PUF protein in fruit flies is essential for early embryogenesis and cell development (Murata and Wharton 1995; Lin and Spradling 1997; Gerber et al. 2006). One of the PUF proteins in worms determines the proliferation of germline stem cells (Crittenden et al. 2002). In humans, two PUF proteins interact with the microRNA system to regulate genes in transcription, the cell cycle, and cell proliferation (Galgano et al. 2008; Morris et al. 2008). The dynamic functions of PUF regulons indicate rich evolutionary history of these posttranscriptional regulatory networks. Our results from this study indeed indicate that the functions of genes that are regulated by the PUF4 posttranscriptional regulon were reprogrammed in different fungal clades during evolution. In the Saccharomycotina subdivision, Puf4p regulates genes in the nucleolus, whereas in the Pezizomycotina subdivision, it regulates genes that function in mitochondria. Together with our previous study on the evolution of Puf3p targets in fungi (Jiang et al. 2010), our results reveal new principles on the evolution of novel posttranscriptional regulation and call for more efforts to investigate the evolution at this critical level of gene regulation.

Materials and Methods

Sequence Data

Sequences for the studied fungal species were from Fitzpatrick et al. (2006) and the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The human, mouse, worm, and fly sequence data were downloaded from the Ensembl database (http://asia.ensembl.org/index.html). The sequence data for Arabidopsis thaliana was downloaded from http://www.plantgdb.org/. Mitochondrial genes for human, mouse, and A. thaliana were downloaded from the MitoP2 database (http://www.mitop.de) (Prokisch et al. 2006). The mitochondrial genes for worm and fly were identified based on their orthology with human mitochondrial genes. The nucleolar genes for human were defined based on the Gene Ontology annotation (GO:0005730). The nucleolar genes for mouse, worm, fly, and A. thaliana were detected by their orthology with nucleolar genes in the human genome.

Definition of Orthologous Genes

Using the InParanoid software package (Remm et al. 2001), orthologs between the budding yeast, S. cerevisiae, and other fungal species were identified. The default parameters were used in detection of PUM-HD repeats detected by HMMER3 (http://hmmer.org/) based on the Hidden Markov Model profile for PUF domain (PF00806) from the Pfam database (http://pfam.sanger.ac.uk/).

Reconstruction of the Fungal Phylogenetic Tree

Using genes in S. cerevisiae as queries, we obtained the ten most conserved orthologs between S. cerevisiae and Cryptococcus neoformans (the most remotely related fungal species to S. cerevisiae) and used these genes to reconstruct a phylogenetic tree for all the studied fungal species. Protein sequences were aligned using MUSCLE (Edgar 2004). The phylogenetic tree was constructed by ClustalW2.0 using distance matrix method and Neighbour-Joining (NJ) clustering method. Finally, the tree was drawn by MEGA5 (Larkin et al. 2007; Tamura et al. 2011).

Detection of Target Genes of PUF4 Posttranscriptional Regulon

As in our previous study (Jiang et al. 2010), we used a Perl script to detect the target motif (P4E) of Puf4p. As shown in Gerber et al. (2004), we allowed flexibility (among A, C, and T) in the fifth and seventh nucleotides and required exact match for the rest nucleotides of the P4E motif during motif detection. For each species, we scanned 250 bp of DNA sequence downstream of all annotated genes to discern the occurrence of the motifs. Genes with the P4E motif in their 250-bp 3′ downstream regions were defined as the target genes of Puf4p.

Enrichment Analysis in the Localization Categories

Sublocalization information for genes in budding yeast was downloaded from the Saccharomyces Genome Database (http://www.yeastgenome.org/) (Huh et al. 2003). The localization of genes in other species was defined based on the localization of their orthologs in S. cerevisiae. If a gene from one species has two orthologs in S. cerevisiae, its localization was defined as the combination of localization for both copies. The hypergeometric test was used to test the enrichment of genes with P4E in each localization category. For multiple comparisons, the false discovery rates are no larger than 0.01 (Benjamini and Hochberg 1995).

Results

The Evolution of PUF Proteins in Fungi

The PUF protein family is conserved throughout the eukaryotic kingdom (Wickens et al. 2002; Spassov and Jurecic 2003). However, the number and function of PUF proteins vary among species (Quenault et al. 2011). In S. cerevisiae, seven proteins with PUM-HD repeats were identified as PUF proteins (Supplementary table 1, Supplementary Material online). In order to investigate the evolution of these PUF proteins in fungi, we identified their orthologous genes in the sequenced fungal species (Supplementary Table 2, Supplementary Material online). In figure 1, we show the distribution of orthologous genes for these seven PUF proteins in different fungal species. Figure 1 shows that PUF2 originated during a whole genome duplication (WGD) event because its orthologs are only present in the post-WGD yeast species (Byrne and Wolfe 2005). The orthologs of PUF5 are present only in the Saccharomycotina subdivision except Yarrowia lipolytica, which is the furthest species from budding yeast in the subdivision. Therefore, both PUF2 and PUF5 genes exist only in some species of the Saccharomycotina subdivision.

FIG. 1.

FIG. 1.

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The evolution of Puf proteins in fungi. The phylogenetic tree for species in fungi was reconstructed based on ten conserved proteins (the bootstrap scores are displayed in Supplementary fig. 1, Supplementary Material online, and the abbreviations for the studied species are shown in Supplementary table 2, Supplementary Material online). The tree was consistent with the results of a previous study (Fitzpatrick et al. 2006). The color in each cell denotes the number of PUM-HD repeats for that gene in the respective species. Gray means that there was no detectable orthologous gene. The arrows indicate the WGD event and the Saccharomycotina clade, respectively.

Since the PUM-HD domain in PUF proteins is essential for binding mRNA, we examined the number of PUM-HD repeats for all of the PUF orthologs we studied. As shown in figure 1, although orthologs of PUF1, PUF6, and NOP9 can be identified in all studied fungal species, the number of PUM-HD repeats in these genes is not conserved, indicating that the functions of Puf1p, Puf6p, and Nop9p may be dramatically changed in different fungal species.

In contrast to the above PUF proteins, most orthologs of Puf3p and Puf4p consist of a canonical PUM-HD domain with eight repeats. Targets of these two PUF proteins also have significant functional enrichment in the budding yeast, warranting further investigation on how their targets evolved in different fungal species. Our previous work has shown that the PUF3 posttranscriptional regulon is highly conserved in fungi, but the dedicated regulation of genes that function in the mitochondria occurs only in the Saccharomycotina subdivision (Jiang et al. 2010). In the following study, we focused on the functional evolutionary process of the PUF4 posttranscriptional regulon in fungal species.

Functional Rewiring of PUF4 Posttranscriptional Regulon in Fungal Species

Previous researches have shown that Puf4p plays an important role in ribosome biogenesis (Gerber et al. 2004; Foat et al. 2005). This motivated us to investigate whether the function of the PUF4 posttranscriptional regulon is conserved among fungal species. Accordingly, we identified all orthologous genes between each studied fungal species and budding yeast, S. cerevisiae. Genes in each species were categorized based on the subcellular localization of their orthologs in the budding yeast (Huh et al. 2003). In each localization category, we defined a gene as a target of the PUF4 regulon if its 3′UTR had the recognized binding element of the PUF4 regulon (P4E). We further estimated the enrichment of target genes in each species (the phylogenetic relationship among species is displayed in Supplementary fig. 1, Supplementary Material online, and the total number of genes in each category are listed in Supplementary table 3, Supplementary Material online). As shown in figure 2A, we confirmed previous experimental results that genes functioning in the nucleolus have enriched Puf4p binding. Indeed, this pattern was observed in most species in the Saccharomycotina subdivision. However, to our surprise, the studied species in the Pezizomycotina subdivision showed significant enrichment of P4E only in genes that are encoded by the nuclear genome but function in mitochondria (therefore called mitochondrial genes for simplicity hereafter), indicating a possible functional rewiring of the PUF4 posttranscriptional regulon during fungal evolution.

FIG. 2.

FIG. 2.

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Functional rewiring of the PUF4 posttranscriptional regulon. (A) Enrichment of P4E in each functional category of genes (column) in each fungal species (row). Colors denote the P values for the enrichment of P4E after multiple test correction, as indicated at the bottom. The phylogenetic tree of the studied fungal species is shown to the left. The Saccharomycotina (pink) and Pezizomycotina (light blue) subdivisions are indicated by arrows. Two other fungal clades, Schizosaccharomyces and Basidiomycota, are indicated as dark blue and green, respectively. Yellow lines separate fungal species into four categories based on the evolutionary pattern of the PUF4 posttranscriptional regulon. (B) The average percentage of nucleolar genes with P4E in each group of fungal species.

Regulation of nucleolar genes by Puf4p also showed clade-specific dynamics in the Saccharomycotina subdivision. All of the species in one of the clades (Group 1) showed functional enrichment of Puf4p binding for the nucleolar genes, whereas only some of the species in the other clade (Group 2) showed a similar pattern (Supplementary fig. 2, Supplementary Material online). As shown in figure 2B, on average, only 2% and 4% of genes in the nucleolus had P4E for species in Group 3 and 4, respectively. Many more genes in the nucleolus had P4E in Group 1 (∼30%, two-tailed t-test P value = 2 × 10−9 comparing with the Groups 3 and 4) and Group 2 species (∼17%, two-tailed t-test P value = 5 × 10−4 comparing with the Groups 3 and 4). The percentage of genes in the nucleolus with P4E in the Group 1 species was also significantly higher than that for the genes in the Group 2 species (two-tailed t-test, P value = 0.001).

Deep Functional Divergence of PUF3 and PUF4 Regulons in Fungal Species

We have shown that mitochondrial genes in the Saccharomycotina subdivision are regulated by the PUF3 posttranscriptional regulon (Jiang et al. 2010). Interestingly, the above results indicate that the mitochondrial genes may be regulated by the PUF4 posttranscriptional regulon for species in the Pezizomycotina subdivision. In order to further understand the evolutionary relationship between these two posttranscriptional regulons, we reconstructed the phylogenetic tree for the orthologs of two regulators, Puf3p and Puf4p, in the studied fungal species. The PUM-HD repeats, which is the functional domain for RNA binding, was used in tree reconstruction. The ortholog of PUF protein in Gardia lamblia, which is considered as the sister group to all other eukaryotes (Sogin et al. 1989; Sun et al. 2010), was used as an out-group for tree reconstruction. As shown in figure 3A, the PUM-HD repeats from all of the Puf3p orthologs were separated from the Puf4p orthologs in the phylogenetic tree, indicating that a duplication event that generated PUF3 and PUF4 genes occurred before the divergence of all the studied fungal species.

FIG. 3.

FIG. 3.

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Deep functional divergence between Puf3p and Puf4p. (A) The phylogenetic tree of the PUM-HD domains from the Puf3p and Puf4p orthologs (bootstrap scores are presented in Supplementary fig. 3, Supplementary Material online). Glam-Puf protein is used as the out-group. The branches to Puf3p and Puf4p in each species were coded in red and blue, respectively. (B) Functional conservation of Puf3p and Puf4p orthologs in fungi. The conserved amino acids for the repeat #3 and #5 of the PUM-HD domain are displayed using ClustalX (Thompson et al. 1997). The red (C ↔ T) and blue (R ↔ C) arrows denote the conserved amino acids in Puf3p and Puf4p that are critical for different motif recognition between these two proteins. (C) Little functional overlapping between Puf3p and Puf4p. The y axis denotes the ratio of genes having both P3E and P4E over the total number of genes with P3E and/or P4E in each species (x axis).

In S. cerevisiae, although both Puf3p and Puf4p have eight PUM-HD repeats, their recognized motif sequences are different (Gerber et al. 2004). The results from Miller and colleagues using protein structures of Puf3p and Puf4p and genetics study showed that two amino acids changes, C ↔ T and R ↔ C, at the third and fifth PUM-HD repeats, respectively, are essential for the recognition difference between Puf3p and Puf4p (Miller et al. 2008). Comparing these two repeats in all orthologs of Puf3p and Puf4p, we found that these two pairs of different amino acids were conserved in all Puf3p and Puf4p orthologs (fig. 3B), indicating that the recognition element by PUM-HD repeats in Puf4p might be conserved and distinct from the recognition element of Puf3p for all the studied fungal species. Genes with P3E or P4E in their 3′UTR regions were further compared in each species. As shown in figure 3C, the possible target genes of PUF3 and PUF4 regulons (genes with P3E and P4E, respectively) has very little overlap (less than 4%, on average), further supporting the conclusion that these two proteins may have different functions in all fungal species.

Posttranscriptional Regulation of Mitochondrial Genes during Fungal Evolution

We further compared the regulatory efficiency of two posttranscriptional regulons for mitochondrial genes in their respective clades. On average, about 12% of the mitochondrial genes are bound by PUF4 genes in the Pezizomycotina subdivision, which is significantly lower than for PUF3 genes in the Saccharomycotina subdivision (∼45%, two-tailed t-test P = 3 × 10−16). Because occurrence of the binding motif is correlated with GC content at the genome level (Jiang et al. 2010), we normalized the frequencies of P3E and P4E in mitochondrial genes by their occurrences in the genome. As shown in figure 4A, on average ∼15% P4E in the whole genome appears in the mitochondrial genes for species in the Pezizomycotina subdivision. This is still significantly lower than that for P3E in the Saccharomycotina subdivision (∼29%, two-tailed t-test P = 4 × 10−8; Supplementary table 3, Supplementary Material online). Since genes that are involved in mitochondrial translation are essential for mitochondrial biogenesis, we investigated the occurrence of PUF motif in mitochondrial ribosomal protein genes and found that about 21% mitochondrial ribosomal protein genes are bound by Puf4p in the Pezizomycotina species, but ∼90% mitochondrial ribosomal protein genes are bound by Puf3p in the Saccharomycotina species (Supplementary fig. 4, Supplementary Material online). Therefore the regulation of mitochondrial genes by the PUF4 regulon in the Pezizomycotina subdivision may be less efficient than that of the PUF3 regulon in the Saccharomycotina subdivision.

FIG. 4.

FIG. 4.

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Posttranscriptional regulation of mitochondrial genes in fungi. (A) The occurrence of P3E and P4E in mitochondrial genes. The x axis denotes each studied fungal species (the Group 4 species, which include the Saccharomycotina and Pezizomycotina subdivisions, were put in the middle of fig. 4). The y axis denotes the percentage of all Puf3p or Puf4p target genes in each genome that are dedicated to mitochondrial regulation. (B) The correlation of Puf3p and Puf4p mitochondrial regulation in fungi. All mitochondrial genes were divided into four groups based on the number of species in the Pezizomycotina subdivision that have the P4E motif. The x axis represents the group of mitochondrial genes. The y axis is the average percentage of species in the Saccharomycotina subdivision that have P3E for each orthologous mitochondrial gene group. The mean and standard deviation for each category are shown.

Because mitochondrial genes in the Pezizomycotina and Saccharomycotina species are regulated by different posttranscriptional regulons, we investigated relationships between these two regulons. We grouped orthologous mitochondrial genes in the Pezizomycotina subdivision based on the number of species in the Pezizomycotina subdivision that have P4E in their 3′UTR regions. For each orthologous gene category, we calculated the percentage of species in the Saccharomycotina subdivision that have PUF3 regulation element. We found that the more species have P4E in mitochondrial orthologs in the Pezizomycotina subdivision, the more species have P3E in the Saccharomycotina subdivision (fig. 4B, analysis of variance, F = 6.5, P value = 3 × 10−4).

Discussion

Functional Innovation of Posttranscriptional Regulons

Our results show that all fungal species in the Pezizomycotina, Schizosaccharomyces, and Basidiomycota subdivisions do not have functional enrichment of PUF4 posttranscriptional regulation for the nucleolar genes. We conducted further analysis for the nucleolar genes in five out-group species, including human, mouse, Caenorhabditis elegans, Drosophila melanogaster, and A. thaliana (fig. 5). Our results show that nucleolar genes do not have an enriched PUF4 regulatory motif in their 3′UTR (fig. 5A). The results for the PUF3 motif are the same (fig. 5B). The parsimony principle suggests that the PUF4 posttranscriptional regulation for the nucleolar genes might be a novel feature that evolved only in the Saccharomycotina species. Based on a similar parsimonious argument, we also concluded that the enriched posttranscriptional regulation by PUF proteins for the mitochondrial genes may also represent evolutionary innovations in the Saccharomycotina and Pezizomycotina subdivisions because mitochondrial genes in the species of Schizosaccharomyces and Basidiomycota subdivisions, and the above five out-group species had no enrichment for either PUF3 or PUF4 posttranscriptional regulation (fig. 5C and D).

FIG. 5.

FIG. 5.

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Evolution of the PUF posttranscriptional regulons in plant and animals. (A) The occurrence of P4E in the nucleolar genes of plant and animals. (B) The occurrence of P3E in the nucleolar genes of plant and animals. (C) The occurrence of P3E in the mitochondrial genes of plant and animals. (D) The occurrence of P4E in the mitochondrial genes of plant and animals. P4E and P3E were detected at the 1,000-bp 3′UTR regions in each species. Green bars denote the percentage of genes with the motif in the studied organelle and blue bars denote the percentage of genes with the motif at the whole-genome level.

The nucleolus is the location for ribosome synthesis. It has been shown that despite extreme functional conservation, ribosomal protein genes had a very dynamic evolutionary process for regulation at the transcriptional level in various fungal species (Tanay et al. 2005). Interestingly, a novel gene transcriptional regulatory network, including a new protein domain in the transcription factor, Rap1p, and a new promoter motif for ribosomal protein genes, evolved in the Group 1 fungal species. Our results show that posttranscriptional regulation of the ribosomal protein genes in the nucleolus also evolved as a novel regulatory module in the same species group. The underlying biological cause for these concurrent regulatory innovations of ribosome biogenesis and regulation during evolution remains to be further investigated.

Functional Rewiring of Posttranscriptional Regulation in Fungi

Our results indicate that the function of the PUF4 posttranscriptional regulon might be rewired from binding mitochondrial genes in the Pezizomycotina clade (Group 3, fig. 2) to binding nucleolar genes in the Saccharomycotina clade (Groups 1 and 2, fig. 2). We also hypothesized that the mitochondrial genes were rewired from being posttranscriptionally regulated by Puf4p in the Pezizomycotina clade to Puf3p in the Saccharomycotina clade because genes functioning in mitochondria are enriched for their respective target 3′UTR motifs in these two clades. The switch of the binding motif in the 3′UTR should have occurred individually in their target mitochondrial genes. We cannot directly rule out the possibility that two regulators, Puf3p and Puf4p, have functional redundancy in the common ancestor of the Pezizomycotina and Saccharomycotina clades. However, several lines of evidence argue against this possibility: First, PUF3 and PUF4 genes were formed by an ancient duplication event, either at or before the common ancestor of all fungal species. Second, two orthologs show clear divergence of amino acids that are functionally critical for different motif recognition in all fungal species. Third, target genes identified by RIP-Chip experiments for Puf3p and Puf4p overlap very little (<1%) in S. cerevisiae (Gerber et al. 2004). And fourth, the target genes with P3E or P4E also overlap very little in all studied fungal species (fig. 3C). We envisioned that it might be adaptive to rewire the mitochondrial posttranscriptional gene regulation in the Saccharomycotina clade for higher efficiency, which may fit the requirement of more efficient regulation of the organelle in this group of yeast species (Hayashi et al. 2011). Another alternative scenario which cannot be ruled out is that Puf3p and Puf4p obtained mitochondrial regulation through parallel evolution in the Saccharomycotina and Pezizomycotina clades, respectively. We are currently conducting further experiments in different fungal species to test these hypotheses.

How genetic novelties are created in nature is a fundamental issue in evolutionary biology. Functional rewiring is one principal mechanism to generate genetic novelties by reusing preexisting modules (Jacob 1977; Jacob 2001; Long 2001; Carroll 2005). In nature, functional rewiring has been frequently used to reshape transcriptional networks (Rodriguez-Caso et al. 2005; Lavoie et al. 2010). Similar evolutionary processes also occurred in reorganizing protein domains (Long and Langley 1993; Bockaert and Pin 1999; Long et al. 2003; Kleine and Leister 2007), reprogramming DNA methylation (Mandrioli 2004), and rewiring gene function by reusing transposable elements (Jordan 2006). Our results show another way to rewire regulation at the posttranscriptional level. Together with our previous study on the evolution of Puf3p targets in fungi (Jiang et al. 2010), our results reveal new principles governing the evolution of novel posttranscriptional regulation and provide a new perspective on understanding how gene regulation evolves in nature.

Supplementary Material

Supplementary figures 1–4 and tables 1–3 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).

Supplementary Data
supp_29_9_2169__index.html (1.3KB, html)

Acknowledgments

We thank Jian Lu and Hong Chen for discussions, and Jun Sun and Brandon Barker for reading the manuscript and their helpful comments. We are also very grateful to the editor and three reviewers for their valuable comments for the earlier version of this paper. This work was supported by startup funds from Cornell University, National Science Foundation grant DEB-0949556, and National Institutes of Health 1R01AI085286 awarded to Z.G.

References

  1. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B. 1995;57:289–300. [Google Scholar]
  2. Bockaert J, Pin JP. Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO J. 1999;18:1723–1729. doi: 10.1093/emboj/18.7.1723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borneman AR, Gianoulis TA, Zhang ZD, Yu H, Rozowsky J, Seringhaus MR, Wang LY, Gerstein M, Snyder M. Divergence of transcription factor binding sites across related yeast species. Science. 2007;317:815–819. doi: 10.1126/science.1140748. [DOI] [PubMed] [Google Scholar]
  4. Byrne KP, Wolfe KH. The Yeast Gene Order Browser: combining curated homology and syntenic context reveals gene fate in polyploid species. Genome Res. 2005;15:1456–1461. doi: 10.1101/gr.3672305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carroll SB. Evolution at two levels: on genes and form. PLoS Biol. 2005;3:e245. doi: 10.1371/journal.pbio.0030245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen K, Rajewsky N. The evolution of gene regulation by transcription factors and microRNAs. Nat Rev Genet. 2007;8:93–103. doi: 10.1038/nrg1990. [DOI] [PubMed] [Google Scholar]
  7. Chritton JJ, Wickens M. Translational repression by PUF proteins in vitro. RNA. 2010;16:1217–1225. doi: 10.1261/rna.2070110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crittenden SL, Bernstein DS, Bachorik JL, Thompson BE, Gallegos M, Petcherski AG, Moulder G, Barstead R, Wickens M, Kimble J. A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans. Nature. 2002;417:660–663. doi: 10.1038/nature754. [DOI] [PubMed] [Google Scholar]
  9. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–1797. doi: 10.1093/nar/gkh340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Emerson JJ, Hsieh LC, Sung HM, Wang TY, Huang CJ, Lu HH, Lu MY, Wu SH, Li WH. Natural selection on cis and trans regulation in yeasts. Genome Res. 2010;20:826–836. doi: 10.1101/gr.101576.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fitzpatrick DA, Logue ME, Stajich JE, Butler G. A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis. BMC Evol Biol. 2006;6:99. doi: 10.1186/1471-2148-6-99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Foat BC, Houshmandi SS, Olivas WM, Bussemaker HJ. Profiling condition-specific, genome-wide regulation of mRNA stability in yeast. Proc Natl Acad Sci U S A. 2005;102:17675–17680. doi: 10.1073/pnas.0503803102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Frankel N, Davis GK, Vargas D, Wang S, Payre F, Stern DL. Phenotypic robustness conferred by apparently redundant transcriptional enhancers. Nature. 2010;466:490–493. doi: 10.1038/nature09158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galgano A, Forrer M, Jaskiewicz L, Kanitz A, Zavolan M, Gerber AP. Comparative analysis of mRNA targets for human PUF-family proteins suggests extensive interaction with the miRNA regulatory system. PLoS One. 2008;3:e3164. doi: 10.1371/journal.pone.0003164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gerber AP, Herschlag D, Brown PO. Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast. PLoS Biol. 2004;2:E79. doi: 10.1371/journal.pbio.0020079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gerber AP, Luschnig S, Krasnow MA, Brown PO, Herschlag D. Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2006;103:4487–4492. doi: 10.1073/pnas.0509260103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Goldstrohm AC, Hook BA, Seay DJ, Wickens M. PUF proteins bind Pop2p to regulate messenger RNAs. Nat Struct Mol Biol. 2006;13:533–539. doi: 10.1038/nsmb1100. [DOI] [PubMed] [Google Scholar]
  18. Grigull J, Mnaimneh S, Pootoolal J, Robinson MD, Hughes TR. Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors. Mol Cell Biol. 2004;24:5534–5547. doi: 10.1128/MCB.24.12.5534-5547.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hayashi M, Schilke B, Marszalek J, Williams B, Craig EA. Ancient gene duplication provided a key molecular step for anaerobic growth of Baker's yeast. Mol Biol Evol. 2011;28:2005–2017. doi: 10.1093/molbev/msr019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hogan DJ, Riordan DP, Gerber AP, Herschlag D, Brown PO. Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system. PLoS Biol. 2008;6:e255. doi: 10.1371/journal.pbio.0060255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hook BA, Goldstrohm AC, Seay DJ, Wickens M. Two yeast PUF proteins negatively regulate a single mRNA. J Biol Chem. 2007;282:15430–15438. doi: 10.1074/jbc.M611253200. [DOI] [PubMed] [Google Scholar]
  22. Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, O'Shea EK. Global analysis of protein localization in budding yeast. Nature. 2003;425:686–691. doi: 10.1038/nature02026. [DOI] [PubMed] [Google Scholar]
  23. Ihmels J, Bergmann S, Gerami-Nejad M, Yanai I, McClellan M, Berman J, Barkai N. Rewiring of the yeast transcriptional network through the evolution of motif usage. Science. 2005;309:938–940. doi: 10.1126/science.1113833. [DOI] [PubMed] [Google Scholar]
  24. Jacob F. Evolution and tinkering. Science. 1977;196:1161–1166. doi: 10.1126/science.860134. [DOI] [PubMed] [Google Scholar]
  25. Jacob F. Complexity and tinkering. Ann N Y Acad Sci. 2001;929:71–73. doi: 10.1111/j.1749-6632.2001.tb05708.x. [DOI] [PubMed] [Google Scholar]
  26. Jiang H, Guan W, Gu Z. Tinkering evolution of post-transcriptional RNA regulons: puf3p in fungi as an example. PLoS Genet. 2010;6:e1001030. doi: 10.1371/journal.pgen.1001030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jordan IK. Evolutionary tinkering with transposable elements. Proc Natl Acad Sci U S A. 2006;103:7941–7942. doi: 10.1073/pnas.0602656103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kadyrova LY, Habara Y, Lee TH, Wharton RP. Translational control of maternal Cyclin B mRNA by Nanos in the Drosophila germline. Development. 2007;134:1519–1527. doi: 10.1242/dev.002212. [DOI] [PubMed] [Google Scholar]
  29. Kaplan T, Li XY, Sabo PJ, Thomas S, Stamatoyannopoulos JA, Biggin MD, Eisen MB. Quantitative models of the mechanisms that control genome-wide patterns of transcription factor binding during early Drosophila development. PLoS Genet. 2011;7:e1001290. doi: 10.1371/journal.pgen.1001290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Keene JD. RNA regulons: coordination of post-transcriptional events. Nat Rev Genet. 2007;8:533–543. doi: 10.1038/nrg2111. [DOI] [PubMed] [Google Scholar]
  31. King MC, Wilson AC. Evolution at two levels in humans and chimpanzees. Science. 1975;188:107–116. doi: 10.1126/science.1090005. [DOI] [PubMed] [Google Scholar]
  32. Kleine T, Leister D. Evolutionary tinkering: birth of a novel chloroplast protein. Biochem J. 2007;403:e13–14. doi: 10.1042/BJ20070312. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Larkin MA, Blackshields G, Brown NP, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23:2947–2948. doi: 10.1093/bioinformatics/btm404. [DOI] [PubMed] [Google Scholar]
  34. Lavoie H, Hogues H, Mallick J, Sellam A, Nantel A, Whiteway M. Evolutionary tinkering with conserved components of a transcriptional regulatory network. PLoS Biol. 2010;8:e1000329. doi: 10.1371/journal.pbio.1000329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lee D, Ohn T, Chiang YC, Quigley G, Yao G, Liu Y, Denis CL. PUF3 acceleration of deadenylation in vivo can operate independently of CCR4 activity, possibly involving effects on the PAB1-mRNP structure. J Mol Biol. 2010;399:562–575. doi: 10.1016/j.jmb.2010.04.034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lin H, Spradling AC. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. Development. 1997;124:2463–2476. doi: 10.1242/dev.124.12.2463. [DOI] [PubMed] [Google Scholar]
  37. Long M. Evolution of novel genes. Curr Opin Genet Dev. 2001;11:673–680. doi: 10.1016/s0959-437x(00)00252-5. [DOI] [PubMed] [Google Scholar]
  38. Long M, Betran E, Thornton K, Wang W. The origin of new genes: glimpses from the young and old. Nat Rev Genet. 2003;4:865–875. doi: 10.1038/nrg1204. [DOI] [PubMed] [Google Scholar]
  39. Long M, Langley CH. Natural selection and the origin of jingwei, a chimeric processed functional gene in Drosophila. Science. 1993;260:91–95. doi: 10.1126/science.7682012. [DOI] [PubMed] [Google Scholar]
  40. Mandrioli M. Epigenetic tinkering and evolution: is there any continuity in the role of cytosine methylation from invertebrates to vertebrates? Cell Mol Life Sci. 2004;61:2425–2427. doi: 10.1007/s00018-004-4184-y. [DOI] [PubMed] [Google Scholar]
  41. Mata J, Marguerat S, Bahler J. Post-transcriptional control of gene expression: a genome-wide perspective. Trends Biochem Sci. 2005;30:506–514. doi: 10.1016/j.tibs.2005.07.005. [DOI] [PubMed] [Google Scholar]
  42. Miller MT, Higgin JJ, Hall TM. Basis of altered RNA-binding specificity by PUF proteins revealed by crystal structures of yeast Puf4p. Nat Struct Mol Biol. 2008;15:397–402. doi: 10.1038/nsmb.1390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Morris AR, Mukherjee N, Keene JD. Ribonomic analysis of human Pum1 reveals cis-trans conservation across species despite evolution of diverse mRNA target sets. Mol Cell Biol. 2008;28:4093–4103. doi: 10.1128/MCB.00155-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Murata Y, Wharton RP. Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos. Cell. 1995;80:747–756. doi: 10.1016/0092-8674(95)90353-4. [DOI] [PubMed] [Google Scholar]
  45. Prokisch H, Andreoli C, Ahting U, Heiss K, Ruepp A, Scharfe C, Meitinger T. MitoP2: the mitochondrial proteome database–now including mouse data. Nucleic Acids Res. 2006;34:D705–711. doi: 10.1093/nar/gkj127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Quenault T, Lithgow T, Traven A. PUF proteins: repression, activation and mRNA localization. Trends Cell Biol. 2011;21:104–112. doi: 10.1016/j.tcb.2010.09.013. [DOI] [PubMed] [Google Scholar]
  47. Remm M, Storm CE, Sonnhammer EL. Automatic clustering of orthologs and in-paralogs from pairwise species comparisons. J Mol Biol. 2001;314:1041–1052. doi: 10.1006/jmbi.2000.5197. [DOI] [PubMed] [Google Scholar]
  48. Rodriguez-Caso C, Medina MA, Sole RV. Topology, tinkering and evolution of the human transcription factor network. FEBS J. 2005;272:6423–6434. doi: 10.1111/j.1742-4658.2005.05041.x. [DOI] [PubMed] [Google Scholar]
  49. Rozpedowska E, Hellborg L, Ishchuk OP, Orhan F, Galafassi S, Merico A, Woolfit M, Compagno C, Piskur J. Parallel evolution of the make-accumulate-consume strategy in Saccharomyces and Dekkera yeasts. Nat Commun. 2011;2:302. doi: 10.1038/ncomms1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Shou C, Bhardwaj N, Lam HY, Yan KK, Kim PM, Snyder M, Gerstein MB. Measuring the evolutionary rewiring of biological networks. PLoS Comput Biol. 2011;7:e1001050. doi: 10.1371/journal.pcbi.1001050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sogin ML, Gunderson JH, Elwood HJ, Alonso RA, Peattie DA. Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science. 1989;243:75–77. doi: 10.1126/science.2911720. [DOI] [PubMed] [Google Scholar]
  52. Spassov DS, Jurecic R. The PUF family of RNA-binding proteins: does evolutionarily conserved structure equal conserved function? IUBMB Life. 2003;55:359–366. doi: 10.1080/15216540310001603093. [DOI] [PubMed] [Google Scholar]
  53. Sun J, Jiang H, Flores R, Wen J. Gene duplication in the genome of parasitic Giardia lamblia. BMC Evol Biol. 2010;10:49. doi: 10.1186/1471-2148-10-49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tam PP, Barrette-Ng IH, Simon DM, Tam MW, Ang AL, Muench DG. The Puf family of RNA-binding proteins in plants: phylogeny, structural modeling, activity and subcellular localization. BMC Plant Biol. 2010;10:44. doi: 10.1186/1471-2229-10-44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–2739. doi: 10.1093/molbev/msr121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tanay A, Regev A, Shamir R. Conservation and evolvability in regulatory networks: the evolution of ribosomal regulation in yeast. Proc Natl Acad Sci U S A. 2005;102:7203–7208. doi: 10.1073/pnas.0502521102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25:4876–4882. doi: 10.1093/nar/25.24.4876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Wickens M, Bernstein DS, Kimble J, Parker R. A PUF family portrait: 3'UTR regulation as a way of life. Trends Genet. 2002;18:150–157. doi: 10.1016/s0168-9525(01)02616-6. [DOI] [PubMed] [Google Scholar]
  59. Woodson JD, Chory J. Coordination of gene expression between organellar and nuclear genomes. Nat Rev Genet. 2008;9:383–395. doi: 10.1038/nrg2348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wray GA. The evolutionary significance of cis-regulatory mutations. Nat Rev Genet. 2007;8:206–216. doi: 10.1038/nrg2063. [DOI] [PubMed] [Google Scholar]
  61. Wray GA, Hahn MW, Abouheif E, Balhoff JP, Pizer M, Rockman MV, Romano LA. The evolution of transcriptional regulation in eukaryotes. Mol Biol Evol. 2003;20:1377–1419. doi: 10.1093/molbev/msg140. [DOI] [PubMed] [Google Scholar]

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