RESEARCH ARTICLE Evolutionary Conservation and Diversification of Puf RNA Binding Proteins and Their mRNA Targets Gregory J. Hogan1,2¤a, Patrick O. Brown1,2¤b*, Daniel Herschlag1,3,4,5* 1 Department of Biochemistry, Stanford University School of Medicine, Stanford, California, United States of America, 2 Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California, United States of America, 3 Department of Chemistry, Stanford University, Stanford, California, United States of America, 4 Department of Chemical Engineering, Stanford University, Stanford, California, United States of America, 5 ChEM-H Institute, Stanford University, Stanford, California, United States of America ¤a Current address: Counsyl, South San Francisco, California, United States of America ¤b Current address: Impossible Foods, Redwood City, California, United States of America * [email protected] (POB); [email protected] (DH) Abstract OPEN ACCESS Reprogramming of a gene’s expression pattern by acquisition and loss of sequences recog- Citation: Hogan GJ, Brown PO, Herschlag D (2015) Evolutionary Conservation and Diversification of Puf nized by specific regulatory RNA binding proteins may be a major mechanism in the evolu- RNA Binding Proteins and Their mRNA Targets. tion of biological regulatory programs. We identified that RNA targets of Puf3 orthologs PLoS Biol 13(11): e1002307. doi:10.1371/journal. have been conserved over 100–500 million years of evolution in five eukaryotic lineages. pbio.1002307 Focusing on Puf proteins and their targets across 80 fungi, we constructed a parsimonious Academic Editor: Jeff Coller, Case Western model for their evolutionary history. This model entails extensive and coordinated changes Reserve University, UNITED STATES in the Puf targets as well as changes in the number of Puf genes and alterations of RNA Received: April 3, 2015 binding specificity including that: 1) Binding of Puf3 to more than 200 RNAs whose protein Accepted: October 23, 2015 products are predominantly involved in the production and organization of mitochondrial Published: November 20, 2015 complexes predates the origin of budding yeasts and filamentous fungi and was maintained for 500 million years, throughout the evolution of budding yeast. 2) In filamentous fungi, Copyright: © 2015 Hogan et al. This is an open access article distributed under the terms of the remarkably, more than 150 of the ancestral Puf3 targets were gained by Puf4, with one line- Creative Commons Attribution License, which permits age maintaining both Puf3 and Puf4 as regulators and a sister lineage losing Puf3 as a regu- unrestricted use, distribution, and reproduction in any lator of these RNAs. The decrease in gene expression of these mRNAs upon deletion of medium, provided the original author and source are Puf4 in filamentous fungi (N. crassa) in contrast to the increase upon Puf3 deletion in bud- credited. ding yeast (S. cerevisiae) suggests that the output of the RNA regulatory network is different Data Availability Statement: All relevant data are with Puf4 in filamentous fungi than with Puf3 in budding yeast. 3) The coregulated Puf4 tar- within the paper and its Supporting Information files. Microarray data are also available from Gene get set in filamentous fungi expanded to include mitochondrial genes involved in the tricar- Expression Omnibus (GEO) under the accession boxylic acid (TCA) cycle and other nuclear-encoded RNAs with mitochondrial function not number GSE50997. bound by Puf3 in budding yeast, observations that provide additional evidence for substan- Funding: This work was supported by grants from tial rewiring of post-transcriptional regulation. 4) Puf3 also expanded and diversified its tar- the National Institutes of Health (NIH RO1 CA77097 gets in filamentous fungi, gaining interactions with the mRNAs encoding the mitochondrial to P.O.B. and PO1 066275 to DH). POB is an electron transport chain (ETC) complex I as well as hundreds of other mRNAs with nonmito- investigator for the Howard Hughes Medical Institute. GJH was supported in part by a Burt and Deedee chondrial functions. The many concerted and conserved changes in the RNA targets of Puf McMurtry Stanford Graduate Fellowship and by the proteins strongly support an extensive role of RNA binding proteins in coordinating gene Howard Hughes Medical Institute. The funders had PLOS Biology | DOI:10.1371/journal.pbio.1002307 November 20, 2015 1 / 47 Evolution of Puf Proteins and mRNA Targets no role in study design, data collection and analysis, expression, as originally proposed by Keene. Rewiring of Puf-coordinated mRNA targets decision to publish, or preparation of the manuscript. and transcriptional control of the same genes occurred at different points in evolution, sug- Competing Interests: The authors have declared gesting that there have been distinct adaptations via RNA binding proteins and transcription that no competing interests exist. factors. The changes in Puf targets and in the Puf proteins indicate an integral involvement Abbreviations: ETC, electron transport chain; FDR, of RNA binding proteins and their RNA targets in the adaptation, reprogramming, and func- false discovery rate; GO, gene ontology; IP, tion of gene expression. immunopurification; MCMC, Markov chain Monte Carlo; NNI, Nearest Neighbor Interchange; PDB, Protein Data Bank; Puf, Pumilio–Fem-3-binding factor; RRM, RNA Recognition Motif; SAM, Significance Analysis of Microarrays; SPR, Subtree Author Summary Pruning and Regrafting; TAP-tag, tandem affinity purification tag; TCA, tricarboxylic acid; UTR, We set out to trace the evolutionary history of an RNA binding protein and how its inter- untranslated region; WAG, Whelan and Goldman. actions with targets change over evolution. Identifying this natural history is a step toward understanding the critical differences between organisms and how gene expression pro- grams are rewired during evolution. Using bioinformatics and experimental approaches, we broadly surveyed the evolution of binding targets of a particular family of RNA binding proteins—the Puf proteins, whose protein sequences and target RNA sequences are rela- tively well-characterized—across 99 eukaryotic species. We found five groups of species in which targets have been conserved for at least 100 million years and then took advantage of genome sequences from a large number of fungal species to deeply investigate the con- servation and changes in Puf proteins and their RNA targets. Our analyses identified mul- tiple and extensive reconfigurations during the natural history of fungi and suggest that RNA binding proteins and their RNA targets are profoundly involved in evolutionary reprogramming of gene expression and help define distinct programs unique to each organism. Continuing to uncover the natural history of RNA binding proteins and their interactions will provide a unique window into the gene expression programs of present day species and point to new ways to engineer gene expression programs. Introduction The phenotypic diversity of life on earth results not only from differences in the proteins encoded by each genome but, perhaps even more, from differences in the programs that specify where, when, under what conditions, and at what levels these proteins are expressed. A grand challenge in biology is to understand these gene expression programs. Uncovering the similari- ties in and differences between gene expression programs in related organisms can help reveal fundamental properties of these programs, how they have evolved, how they may be wired and rewired, and ultimately how they can be engineered. The seminal step in gene expression and the focus of much current effort is the initiation of transcription through transcription factors that bind in proximity to genes and regulate the timing and magnitude of RNA synthesis (see [1–7] for reviews). Each transcription factor regu- lates a set of genes, numbering a few to thousands, specified by short DNA sequences that are in proximity to those genes and are recognized by that transcription factor. One major mecha- nism for diversification of gene expression programs is the loss or gain of regulation by individ- ual transcription factors, due to mutations that, respectively, disrupt or create the proximal recognition sequences (see [8–13] for reviews). The binding specificity, regulation, and targets of a transcription factor tend to be conserved over a short evolutionary timescale, but each of these properties has changed over evolution, allowing the regulatory roles of orthologous tran- scription factors to diverge and diversify. PLOS Biology | DOI:10.1371/journal.pbio.1002307 November 20, 2015 2 / 47 Evolution of Puf Proteins and mRNA Targets Evolutionary changes in regulation at the next level of gene expression are virtually unex- plored. After transcription, each messenger RNA (mRNA) undergoes a functional odyssey and can be regulated at steps that include splicing, transport, localization, translation, and decay [14]. RNA binding proteins function in each step, and each mRNA interacts with many RNA binding proteins over its lifetime [15–22]. Each RNA binding protein can recognize a few to thousands of mRNAs, and the target sets of each individual RNA binding protein often share functional themes, encoding proteins involved in a particular biological process or localized to the same part of the cell [15,23–37]. These effects can be described in terms of a model origi- nally referred to as the “RNA operon”