Comparative Analysis of mRNA Targets for Human PUF- Family Proteins Suggests Extensive Interaction with the miRNA Regulatory System Alessia Galgano 1 , Michael Forrer 1 , Lukasz Jaskiewicz 2 , Alexander Kanitz 1 , Mihaela Zavolan 2 , Andre ´ P. Gerber 1 * 1 Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland, 2 Biozentrum, University of Basel, Basel, Switzerland Abstract Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 39-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 39-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs. Citation: Galgano A, Forrer M, Jaskiewicz L, Kanitz A, Zavolan M, et al. (2008) Comparative Analysis of mRNA Targets for Human PUF-Family Proteins Suggests Extensive Interaction with the miRNA Regulatory System. PLoS ONE 3(9): e3164. doi:10.1371/journal.pone.0003164 Editor: Ju ¨rg Ba ¨hler, Wellcome Trust Sanger Institute, United Kingdom Received July 11, 2008; Accepted August 18, 2008; Published September 8, 2008 Copyright: ß 2008 Galgano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: L.J. was supported by SNF grant #3100A0-114001 to M.Z. A.K. was supported by a grant from the Bonizzi-Theler Foundation to A.P.G. A.P.G. is the recipient of a Career Development Award (CDA0048/2005) from the International Human Frontier Science Program Organization (HFSP). Competing Interests: The authors have declared that no competing interests exist. * E-mail: andre ´ .gerber@pharma.ethz.ch Introduction Gene expression is regulated at multiple levels to ensure coordinated synthesis of the cells’ macromolecular components. Besides transcriptional regulation, it is becoming increasingly recognized that control of the post-transcriptional steps has substantial impact on gene expression with widespread physiolog- ical implications [1,2]. This regulation is mediated by hundreds of RNA-binding proteins (RBPs) that are encoded in eukaryotic genomes and bind to sequence/structural elements in mRNAs, and thereby regulate the localization, translation or decay of messages [3–7]. On the other hand, microRNAs (miRNAs), ,22 nucleotide (nt) long RNA molecules, can repress gene expression by base- pairing with sequences in 39-untranslated regions (39-UTRs) of messages and thus inhibit their translation or promote decay [8,9]. The PUmilio-Fem-3-binding factor (PUF) proteins comprise an evolutionarily conserved family of RNA-binding proteins that are implicated in various physiological processes [10,11]. They are defined by the presence of an RNA-binding domain, termed Pumilio- homology domain (Pum-HD), which consists of eight repeats, each of which makes contact with a different RNA base [12–15]. PUF proteins bind to an RNA element that comprises a core ‘UGUR’ tetranucleotide followed by 39-UTR sequences that vary among PUF proteins. In concert with other factors, PUFs repress gene expression by inhibiting translation or promoting decay [16,17,18]. The study of PUF proteins in diverse model organisms revealed widespread roles for these proteins in embryonic development, stem-cell maintenance and neurogenesis [10,11]. In the fruit fly Drosophila melanogaster, Pumilio (Pum) is required for proper anterior/posterior patterning during early embryogenesis by repression of the translation of hunchback mRNA [19]. Further- more, Pum is also involved in the development and migration of primordial germ cells [20,21,22], and it may be implicated in long- term memory formation and neuronal excitability [23,24,25]. In the nematode Caenorhabditis elegans, Fem-3 mRNA Binding Factors 1 and 2 (FBF-1, FBF-2) regulate the germline switch from spermatogenesis to oogenesis by repressing fem-3 mRNA transla- tion [26]. The six yeast Saccharomyces cerevisiae PUF proteins (Puf1p– Puf6p) regulate aging, mating-type switching and mitochondrial function [10,27,28]. Much less is known about the functions of PUF homologs in vertebrates. Two paralogous PUF proteins exist in human, termed Pumilio homolog 1 (PUM1) and Pumilio homolog 2 (PUM2). PUM1 and PUM2 are often co-expressed in diverse tissues PLoS ONE | www.plosone.org 1 September 2008 | Volume 3 | Issue 9 | e3164