Published online 12 August 2010 Nucleic Acids Research, 2010, Vol. 38, No. 22 8039–8050 doi:10.1093/nar/gkq686 Identifying eIF4E-binding protein translationally- controlled transcripts reveals links to mRNAs bound by specific PUF proteins Andrew G. Cridge 1 , Lydia M. Castelli1, Julia B. Smirnova 1 , Julian N. Selley1, William Rowe 1 , Simon J. Hubbard1, John E.G. McCarthy2, Mark P. Ashe 1 , Christopher M. Grant1 and Graham D. Pavitt1,* 1The Michael Smith Building and 2Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, Manchester, UK Received July 15, 2009; Revised July 12, 2010; Accepted July 20, 2010 ABSTRACT eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed transla- tional profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global com- parison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs re- vealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1D and caf20D cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chroma- tography experiments to uncover specific RNA- stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 30-UTR-binding proteins mediates mRNA- specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought. INTRODUCTION It has become increasingly evident that the translation process for a large number of mRNAs is controlled at the level of initiation in a wide variety of cells. The selec- tion of mRNAs for translation is mediated by the binding of eukaryotic initiation factor (eIF) 4F complex to the 50-end of each mRNA. The eIF4F complex is composed of eIF4E that binds to the 50 7-methyl guanosine mRNA cap, eIF4A an RNA helicase, and eIF4G a scaffolding protein capable of bridging contacts between eIF4E and 40S ribosomal subunits. eIF4G and the eIF4E-binding proteins (4E-BPs) share a motif (YXXXXLf, where X is any residue and f is hydrophobic) that is critical for eIF4E interaction. Hence 4E-BP bound mRNAs exclude eIF4G and are translationally silenced (1,2). Mammalian 4E-BP1 phosphorylation status modulates its afﬁnity for eIF4E. mTOR dependent hyperphosphorylation of 4E-BP1 causes its dissociation from eIF4E relieving re- pression and promoting eIF4F assembly, translation and cell growth (3,4). A second class of 4E-BP has been identiﬁed from studies of early development, e.g. in Xenopus and Drosophila where asymmetric expression of proteins is important for determining the body plan of the embryo (2,5). For these 4E-BPs additional protein–protein and protein–mRNA contacts tether the 4E-BP to speciﬁc mRNA targets. Thus Xenopus Maskin is a 4E-BP that also binds to the cytoplasmic polyadenylation element-binding (CPEB) protein which itself interacts with sequences within mRNA 30-UTRs. Together and in combination with other proteins, these factors mediate circularization and repression of transcripts until developmentally *To whom correspondence should be addressed. Tel: (+44/0) 161 306 4477; Fax: (+44/0) 161 275 5082; Email: graham.pavitt@manchester.ac.uk Present address: Andrew G. Cridge, Massey University, Institute of Natural Sciences, Albany, New Zealand. The authors wish it to be known that, in their opinion, the ﬁrst three authors should be regarded as joint First Authors. The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.