Nucleic Acids Research Advance Access published October 7, 2016 Nucleic Acids Research, 2016 1 doi: 10.1093/nar/gkw907 Analysis of translation using polysome profiling Helo´ ¨ıse Chasse´ 1,2,†, Sandrine Boulben1,2,†, Vlad Costache1,2, Patrick Cormier1,2 and Julia Morales1,2,*
1CNRS, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, F-29688, Roscoff cedex, France and 2Sorbonne Universites,´ UPMC Univ Paris 06, UMR 8227, Integrative Biology of Marine Models, Station Biologique de Roscoff, CS 90074, F-29688, Roscoff cedex, France
Received September 16, 2015; Revised September 7, 2016; Accepted September 30, 2016
ABSTRACT of the genome at a given time in a given cell type. There are various techniques for specifically studying translated During the past decade, there has been growing in- mRNAs, of which polysome profiling is the most common. terest in the role of translational regulation of gene Polysome profiling is based on sucrose-gradient separation expression in many organisms. Polysome profiling of translated mRNAs, which are associated with polysomes, Downloaded from has been developed to infer the translational status from untranslated ones. More recently, other techniques of a specific mRNA species or to analyze the trans- have emerged such as ribosome profiling and translating ri- latome, i.e. the subset of mRNAs actively translated bosome affinity purification (TRAP) (reviewed8 in( )). Ri- in a cell. Polysome profiling is especially suitable for bosome profiling measures translation by deep-sequencing emergent model organisms for which genomic data ribosome-protected mRNA fragments. This technique also http://nar.oxfordjournals.org/ are limited. In this paper, we describe an optimized determines the position of the ribosome at codon resolu- protocol for the purification of sea urchin polysomes tion, allowing discoveries of new coding transcripts and protein isoforms as well as accurate measurement of trans- and highlight the critical steps involved in polysome lation rates (9). TRAP is suitable for analyzing translation purification. We applied this protocol to obtain ex- in a specific cell type. Genetically modified cells or organ- perimental results on translational regulation of mR- isms are engineered to express a tagged ribosomal protein in NAs following fertilization. Our protocol should prove vivo, under the control of a tissue-specific promoter. Tagged useful for integrating the study of the role of transla- ribosomes are affinity-purified and associated mRNAs are tional regulation in gene regulatory networks in any identified by microarray or deep-sequencing (10). by guest on October 7, 2016 biological model. In addition, we demonstrate how to Among these three main methods for mRNA translation carry out high-throughput processing of polysome analysis, polysome profiling has several interesting appli- gradient fractions, for the simultaneous screening of cations. Polysome profiling can be used for targeted anal- multiple biological conditions and large-scale prepa- yses on specific mRNAs (11–13) as well as for holistic anal- ration of samples for next-generation sequencing. yses (14–17). Furthermore, this technique gives access to the full-length translated mRNAs, including the untrans- lated regions (UTRs), in contrast to ribosome profiling in INTRODUCTION which the protected fragments map to coding sequences Gene expression is regulated at many levels: at the epi- only. UTRs harbor cis-acting elements that regulate selec- genetic level, at the transcriptional level, at the transla- tive translation (18). Identification of UTRs and conserved tional level and at the post-translational level. Among these cis-acting elements corresponding to translated mRNA iso- levels, translational regulation governs protein production forms are essential for a mechanistic analysis of gene expres- in response to a number of physiological and pathologi- sion regulation. In addition, polysome profiling can be com- cal situations (1,2). Accordingly, almost half of the varia- bined with immunoblotting and/or proteomics to monitor tion of protein concentration is due to translational con- proteins associated with ribosomes and/or initiation com- trol (3). Studying translation leads to a better understand- plexes. Lastly, polysome profiling is especially suitable for ing of gene expression regulation and can explain the dif- model organisms for which genomic data and/or tools are ferences between transcriptome and proteome analyses (4– scarce, and/or for which genetic manipulation is not pos- 7), because the subset of mRNAs actively engaged in trans- sible. However, the broad application of polysome profil- lation (i.e. the translatome) reflects the functional readout ing has been slowed by its complexity and the difficulty of
*To whom correspondence should be addressed. Tel: +33 298 292 369; Fax: +33 298 292 324; Email: [email protected] †These authors contributed equally to the paper as first authors. Present address: Vlad Costache, Laboratoire Biologie du Developpement,´ UMR7009, CNRS, UPMC, 181 Chemin du Lazaret, Observatoire Oceanologique,´ 06230 Villefranche-sur-Mer, France.