Translatome profiling: methods for genome-scale analysis of mRNA translation

Abstract

During the past decade, there has been a rapidly increased appreciation of the role of translation as a key regulatory node in gene expression. Thereby, the development of methods to infer the translatome, which refers to the entirety of mRNAs associated with ribosomes for protein synthesis, has facilitated the discovery of new principles and mechanisms of translation and expanded our view of the underlying logic of protein synthesis. Here, we review the three main methodologies for translatome analysis, and we highlight some of the recent discoveries made using each technique. We first discuss polysomal profiling, a classical technique that involves the separation of mRNAs depending on the number of bound ribosomes using a sucrose gradient, and which has been combined with global analysis tools such as DNA microarrays or high-throughput RNA sequencing to identify the RNAs in polysomal fractions. We then introduce ribosomal profiling, a recently established technique that enables the mapping of ribosomes along mRNAs at near-nucleotide resolution on a global scale. We finally refer to ribosome affinity purification techniques that are based on the cell-type-specific expression of tagged ribosomal proteins, allowing the capture of translatomes from specialized cells in organisms. We discuss the advantages and disadvantages of these three main techniques in the pursuit of defining the translatome, and we speculate about future developments.

nanoCAGE reveals 5′ UTR features that define specific modes of translation of functionally related MTOR-sensitive mRNAs

“…In polysome-profiling, translation efficiency is directly determined by separating efficiently (commonly defined as mRNAs associated with more than three ribosomes) and not efficiently translated mRNAs (defined as mRNAs associated with three or less ribosomes) (Gandin et al 2014). In contrast, with ribosomeprofiling translational efficiency is inferred based on the number of RNA sequencing reads corresponding to ribosome-protected fragments (Gandin et al 2014;Ingolia 2014;King and Gerber 2014). TOP mRNAs are highly expressed (Fig.…”

“…However, there are a number of challenges regarding interpretation of ribosome-profiling (Guttman et al 2013;Gerashchenko and Gladyshev 2014;King and Gerber 2014;Lareau et al 2014;Pelechano et al 2015). The relative performance of ribosome-vs. polysome-profiling to study changes in translation efficiency also remains unclear.…”

Linking transcriptomics and proteomics in spermatogenesis

“…Interestingly, the expression of these tagged proteins can be driven by a tissue/cell-specific promoter, such as the Cre-lox system in mice, which enables the capture of tagged ribosomes from an entire organ or tissue without the need to isolate the cells of interest (for review, see King & Gerber (2014)). These methodologies, initially called translating ribosome affinity purification (TRAP) in the mouse (Doyle et al 2008, have been used several times to capture the translatome of various testicular cell types.…”

Super-resolution ribosome profiling reveals unannotated translation events inArabidopsis

“…Until recently, genome-wide mapping of translation has relied primarily on polysome profiling (1). This involves isolation and separation of polysome-associated mRNA through differential centrifugation and fractionation.…”