Novel proteomic approach (PUNCH-P) reveals cell cycle-specific fluctuations in mRNA translation

Abstract

Monitoring protein synthesis is essential to our understanding of gene expression regulation, as protein abundance is thought to be predominantly controlled at the level of translation. Mass-spectrometric and RNA sequencing methods have been recently developed for investigating mRNA translation at a global level, but these still involve technical limitations and are not widely applicable. In this study, we describe a novel system-wide proteomic approach for direct monitoring of translation, termed puromycin-associated nascent chain proteomics (PUNCH-P), which is based on incorporation of biotinylated puromycin into newly synthesized proteins under cell-free conditions followed by streptavidin affinity purification and liquid chromatography-tandem mass spectrometry analysis. Using PUNCH-P, we measured cell cycle-specific fluctuations in synthesis for >5000 proteins in mammalian cells, identified proteins not previously implicated in cell cycle processes, and generated the first translational profile of a whole mouse brain. This simple and economical technique is broadly applicable to any cell type and tissue, enabling the identification and quantification of rapid proteome responses under various biological conditions.[Keywords: cell cycle; proteomics; translation; protein synthesis; puromycin; PUNCH-P] Supplemental material is available for this article. mRNA translation is a key step in gene expression that attracts increasing attention at the systems biology level. In past decades, major efforts were invested in studying transcription regulation, while research focusing on posttranscriptional control has lagged behind. Although mRNA levels are commonly used as a proxy of protein amounts, comparative genomic and proteomic analyses of different species and cell types have shown that mRNA and protein levels do not correlate perfectly, thereby stressing the important contribution of translation control and protein stability to gene expression (Vogel and Marcotte 2012). This provides cells with the plasticity needed to rapidly modulate gene expression in response to changes in environmental conditions (e.g., cellular stress) and also for finetuning of protein levels during cell cycle progression, proliferation, and differentiation (Calkhoven et al. 2002;Holcik and Sonenberg 2005). The ability to identify and quantify the proteins produced in a population of cells under various conditions is therefore essential to our understanding of the processes underlying gene expression.Traditionally, translation rates have been monitored by metabolic labeling of cells with radioactive amino acids. For high-resolution identification and quantification of proteins, metabolic labeling was combined with mass spectrometric (MS) analysis, and the radioactive amino acids were replaced by stable isotope-labeled (SILAC [stable isotope labeling by amino acids in cell culture]) amino acids. To distinguish newly synthesized from preexisting proteins, cells are pulse-labeled with SILAC amino acids, allowing only proteins produced during th...

Next-generation analysis of gene expression regulation – comparing the roles of synthesis and degradation

“…For mammalian cells, both a computational study in 2010 and an experimental approach in 2011 concluded that “transcription is only half the story” 37 and protein-level regulation may be as important as that of the RNA-level 23, 38 . Similarly, in synchronized cells at different cell cycle stages, post-transcriptional regulation has been observed for much of the proteome 39 . However, these findings have been disputed, and reanalysis of the 2011 data showed that transcription may indeed do the majority of the regulatory workload, accounting for 56 to 81% of the overall variation in gene expression 40 .…”

Dynamic profiling of the protein life cycle in response to pathogens

“…, time point of rate change). This would require finer-resolution data, such as from ribosome profiling (49, 54, 55), puromycin-associated nascent chain proteomics (56), or combining pulsed-SILAC labeling with pulse-labeling using the methionine analogue azidohomoalanine (33, 57). Such enhanced methods will provide a framework to study the contributions of the protein life cycle in diverse dynamic systems and help identify new key regulators of these responses.…”

Mitotic Phosphorylation of Eukaryotic Initiation Factor 4G1 (eIF4G1) at Ser1232 by Cdk1:Cyclin B Inhibits eIF4A Helicase Complex Binding with RNA

“…Thus, our approach almost completely replaces endogenous eIF4G with a mutated variant at constant expression levels. Second, we assessed global translation by labeling with the translation elongation inhibitor puromycin (PMY [42]). Conventional metabolic labeling with Met analogs requires Met starvation, which elicits stress responses with effects on protein synthesis.…”