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: PUNCH-P; cell cycle; protein synthesis; proteomics; puromycin; translation.

Figure 1.

Experimental setup of PUNCH-P. Ribosomes are extracted from cultured cells by ultracentrifugation on a sucrose cushion. Ribosomes are then incubated with the labeling reagent Biot-PU, and labeled newly synthesized proteins are isolated by streptavidin affinity purification and analyzed by LC-MS/MS.

Figure 2.

Biochemical characterization of PUNCH-P. (A,B) Newly synthesized proteins were labeled by Biot-PU for the indicated times and under the indicated conditions and detected by Western blotting using streptavidin-HRP. (C) Newly synthesized proteins labeled by Biot-PU were fractionated on a linear 10%–50% sucrose gradient, and fractions corresponding to the entire range of monosomes to heavy polysomes were collected and probed by Western blotting. (Top panel) rRNA absorption at 254 nm, showing the positions of 80S monosome and polysomes. (Bottom panel) Western blotting of gradient fractions using streptavidin-HRP or anti-ribosomal protein L26 antibody. Vertical line traces on the right represent the densitometry of lane 5 (light polysomes) and lane 10 (heavy polysomes).

Figure 3.

PUNCH-P analysis of cycling HeLa cells. (A) Puromycylation and control samples were incubated with streptavidin beads under stringent conditions, and puromycylated newly synthesized proteins were eluted following extensive washing. Equal volumes of starting material (input), flowthrough (FT), wash, and eluate were analyzed by Western blotting. Ribosomal protein L26 is shown as control for removal of ribosomes from the eluate. (B) Volcano plot of proteins identified by MS in puromycylated compared with control samples. (C) Scatter plot showing the high technical reproducibility of PUNCH-P.

Figure 4.

Comparison of PUNCH-P, 10-h pSILAC, and ribo-seq. (A) Heat map showing Pearson correlation between the methods for measuring protein synthesis and HeLa whole-cell proteome. (B) Representative scatter plots show the correlation between HeLa whole-cell proteome and PUNCH-P, 10-h pSILAC, and ribo-seq.

Figure 5.

Cell cycle-related dynamics in protein synthesis. Hierarchical clustering of proteins was performed on logarithmized intensities after Z-score normalization of the data. The heat map shows proteins with statistically significant differences in synthesis throughout the cell cycle. Selected proteins from each cluster are indicated on the right. Flow cytometry analysis of DNA content using propidium iodide is shown in the bottom panel. Respective cell cycle stages are indicated, with time after release from second thymidine block in parenthesis and percentage of cells in each phase shown below.

Figure 6.

qPCR validation of PUNCH-P results. (A, top panel) Polysome profiles of HeLa cells synchronized to S and M phase by double-thymidine block. (Bottom panel) Total RNA extracted from each of the polysomal fractions visualized by ethidium bromide staining. (B,C) Polysomal association of nonfluctuating mRNAs encoding for CALR (B) and CYCS (C). (D) Polysomal association of mRNAs encoding for proteins that were elevated in S phase according to PUNCH-P. (E) Polysomal association of mRNAs encoding for proteins that were elevated in M phase according to PUNCH-P. Graphs show mean ± SD of qPCR replicates for one of three independent experiments with similar results. C–E represent qPCR results normalized to CALR.