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Fig. 3 | Genome Biology

Fig. 3

From: A rare codon-based translational program of cell proliferation

Fig. 3

Rare codons confer increased translation efficiency in rapidly dividing cells. a Starting from the rapid turnover rate protein d2eGFP [48], we designed two fluorescent reporters, encoded with codons that are more frequently used in either G1- or G2/M-enriched mRNAs. Yellow box corresponds to the PEST sequence attached to the C-terminal region of the eGFP protein. b G2M/G1 codon score (Fig. 1c) for each codon of the d2eGFP_G1 (top) and d2eGFP_G2M (bottom) reporters. The first and last 17 codons (51 nucleotides) of eGFP, as well as the PEST sequence, were left unchanged (gray regions) to avoid effects on translation initiation and termination. c Mean (± s.e.m.) G2M/G1 codon score for the re-coded region in the 2 fluorescent reporters. d Mean (± s.e.m.) percentage of cells in the G1 and G2/M phases of the cell cycle, as determined by Hoechst staining for DNA content, for the d2eGFP_G1 and d2eGFP_G2M cell lines grown in either 1% or 10% FCS (n = 6). e Representative result of the GFP fluorescence intensity measurement by flow cytometry in the 2 cell lines grown in either 1% or 10% FCS (left). Mean (± s.e.m.) ratio of fluorescence intensity for cells grown in 10% relative to 1% FCS for the two reporters (n = 6) (right). Shown is the P value determined by the non-parametric Mann-Whitney U test. f Mean (± s.e.m.) ratio of transcript abundance, as measured by qRT-PCR, for cells grown in 10% relative to 1% FCS for the 2 reporter cell lines (n = 4) (right). Shown is the P value determined by the non-parametric Mann-Whitney U test. g Representative result of HPG incorporation measurement (a.u., arbitrary units) by flow cytometry in the 2 cell lines growing in either 1% or 10% FCS (left). Quantification of the mean (± s.e.m.) ratio of HPG incorporation in 10% compared to 1% FCS for the 2 reporter cell lines (n = 5) (right). For control, we also incubated wild-type NIH-3T3 cells with the translation inhibitor cycloheximide (CHX). Shown are the P values determined by Dunn’s multiple comparisons test post hoc and the non-parametric Kruskal-Wallis test (P = 0.0018)

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