Fig. 6

Polysome profiles of proliferating, quiescent, and differentiated muscle cells reveal stalled polysomes in G0. Translational profiles of myoblasts (a), myotubes (b), and G0 cells (c) using polysome display on sucrose gradients. Panels on left depict profiles derived from cells briefly treated with CHX to ‘fix’ ribosomes in the act of translation, while panels on the right depict profiles derived from cells treated with Puro to disrupt translation by mRNA release. Western blotting of proteins isolated from 9 individual 1-ml fractions from the sucrose gradients (equal volumes loaded) reveals (i) distribution of ribosomes in each fraction based on ribosomal protein P0 (middle) and the extent of association of decay complex based on Xrn1 (top), and translation inhibitory complex based on Fmrp (botttom) with each fraction. Comparison of the profiles and distribution of individual proteins reveals very poor translation in G0, correlating with the OPP incorporation in Fig. 5. The presence of puromycin-insensitive complexes in G0 arrested cells, suggests polysome stalling. d Analysis of transcript distribution in polysome profiles correlates with rate of protein synthesis and suggests low mRNA utilization in G0. qRT-PCR analysis of selected transcripts (GAPDH, Cyclin D1, MyoD, Myf5, and p27) from RNA isolated from the mRNP-, monosome-, and polysome-containing fractions of profiles depicted in Fig. 6a–c. All transcripts tested show substantial enrichment in the mRNP and monosome compartment in G0 compared with the monosome and polysome fraction, suggesting a severe suppression of protein synthesis consistent with the OPP incorporation study (Fig. 5b, c). Notably, none of the transcripts tested show appreciable enrichment in the mRNP fraction in MB and MT, indicating their robust translational utilization in the polysomal compartment. Values represent the mean + SD of transcript levels in fractions from two independent polysome profiles for each condition