Characterizing inactive ribosomes in translational profiling

Abstract

The broad impact of translational regulation has emerged explosively in the last few years in part due to the technological advance in genome-wide interrogation of gene expression. During mRNA translation, the majority of actively translating ribosomes exist as polysomes in cells with multiple ribosomes loaded on a single transcript. The importance of the monosome, however, has been less appreciated in translational profiling analysis. Here we report that the monosome fraction isolated by sucrose sedimentation contains a large quantity of inactive ribosomes that do not engage on mRNAs to direct translation. We found that the elongation factor eEF2, but not eEF1A, stably resides in these non-translating ribosomes. This unique feature permits direct evaluation of ribosome status under various stress conditions and in the presence of translation inhibitors. Ribosome profiling reveals that the monosome has a similar but not identical pattern of ribosome footprints compared to the polysome. We show that the association of free ribosomal subunits minimally contributes to ribosome occupancy outside of the coding region. Our results not only offer a quantitative method to monitor ribosome availability, but also uncover additional layers of ribosome status needed to be considered in translational profiling analysis.

Eukaryotic elongation factor 2 kinase upregulates the expression of proteins implicated in cell migration and cancer cell metastasis

“…b ), likely reflecting the expected acceleration of elongation and thus ribosomal “run‐off” when eEF2K is knocked down. Consistent with recent findings, eEF2 is mainly associated with inactive ribosomes, although this binding was unaffected by its phosphorylation status (Figs. c and d ; Supporting Information Fig.…”

Polysome fractionation analysis reveals features important for human nonsense-mediated mRNA decay

“…We then estimated the proportion of rRNA to non-ribosomal RNA molecules for each fraction with these RNA lengths as follows: For the monosome fraction, it should be one ribosome on each non-ribosomal RNA transcript. However, approximately half of monosomes are empty (Liu & Qian, 2016), therefore we estimate two ribosomes to each transcript. The same ratio is applied to the polysome 2 fraction.…”

Regulation of eIF6 through multisite and sequential phosphorylation by GSK3β

“…The metabolic response to starvation is to conserve energy and limit processes with high-energy requirements such as translation and ribosome biogenesis [22][23][24][25][26][27][28][29][30][31] . Here, we provide a novel link between starvation response and regulation of both translation initiation and ribosome availability through the control of translation initiation factor eIF6.…”

“…In terms of mechanism, the increased formation of inactive 80S complexes in the eIF6+/-cells is attributed to an impairment of its anti-association function in the cytoplasm [17][18][19] . Interestingly, such an accumulation of inactive 80S monosomes is commonly observed in cells subjected to stress, especially stress induced by nutrient deprivation or limitation [22][23][24] . Starvation or nutrient limitation in yeast and mammalian cells invokes an adaptive metabolic response that conserves energy by restricting global protein synthesis and leads to an accumulation of inactive (empty) 80S ribosomes and can also increase the pool of free 60S subunits [22][23][24][25][26][27][28][29][30][31][32][33] .…”