Translational Activity Controls Ribophagic Flux and Turnover of Distinct Ribosome Pools

Abstract

SummaryRibosomes are among the most abundant and complex machineries in the cell, however, the turnover of their subunits remains poorly understood. Here, we apply proteomic flux and cryo-electron microscopy analyses to interrogate the ribosome life cycle in human cells. We show that subpopulations of ribosomal subunits coexist, which vary in turnover kinetics and structure. Specifically, 80S ribosomes have a much longer half-life than free 40S and 60S ribosomal subunits, indicating that they represent distinct subunit pools that poorly intermix. Translation inhibition starkly increases the pool-size of 80S ribosomes in a translationally idle state and induces ribophagy of old ribosomes, ultimately rejuvenating the ribosome fleet. Our findings provide a comprehensive model for ribosome turnover and its regulation via translational activity.

PEPseq Quantifies Transcriptome-Wide Changes in Protein Occupancy and Reveals Selective Translational Repression After Translational Stress

“…Indeed, we observed a strong 2.5 fold shift from 21 nucleotide reads (pre-translocation) to 28 nucleotide reads (post-translocation) upon arsenite treatment in the published Ribo-seq data (two-sided Kolmogorov-Smirnov test p<2E-16, Figure 3A). This aligned with our cryo-EM findings [19], and overall suggested that arsenite stress led to an accumulation of 80S ribosomes trapped in the post-translocation conformation across the CDS. Next, we turned our attention to the ribosomes themselves and asked if there were any indications in our PEPseq data for changes in protein-RNA interactions towards ribosomal RNA (rRNA).…”

“…Conversely, stalling ribosomes and the repair machinery they attract might explain increased UV-crosslinked protein in the CDS and increased PEPseq signal in the CDS upon arsenite stress. Using cryo-electron microscopy we recently reported that 80S ribosomes from arsenite-treated MCF7 cells adopt almost exclusively a post-translocation conformation, indicating that their normal ratcheting motion becomes impaired, potentially leading to stalling of ribosomes on mRNA [19]. In yeast and human cells it was demonstrated that in ribosome profiling (Ribo-seq) the length of a ribosome footprint informs about the conformation of the ribosome during its ratcheting motion on mRNA [20].…”

“…Using polysome profiling in MCF7 cells we recently demonstrated that after 30 minutes of arsenite stress at the identical concentration applied here, almost no polysomes remain on mRNA indicating that very little residual translation occurs under this condition [19]. We hypothesized that if a biological program was prepared by sorting mRNAs into or outside of stress granules, this program would probably not be put into action during arsenite stress because independent of where an mRNA was located very little protein would be produced from it.…”

The Emerging Role of Autophagy as a Target of Environmental Pollutants: An Update on Mechanisms

“…During reticulophagy, the ER recognizes autophagy adaptors autophagy-related 40 (ATG40) and the ER-anchored autophagy receptor (reticulophagy regulator 1) [ 23 ]. In ribophagy, autophagy selectively degrades ribosomes, which bind to nuclear fragile X mental retardation-interacting protein 1 (NUFIP1), an autophagy receptor-like protein [ 24 ]. Midbody degradation is the selective destruction of midbody rings produced during cytokinesis via autophagy [ 25 ].…”

Visualization of translation reorganization upon persistent collision stress in mammalian cells

“…The pulling force of the ASCC on the mRNA to split the stalled ribosome, may have detached the mRNA from the A/P tRNA in the second collided ribosome ( 17 ). Further studies will be required to investigate the cellular impact of the accumulation of these ribosomes and their possible elimination mechanisms such as autophagic recycling ( 31 ).…”