Ribosome profiling analysis of eEF3-depletedSaccharomyces cerevisiae

Abstract

In addition to the standard set of translation factors common in eukaryotic organisms, protein synthesis in the yeast Saccharomyces cerevisiae requires an ABCF ATPase factor eEF3, eukaryotic Elongation Factor 3. eEF3 is an E-site binder that was originally identified as an essential factor involved in the elongation stage of protein synthesis. Recent biochemical experiments suggest an additional function of eEF3 in ribosome recycling. We have characterised the global effects of eEF3 depletion on translation using ribosome profiling. Depletion of eEF3 results in decreased ribosome density at the stop codon, indicating that ribosome recycling does not become rate limiting when eEF3 levels are low. Consistent with a defect in translation elongation, eEF3 depletion causes a moderate redistribution of ribosomes towards the 5’ part of the open reading frames. We observed no E-site codon-or amino acid-specific ribosome stalling upon eEF3 depletion, supporting its role as a general elongation factor. Surprisingly, depletion of eEF3 leads to a relative decrease in P-site proline stalling, which we hypothesise is a secondary effect of generally decreased translation and/or decreased competition for the E-site with eIF5A.

Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation

“…These in vitro findings are in strong agreement with the in vivo ribosome profiling experiments. Similar to previous studies where eEF3 was depleted in the cell (Kasari et al, 2019a), we observe general defects in translation elongation. However, our application of high-resolution ribosome profiling, using combinations of elongation inhibitors for library preparation, allowed us to follow ribosomes trapped in distinct functional states and thereby identify the specific elongation defect (Fig 3).…”

eEF3 promotes late stages of tRNA translocation on the ribosome

“…These in vitro findings are in strong agreement with the in vivo ribosome profiling experiments. Similar to previous studies where eEF3 was depleted in the cell (Kasari et al, 2019a), we observe general defects in translation elongation. However, our application of high-resolution ribosome profiling, using combinations of elongation inhibitors for library preparation, allowed us to follow ribosomes trapped in distinct functional states and thereby identify the specific elongation defect (Figure 3).…”

Multifaceted deregulation of gene expression and protein synthesis with age

“…Thus, under conditions when translation initiation is inhibited, ribosome density along the coding sequence should be redistributed from the 5′ proximal to the distal part of the transcript, while inhibition of termination and/or of ribosome recycling should increase the density in its 3′ proximal part. It should be noted, however, that elevated elongation speed and perhaps other factors could also lead to a similar pattern of metagene profile changes with age (53).…”