Translation inhibitors cause abnormalities in ribosome profiling experiments

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Ribosome profiling and high-throughput sequencing offer insights into mRNA translation.
• Previous studies suggested roles for upstream reading frames and stress-induced elongation changes.
• The impact of translation inhibitors like cycloheximide on ribosome profiles was noted.

Purpose of the Study:

• To investigate the effect of varying cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae.
• To determine if stress conditions (oxidative, heat shock, amino acid starvation) affect translation elongation.
• To clarify the role of short upstream reading frames in translational control under stress.

Main Methods:

• Ribosome profiling was performed on Saccharomyces cerevisiae under different cycloheximide concentrations.
• Cells were subjected to various stress conditions (oxidative stress, heat shock, amino acid starvation).
• Ribosome coverage profiles were analyzed to assess translation elongation and upstream reading frame activity.

Main Results:

• Increasing cycloheximide concentration mitigated some artifacts observed in ribosome profiles.
• Stress conditions did not significantly affect translation elongation.
• Observed effects in prior studies were attributed to cycloheximide-induced artifacts, not stress.
• Limited evidence supports widespread regulation by short upstream reading frames under stress.

Conclusions:

• Cycloheximide concentration significantly influences ribosome profiling results, potentially creating artifacts.
• Stress responses do not appear to alter translation elongation as previously suggested.
• Short upstream reading frames are unlikely to be major regulators of protein synthesis during stress.
• Standardization of ribosome profiling methods, particularly regarding translation inhibitors, is essential for reliable data.