Cycloheximide can distort measurements of mRNA levels and translation efficiency

Abstract

Regulation of the efficiency with which an mRNA is translated into proteins represents a key mechanism for controlling gene expression. Such regulation impacts the number of actively translating ribosomes per mRNA molecule, referred to as translation efficiency (TE), which can be monitored using ribosome profiling and RNA-seq, or by evaluating the position of an mRNA in a polysome gradient. Here we show that in budding yeast, under nutrient limiting conditions, the commonly used translation inhibitor cycloheximide induces rapid transcriptional upregulation of hundreds of genes involved in ribosome biogenesis. Cycloheximide also prevents translation of these newly transcribed messages, leading to an apparent drop in TE of these genes under conditions that include key transitions during the yeast metabolic cycle, meiosis, and amino acid starvation; however, this effect is abolished when cycloheximide pretreatment is omitted. This response requires TORC1 signaling, and is modulated by the genetic background as well as the vehicle used to deliver the drug. The present work highlights an important caveat to the use of translation inhibitors when measuring TE or mRNA levels, and will hopefully aid in future experimental design as well as interpretation of prior results.

Codon bias confers stability to human mRNA s

“…Treatment with CHX improved the stability of both REL-OPT and REL-WT transcripts compared to the control group. It should be noted that an important caveat to the use of global translation inhibitors, CHX in particular, is that they have been reported to potentially distort mRNA level measurements as well as translation efficiency [38][39][40]. We repeated our experiments using the IL6 reporters and found that in a similar fashion, CHX-treated IL6-DE transcripts were stabilized, albeit, not to the same extent as CHXtreated IL6-OPT ( Fig 4B).…”

“…Following this, we repeated our experiments using a different translation inhibitor, anisomycin (ANI), and obtained similar results ( Fig 4C and D), suggesting that a translation-independent mRNA degradation pathway could also be present. It should be noted that an important caveat to the use of global translation inhibitors, CHX in particular, is that they have been reported to potentially distort mRNA level measurements as well as translation efficiency [38][39][40].…”

The translation inhibitor cycloheximide affects ribosome profiling data in a species-specific manner

“…CHX is a small molecule that inhibits translation elongation by binding to the ribosomal E-site (29,30). Different studies, in particular in Saccharomyces cerevisiae, have reported biases triggered by CHX pre-treatment (31)(32)(33)(34)(35)(36). For example, CHX appears to reversibly interact with ribosomes, allowing them to move away from their initial position on mRNA (33).…”

Global translation inhibition yields condition-dependent de-repression of ribosome biogenesis mRNAs

“…The rapid decline in TE that we observed in meiotic cells was reminiscent of translational repression seen by the same ribosome profiling protocol applied to yeast cells that were transferred from rich media to media lacking amino acids (32). In addition, we became aware that a similar type of time-interval-specific apparent translational repression was detected by another group during amino acid starvation and metabolic cycling in yeast, and that it appeared to be dependent on the translation inhibitor, CHX, due to a resultant increase in RiBi mRNAs (personal communication, Dan Santos; reported in accompanying study (33)). CHX is a chemical that is frequently used to pre-treat cells prior to harvesting for ribosome profiling (32,34).…”