Expression of transgenes enriched in rare codons is enhanced by the MAPK pathway

Jackson Peterson,Ozgun Erdogan,Erin Kaltenbrun,Christopher M. Counter,Siqi Li

doi:10.1038/s41598-020-78453-5

Jackson Peterson, Ozgun Erdogan + Show 3 more

Open Access

https://doi.org/10.1038/s41598-020-78453-5

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Journal: Scientific Reports	Publication Date: Dec 1, 2020
Citations: 15	License type: open-access

Affiliation: Duke University, Durham University

Abstract

The ability to translate three nucleotide sequences, or codons, into amino acids to form proteins is conserved across all organisms. All but two amino acids have multiple codons, and the frequency that such synonymous codons occur in genomes ranges from rare to common. Transcripts enriched in rare codons are typically associated with poor translation, but in certain settings can be robustly expressed, suggestive of codon-dependent regulation. Given this, we screened a gain-of-function library for human genes that increase the expression of a GFPrare reporter encoded by rare codons. This screen identified multiple components of the mitogen activated protein kinase (MAPK) pathway enhancing GFPrare expression. This effect was reversed with inhibitors of this pathway and confirmed to be both codon-dependent and occur with ectopic transcripts naturally coded with rare codons. Finally, this effect was associated, at least in part, with enhanced translation. We thus identify a potential regulatory module that takes advantage of the redundancy in the genetic code to modulate protein expression.

Highlights

In response, viral proteins appear to evolve codon usage that matches the tRNA expression profiles of interferon-treated cells[32]
The resultant two populations were subjected to Fluorescence-Activated Cell Sorting (FACS) analysis to separate cells based on the level of their Green Fluorescent Protein (GFP) fluorescence
G FPrare exhibited approximately 100-fold lower Mean Fluorescent Intensity (MFI) when compared to GFPcom. Despite this obvious difference in expression, GFPrare-expressing cells were not fully resolvable from their G FPcom counterparts (Supplementary Fig. 2b). To overcome this limitation, mCherrycom cDNA encoding the fluorescent protein mCherry enriched in common codons (Supplementary Figs. 1c, 2c) was inserted into these vectors to allow for normalization of expression levels. 293T cells transfected with the m Cherrycom:GFPrare or m Cherrycom:GFPcom reporter constructs were again subjected to FACS analysis, revealing a linear relationship between mCherrycom and GFPrare or GFPcom fluorescence (Fig. 1a and Supplementary Fig. 2d,e)

Summary

Introduction

In response, viral proteins appear to evolve codon usage that matches the tRNA expression profiles of interferon-treated cells[32]. Given the effect of rare codon bias on gene function, the increasing number of examples of rare codon-enriched transcripts highly expressed, and at least one tangible example of a mammalian protein regulating translation in a rare codon-dependent manner, we sought to identify pathways that affect rare codon-dependent expression. To this end, we hypothesized that a fluorescent protein coded with rare codons could be used to detect rare codondependent expression. To identify modifiers of rare codon-dependent expression in mammals we screened a cDNA library encoding common cancer-related proteins for their ability to preferentially increase the expression of a fluorescent reporter enriched in rare codons in human cells

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