Distinct responses to rare codons in select Drosophila tissues

  1. Scott R Allen
  2. Rebeccah K Stewart
  3. Michael Rogers
  4. Ivan Jimenez Ruiz
  5. Erez Cohen
  6. Alain Laederach
  7. Christopher M Counter
  8. Jessica K Sawyer
  9. Donald T Fox

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    The results in this report are intriguing in providing evidence that differences in codon optimality in mRNAs can underlie tissue-specific differences in expression and that this phenomenon operates in restricting expression of an evolutionarily young endogenous genes to the testis versus ovaries in a manner important for female fertility in the fruit fly. The scientific quality of the work would be further enhanced by additional experiments to determine whether the differences in expression arise primarily at the translational level or also/rather involve altered mRNA turnover or transcription in response to poor codon usage outside of testis and brain.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Codon usage bias has long been appreciated to influence protein production. Yet, relatively few studies have analyzed the impacts of codon usage on tissue-specific mRNA and protein expression. Here, we use codon-modified reporters to perform an organism-wide screen in Drosophila melanogaster for distinct tissue responses to codon usage bias. These reporters reveal a cliff-like decline of protein expression near the limit of rare codon usage in endogenously expressed Drosophila genes. Near the edge of this limit, however, we find the testis and brain are uniquely capable of expressing rare codon-enriched reporters. We define a new metric of tissue-specific codon usage, the tissue-apparent Codon Adaptation Index (taCAI), to reveal a conserved enrichment for rare codon usage in the endogenously expressed genes of both Drosophila and human testis. We further demonstrate a role for rare codons in an evolutionarily young testis-specific gene, RpL10Aa . Optimizing RpL10Aa codons disrupts female fertility. Our work highlights distinct responses to rarely used codons in select tissues, revealing a critical role for codon bias in tissue biology.

Article activity feed

  1. Version published to 10.7554/elife.76893 on eLife
  2. eLife

    Evaluation Summary:

    The results in this report are intriguing in providing evidence that differences in codon optimality in mRNAs can underlie tissue-specific differences in expression and that this phenomenon operates in restricting expression of an evolutionarily young endogenous genes to the testis versus ovaries in a manner important for female fertility in the fruit fly. The scientific quality of the work would be further enhanced by additional experiments to determine whether the differences in expression arise primarily at the translational level or also/rather involve altered mRNA turnover or transcription in response to poor codon usage outside of testis and brain.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)

  3. eLife

    Reviewer #1 (Public Review):

    By examining expression of GFP reporters containing different proportions of rare codons, all expressed from the same promoter and genomic locus, in different transgenic flies, these authors have uncovered a surprising sudden elimination of expression when rare codon proportions exceeded 50%, which is near the upper limit of rare codon usage among native mRNAs. Examining a reporter near the boundary of this limit, they observed high expression in testes and brain and little/no expression in other tissues; and within the testis, they found high expression in germ cells and somatic cells of the germline stem cell niche but no expression in the somatic cyst cells. Measuring both reporter protein and mRNA expression suggested that impaired translation dominates, and increased mRNA turnover contributes, to the failure to express reporters exceeding 50% rare codons. Developing a new metric for tissue-specific codon usage, taCAI, they found that endogenous genes in the testis of both Drosophila and humans show an abundance of rare codon-enriched mRNAs, suggesting that rare codon usage may play an important role in testis-specific gene expression. They went on to provide evidence that evolutionarily young genes in duplicated gene pairs generated by transposon insertions tend to have higher rare codon usage, particularly among testis mRNAs and provided evidence that for one such young gene, Rpl10a, decreasing its proportion of rare codons in a transgene led to aberrant increased expression in ovaries versus testis in a manner that severely reduced fertility specifically in females.

  4. eLife

    Reviewer #2 (Public Review):

    Codon usage has been shown to be an important determinant of gene expression levels in diverse organisms but the impact of codon usage on tissue-specific gene expression is unclear. In this study, the authors examined tissue-specific expression of a series of codon usage-modified GFP/mCherry-GFP reporters in Drosophila. The results showed that testis and brain can specifically allow the expression of reporter GFP protein encoded by mRNA enriched with rare codons. The authors also developed a new tissue-specific codon usage metrics (taCAI) and revealed that genes specifically expressed in both Drosophila and human testis are enriched for those with low codon usage biases. Codon optimization of RpL10Aa, which is an evolutionarily young and testis-specific gene enriched with rare codons, results in its upregulation in the ovary. Together, these results demonstrate the tissue-specific impacts of codon usage on gene expression in Drosophila and suggest a role for codon usage bias in restricting gene expression to specific tissues.

    The study addresses an important biological problem and the results suggest a role for codon usage in tissue-specific gene expression in Drosophila and possibly in mammals.

    However, the cliff-like decrease of GFP reporter expression level in Figure 1 appeared to suggest that codon usage bias does not have much impact on gene expression level unless codon usage biases are very poor. This is not consistent with results of many other studies in different organisms including those in Drosophila (Carlini & Stephan Genetics 2003; Fu et al., 2016 Genes & Dev). It is important to note that the codon usage profile range of the reporters designed was not a good representation of the codon usage bias range of endogenous genes. As shown by Figure 1S, 14/16 reporter genes have extremely poor codon usage biases. The authors should clarify this point to avoid misinterpretation of the results. To understand the mechanism underlying the abolished GFP expression, I suggest that the gfp mRNA levels should be determined for at least some reporters. As suggested by previous studies in Neurospora and in Drosophila cells (Zhou et al., 2018 eLife, Yang et al., Nucleic Acids Research 2019), the dramatic decrease of protein expression by codon de-optimization could be due to premature termination of transcription or translation. Mechanisms that can cause such a cliff-like behavior should be discussed in the paper.

  5. eLife

    Reviewer #3 (Public Review):

    61 codons encode 20 amino acids, and synonymous codons are used at different frequencies across different species. The inclusion of common or rare codons within a transcript has important functional consequences for the expression levels of a gene product. High frequencies of rare codons within messages are thought to cause ribosome stalling, ribosome collisions, and mRNA degradation. Recent work by others in the field suggests that the percentage of common and rare codons within a gene can also influence its transcription.

    This current study seeks to explore how the inclusion of rare codons influences gene expression in a tissue specific manner. Using Drosophila as a model, the authors generate a series of GFP-based reporters that contain a range of rare codons. The positions of these rare codons were either randomly selected or clustered. Examining developing larvae, the authors observe that reporters containing greater than 50% dispersed rare codons exhibit a dramatic drop in GFP fluorescence compared to controls and other reporters that contain less then 50% rare codons. Reporters that contain clustered rare codons behave in a similar manner. The authors then use a Codon Adaptation Index (CAI) to provide evidence that endogenous Drosophila genes may also exhibit a drop off of protein expression when the rare codon percentage within a given transcript exceeds a certain threshold. Based on these data, they examine another clustered reporter (GFP54C3'), which falls near the limit of CAI for endogenously expressed Drosophila genes. Strikingly, expression of this reporter in developing larvae was only observed in the testes and brain. Further observations indicate that expression within the testis is enriched in the somatic hub and germ cells but was largely absent from somatic cyst cells.

    Previous work in the field shows that the presence of rare codons can influence transcription and mRNA stability. Detailed analysis of public datasets showed that Drosophila testes accumulate high levels of endogenous mRNAs enriched for rare codons relative to other tissues, consistent with their reporter assays. Analysis of Human Protein Atlas (HPA) project data shows this trend appears to hold true for human testes as well.

    Lastly, the authors show that Drosophila RpL10a, a newly evolved gene with a high percentage of rare codons, shows enriched expression in the testis. Codon optimizing RpL10a results in higher protein expression levels relative to a transgene with the endogenous compliment of rare codons. Moreover, the codon optimized transgene is expressed in the ovary, resulting in reduced female fertility. These results suggest that limiting expression of RpL10a protein to the testis is functionally significant.

    Strengths
    The paper is well-written, and the data are clear. The significance of the study builds on previous work and now shows that the presence of rare codons within transcripts influences their tissue specific protein expression. This paper will have broad appeal to those interested in gene regulation, mRNA translation, codon usage, evolution of new genes, and fertility.

    Weaknesses
    Although this study presents several very interesting observations, few mechanistic insights are provided. At the very least, comparison of the mRNA levels between the various reporters should be directly evaluated throughout the study.

  6. Version published to 10.1101/2022.01.06.475284v1 on bioRxiv