Exploiting codon usage identifies intensity-specific modifiers of Ras/MAPK signaling in vivo

Abstract

Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. Our screen enriched for regions of the genome not previously connected with Ras phenotypic modification. We mapped the underlying gene from one modifier to the ribosomal gene RpS21. In multiple contexts, we show that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.

Ras protein abundance correlates with Ras isoform mutation patterns in cancer

“…Rare codons limit protein translation efficiency 14,15 and optimizing codons in the KRAS gene locus did indeed result in higher KRAS protein expression 13 . Consistent with the sweet-spot model, higher KRAS expression reduced carcinogen-induced tumorigenesis in mice and altered engagement with cancer signaling pathways 13,16,17,18 . As a result, it was suggested that KRAS expression is optimal in most contexts, whereas HRAS and NRAS expression is too high 4,13 .…”

Distinct responses to rare codons in select Drosophila tissues

“…This locus is well-established to express transgenes at robust and reproducible levels (Markstein et al, 2008). As we have done previously (Sawyer et al, 2020), we placed all our transgenic reporters in the same vector backbone, which uses a ubiquitin promoter sequence for robust gene expression (Methods). In this way, we control for the effects of chromatin environment on gene expression and ensure that any differences in protein production are due to differences in coding sequence.…”

“…Our previous study of codon-dependent effects of Ras signaling in Drosophila identified profound phenotypic differences of a 2-fold, codon-dependent increase in protein expression (Sawyer et al, 2020). We next examined the impact of increased, codon-dependent protein expression of RpL10Aa.…”

“…Protein samples were prepared as in Sawyer et al, 2020. Briefly, tissues or whole animals were homogenized in Laemmli buffer (10 tissues in 50ul or 5 animals in 100ul) on ice, then boiled for 5 min.…”

Correlations Between the Characteristics of Alternative Splicing Events, Prognosis, and the Immune Microenvironment in Breast Cancer

“…A single SF may regulate multiple AS events, and an AS event may also be regulated by multiple SFs. Ribosomal protein s21 (RPS21) is a ribosome-related protein, which has been shown to play an important role in ribosomal biogenesis and may affect the occurrence and development of osteosarcoma and prostate cancer through the RAS/MAPK pathway ( Arthurs et al, 2017 ; Fan et al, 2018 ; Liang et al, 2019 ; Sawyer et al, 2020 ; Wang et al, 2020 ). Through the query of the database GEPIA2, the expression of RPS21 gene is not significantly different in tumor and normal tissues ( Supplementary Figure 2C ), so its potential function in breast cancer has not been studied so far, but according to our research, one of the transcripts of RPS (AA- RPS21) is differentially expressed in cancer and normal tissues of breast cancer patients, which suggests that this transcript may play a tumor-driving role in breast cancer, so it is worthy of further investigation.…”