Extended Data Fig. 9: EXSISERS enables non-invasive protein-level quantification of co-translation regulation.
From: Non-invasive and high-throughput interrogation of exon-specific isoform expression
a, The antizyme Oaz1 is regulated co-translationally by ribosomal frameshifting, which strongly depends on polyamine levels such as spermidine and spermine. Increasing polyamine levels lead to a +1 frameshift and skipping of the in-frame stop codon providing the full-length Oaz1 antizyme. The usage of the in-frame stop codon otherwise will lead to truncated non-functional Oaz1. Full-length Oaz1 binds and inactivates the enzyme Odc, the rate-limiting enzyme in the polyamine biosynthesis pathway, resulting in a product-mediated closed-loop homeostatic regulation of polyamine levels. b, EXSISERSOaz1-mTagBFP2 and EXSISERSOaz1-mNeonGreen were inserted into a plasmid harboring the full-length Oaz1 gene up- and downstream of the regulatory hairpin with the in-frame stop codon. c, FACS analysis of EXSISERSOaz1-transfected cells treated with different polyamine levels. The first gate (blue) ensured analysis of transfected cells only (Supplementary Information). Transfected cells were analyzed by counting the fraction of cells passing the green gate, which was set to contain the greenest 25% in the untreated condition (0 mM). Data points represent the mean values ± s.d. (n = 3 biological replicates). d, Immunoblot from the lysate of the corresponding samples shown in c. ***, and **** denotes p < 0.001, and p < 0.0001, one-way ANOVA, of Bonferroni MCT against vehicle control (full statistical results are available in Supplementary Table 1). Unprocessed blots and numerical source data are provided in Source Data Extended Data Fig. 9.
