Mitotic checkpoint gene expression is tuned by codon usage bias (2022)
Figure 7

Figure 7. Mad1 homodimers assemble co-translationally.

(A) Top: Immunoprecipitation (IP) with anti-GFP or anti-Mad1 from extracts of haploid S. pombe cells expressing both untagged and GFP-tagged Mad1, probed with antibodies against Mad1 and tubulin; in = input (2.5 % of extract for IP), sup = supernatant after IP. Bottom: Comparison between the observed (obs.) and the expected (expect.) ratio between Mad1-GFP and untagged Mad1 in the IP given their ratio in the input (see Fig EV6A); two and one experiment(s), respectively. One more GFP-IP from the same strain was unquantifiable, because no second band was visible in the IP.

(B) Schematic illustrating that Mad1-Mad1 complex assembly likely takes place co-translationally with only proteins synthesized from the same mRNA being combined.

(C) Top: Anti-GFP immunoprecipitation (IP) and Strep pull-down (PD) from extracts of diploid cells expressing Mad1-GFP and Mad1-Strep from the two endogenous loci; membrane probed with anti-Mad1; in = input (7 % of extract for IP/PD), sup = supernatant after IP/PD. Bottom: as in (A), 2 experiments each. See Fig EV6 for a quantified experiment. The experiment shown at the top and two more GFP-IPs from the same strain were unquantifiable, because no second band was visible in the IP.

(D) In vitro translation (IVT) of Mad1-flag-His and untagged Mad1 in the presence of 35S-labelled Methionine and Cysteine, followed by Mad1 immunoprecipitation (IP) or His pull-down (PD); in = input (9.5 % of extract for IP/PD), sup = supernatant after IP/PD. An IVT with only untagged Mad1 was used to check for specificity of the His PD (right side). Shown is the autoradiograph after SDS-PAGE with quantification of the Mad1-flag-His to untagged Mad1 ratio in select lanes.

(E) Test for mRNA dimerization by single molecule mRNA FISH; probes against mad1+ and GFP. Top: Schematic of genotypes. Example pictures in Fig EV6. Bottom left: Intensity of cytoplasmic mad1+ mRNA spots in the different strains. For the 2 copy strain, a mad1+ spot was classified as mad1+-GFP if it was colocalizing with a GFP spot, and as mad1+ otherwise. Colors as indicated in the schematic. Vertical solid line: peak of each density plot; dashed line: theoretical position of a double-intensity peak. Number of spots analyzed: mad1+ (1 copy strain) = 921, mad1+ (2 copy strain) = 637, mad1+-GFP (2 copy strain) = 982, mad1+-GFP (1 copy strain) = 1699. Bottom right: Counts of cytoplasmic mad1+ or mad1+-GFP mRNA from the same experiment with generalized linear mixed model fits as lines. Number of cells: 1 copy strain mad1+ = 478, 2 copy strain = 327, 1 copy strain mad1+-GFP = 466.

(F) Experiment similar to (E), except that cells expressing both mad1+-GFP and mad3+-GFP from the respective endogenous locus were probed with FISH probes against mad1+ and GFP mRNA. A GFP spot was classified as mad1+-GFP if it was colocalizing with a mad1+ spot (arrowheads), and as mad3+-GFP otherwise. The intensity of GFP spots was quantified. Vertical solid line: peak of each density plot; dashed line: theoretical position of a double-intensity peak. Number of spots analyzed: mad1+-GFP = 987, mad3+-GFP = 1299.

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