Fig. 4
Increasing protein production by reducing or deleting 5´ UTR poly(A) with the guidance of the machine-learning model. (A) Summary of 5´ UTR mutants. The mutants were divided into two groups: one with changes in poly(A) length, and the other with shifts of the poly(A) position. (B) Plot representing the measured versus predicted relative GFP abundance of 5´ UTR mutants in (A). The value of R2 was shown in the plot. Standard deviations (SD) of measured abundance were shown (n = 3). Mutants that changed the poly(A) length and position were labelled in red and green, respectively. (C) Ratio between relative GFP abundance caused by mutant 5´ UTRs and that caused by wild-type 5´ UTRs. The mutants were ranked in descending order based on the ratios. Four types of mutants were distinguished by different colors. Mean ± SD was shown (n = 3). (D) Plot representing the measured AnFaeA activity versus predicted AnFaeA production in poly(A) mutants of INU1 5´ UTR. The value of R2 was shown in the plot. SD of measured abundance were shown (n = 3). The point representing the wild-type 5´ UTR of INU1 was colored blue. (E) Plot representing the measured versus predicted relative GFP abundance caused by 5´ UTR Δpoly(A) mutants. The value of R2 was shown in the plot. Mean ± SD of measured abundance was shown (n = 3). (F) Comparison between the relative GFP abundance caused by 5´ UTR Δpoly(A) mutants and that caused by the wild-type 5´ UTRs. Mean ± SD was shown (n = 3). The significance was assessed by a two-tailed t-test. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns > 0.05. (G) Comparison between the AnFaeA activity caused by 5´ UTR Δpoly(A) mutants and that caused by the wild-type 5´ UTRs. Enzymatic activity was measured after culturing for 72 h. Mean ± SD was shown (n = 3). The significance was assessed by a two-tailed t-test. * p < 0.05. (H, I) Comparison between relative mRNA levels of GFP (H) or AnFaeA (I) expressed by 5´ UTR Δpoly(A) mutants and those expressed by wild-type 5´ UTRs. mRNA was extracted from samples described in (E) and (F) and subjected to qPCR analysis. The mRNA levels of GFP or AnFaeA were normalized with the mRNA level of SWC4. Mean ± SD was shown (n = 3). The significance was assessed by a two-tailed t-test. **** p < 0.0001; ** p < 0.01; * p < 0.05; ns > 0.05