Figure 6. mRNAs showing relative TE increases in response to deletion of TMA64/TMA20, increased eIF2α phosphorylation, or 40S subunit depletion have a strong propensity to form the closed-loop intermediate.

(A–F) Cumulative distribution function (CDF) plots of changes in TEs (log₂∆TE) (A, C, E) or mRNA levels (log₂∆mRNA) (B, D, F) for all mRNAs (black) or the 386 SCL mRNAs (red) in the tma∆∆ mutant versus WT cells (A–B), in SM-treated versus untreated WT cells (C–D), and in the rps26∆∆ mutant versus WT cells (E–F). p values were calculated using the Kolmogorov-Smirnov test.

Figure 6—figure supplement 1. Strong Closed-Loop (SCL) mRNAs show attributes of highly translated mRNAs.

(A, C, E, and F) Cumulative distribution function (CDF) plots of log₂WT TE values (A), 5’UTR length (C), mRNA abundance in molecules per picogram of dry cellular weight (pgDW) (E), and mRNA half-life (F), for all mRNAs (black) and the 386 SCL mRNAs (red). (B) Frequency distribution plots of CDS length for all mRNAs and the SCL mRNAs. (D) Notched box plots of context scores calculated for positions −3 to −1 and +4 of main CDS AUGs for all mRNAs (white) and the SCL mRNAs (red). p values were calculated using the Mann-Whitney U test. (G) Venn diagrams of overlaps between the SCL mRNAs and RPG mRNAs, with the p value calculated using the Fisher's exact test.

Figure 6—figure supplement 2. The tma∆∆ mutations exacerbate the inhibitory effect of cap-proximal stem-loop structures on expression of luciferase reporter mRNAs.

Luciferase reporter mRNAs shown schematically on the left were expressed from low-copy number plasmids in WT and the tma∆∆ mutant. Eight independent transformants for each strain/plasmid were cultured for approximately three cell doublings in SC-Ura at 30°C and luciferase activity (relative light units, RLUs) and total protein concentration were measured in WCEs. Plotted on the right are the RLUs normalized for total protein, with error bars indicating ± SEMs and p values calculated using the student’s t-test. Stem-loops of the indicated predicted stabilities (in kcal/mol) were inserted into a 70 nt unstructured 5′ UTR at either 5 nt or 44 nt from the capped mRNA 5’ end (Left). The parental reporter (first row) contains the RPL41A promoter and first 5 nt of RPL41A mRNA followed by a tandem array of 18 CAA repeats, expected to produce a 5’UTR largely devoid of secondary structure, attached to the firefly luciferase CDSs, and an internally truncated RPL41A 3’UTR sequence (Sen et al., 2016).