Figure 2
Silvestrol-sensitive mRNAs are enriched with complex 5′ UTRs. (a) Box plot showing distribution of free energy values for 5′ UTRs of Silvestrol-sensitive genes (Decreased, n =284, z-score below -1.5), genes increased in Silvestrol (n =146 genes, z-score >1.5), and genes insensitive to Silvestrol (n =6,303). ***P =2.53 × 10-67, **P =1.097 × 10-27 and *P =4.25 × 10-7. Energy values were predicted using the CONTRAfold algorithm [25]. (b) Box plot showing distribution of 5′ UTR length, L: ***P =1.39 × 10-133, **P =3.1 × 10-44 and *P =4.83 × 10-10. (c) Box plot showing distribution of GC content: ***P =1.02 × 10-47, **P =3.35 × 10-23 and *P =8.08 × 10-6. For genes with decreased TE: n =284, ΔGmean = -103.9 kcal/mol, Lmean =493.4 nucleotides, GCmean =59.8%. For insensitive genes: n =6,303, ΔGmean = -54.72 kcal/mol, Lmean =223.6 nucleotides, GCmean =66.9%. For genes with increased TE: n =142, ΔGmean = -45.93 kcal/mol, Lmean =171.8 nucleotides, GCmean =71.23%. Significance values were determined using the two-tailed Mann-Whitney U test (d) RPKM and TE values from ribosome profiling data for genes used in 5′ UTR analyses of CyclinD1, ARF6, ROCK1 and PFN2. (e) Luciferase reporter constructs, stably transfected into 293 T cells, were treated with increasing concentrations of Silvestrol and luciferase expression was measured after 40 minutes; constructs bearing 5′ UTRs from Silvestrol-sensitive genes (CyclinD1, ROCK1, ARF6) or insensitive genes (PFN2 or CMV alone) were compared. Triplicate values were obtained in each experiment; data presented were obtained from four independent experiments. (f) Luciferase reporter expression in stably transfected 293 T cells treated with increasing concentrations of Silvestrol; constructs bearing ARF6wt 5′ UTR or ARF6mut 5′ UTR were compared; data presented were obtained from two independent experiments with measurements taken in triplicate.