SL1 trans-splicing removes the uAUG and attenuates secondary structures in the 5′ UTR to enhance translational efficiency. The 5′ UTR length is negatively correlated with translational efficiency in both (A) SL1 trans-spliced genes (N = 3373) and (B) non-trans-spliced genes (N = 1404). Linear regression lines and their 95% confidence intervals are shown. (C) Before trans-splicing, the 5′ UTRs of SL1 trans-spliced genes (pre-SL1) are longer than those of non-trans-spliced genes (Non), whereas after trans-splicing, they (SL1) are shorter than those of non-trans-spliced genes. P-values were given by the Mann-Whitney U test. (D) Genes without an uAUG exhibit higher translational efficiencies among both SL1 trans-spliced and non-trans-spliced genes. P-values were given by the Mann-Whitney U test. (E) The translational efficiencies of genes with at least one in-frame uORF that terminates at an upstream stop codon (USC) are significantly lower than those with in-frame uORFs that share the stop codons (SSC) with the annotated ORFs (P-value was given by the Mann-Whitney U test). In addition, genes with an in-frame uAUG exhibit higher translational efficiencies than those with an out-of-frame uAUG (P = 7 × 10−5, Mann-Whitney U test). (F) The proportion of genes without an uAUG in non-trans-spliced genes is larger than that in pre-SL1 transcripts but is smaller than that in SL1 transcripts (Fisher's exact test). Error bars represent standard errors estimated from a binomial distribution. (G,H) The smallest MFE (sMFE) was used to estimate the free energy (in units of kcal/mol) of the most stable secondary structure in the 5′ UTR. The sMFE is positively correlated with translational efficiency in both (G) SL1 trans-spliced genes (N = 3373) and (H) non-trans-spliced genes (N = 1055). Schematic secondary structures are shown on the x-axis. (I) The sMFE in non-trans-spliced genes is slightly higher than that in pre-SL1 transcripts but is much smaller than that in SL1 transcripts. P-values were given by the Mann-Whitney U test.
