Melting temperature highlights functionally important RNA structure and sequence elements in yeast mRNA coding regions

Abstract

Secondary structure elements in the coding regions of mRNAs play an important role in gene expression and regulation, but distinguishing functional from non-functional structures remains challenging. Here we investigate the dependence of sequence-structure relationships in the coding regions on temperature based on the recent PARTE data by Wan et al. Our main finding is that the regions with high and low thermostability (high Tm and low Tm regions) are under evolutionary pressure to preserve RNA secondary structure and primary sequence, respectively. Sequences of low Tm regions display a higher degree of evolutionary conservation compared to high Tm regions. Low Tm regions are under strong synonymous constraint, while high Tm regions are not. These findings imply that high Tm regions contain thermo-stable functionally important RNA structures, which impose relaxed evolutionary constraint on sequence as long as the base-pairing patterns remain intact. By contrast, low thermostability regions contain single-stranded functionally important conserved RNA sequence elements accessible for binding by other molecules. We also find that theoretically predicted structures of paralogous mRNA pairs become more similar with growing temperature, while experimentally measured structures tend to diverge, which implies that the melting pathways of RNA structures cannot be fully captured by current computational approaches.

Figure 1.

Variation of the distance between secondary structures of paralogous mRNA pairs along the temperature ladder. Points are the median levels of the distance at each temperature. (A) Distance between PARTE structures. (B) Distance between predicted structures.

Figure 2.

Sequence–structure relationships in the high/low T_m regions of paralogous mRNA pairs. For high T_m regions, the distance between structures shows a linear dependence from sequence identity for sequence identity values over 80% (correlation coefficient −0.54, P-value = 2.0e-7). For low T_m regions, the distance between structures shows a linear dependence from sequence identity for the sequence identity values over 90% (correlation coefficient −0.69, P-value = 2.1e-11). Linear regression for each 10% range of sequence identity is shown by a dashed line with the corresponding color. PARTE structures at 23°C were used.

Figure 3.

Low T_m regions in paralogous mRNA pairs are more conserved in sequence while high T_m regions are more conserved in RNA secondary structure. (A) Sequence identity between mRNAs (Mann–Whitney–Wilcoxon test, P-value = 1.9e-21). (B) Distance between RNA secondary structures (PARTE structures at 23°C; Mann–Whitney–Wilcoxon test, P-value = 4.6e-3). The differences are significant according to Mann–Whitney–Wilcoxon test. Error bars indicate standard error. The investigation of structural distances was effected upon controlling for sequence identity. Only regions with sequence identity 85–95% were considered in this analysis, while the regions with sequence identity < 85%, for which the distance between structures does not differ from randomly selected mRNA pairs as well as the regions with sequence identity > 95%, among which almost no high T_m regions exists, were excluded from consideration.

Figure 4.

Schematic illustration of the conservation levels of high/low T_m regions and thermo-stable/meltable positions. The alignment of two mRNAs is shown on the top. The low T_m region displays a higher sequence identity than the high T_m region (92 versus 60%). When all thermo-stable and meltable positions are considered together, the thermo-stable positions show a higher conservation level than the meltable positions (75 versus 71.9%). When the thermo-stable and the meltable positions located in high T_m and low T_m regions are considered separately, the meltable positions in the low T_m region are most conserved (90%), followed by the thermo-stable positions in the high T_m region (80%), while the meltable positions in the high T_m region are least conserved (40%).

Figure 5.

Conservation levels of thermo-stable and meltable positions in high/low T_m regions. Positions in high T_m and low T_m regions are examined separately. In high T_m regions the thermo-stable positions exhibit higher sequence conservation than meltable positions (Z-test for two proportions, P-value = 1.3e-6), while in low T_m regions meltable positions display a very high conservation level (Z-test for two proportions, P-value = 2.3e-37) and thermo-stable position are completely absent.

Figure 6.

Most low T_m regions exhibit negative ΔpS values while most high T_m regions exhibit positive ΔpS values. The differences are significant according to chi-squared test. Error bars indicate standard error.

Figure 7.

Melting temperatures of high T_m regions (boxplot and grey dots) and experimentally validated structure elements (black triangles).

Figure 8.

(A) Summary of the findings about high T_m and low T_m regions. (B) Inferences based on these findings.