A Systematic Analysis Of Roles Of MRNA Secondary Structures During Translation

mRNA is a single-strand molecule with a strong potential to fold back on itself, if no ribosome binds to it. The resulting so-called m RNA secondary structure emerges to be a key player in the regulations of gene expression, including translation initiation and localization of m RNA or protein. During the past decade, there has been sharply increasing evidence showing that m RNA secondary structure participates in translation regulation through structural conformation, and thus many studies aimed at identifying the functional structures by combining with conservation analysis of structures. However, recent studies revealed that structural stability has important roles in translation regulation. Thereby many functionally structural regions have not been analyzed by previous studies. For this reason, in the current work, I focus on the stability of m RNA secondary structure. Using computational methods, I analyzed the functions of the regions with high structural stability, and the mechanisms by which structural stability is maintained during evolution. Specifically, I explained how m RNA secondary structure regulates translation rate through the dynamic change of structural stability. The current work includes four parts as follows.Part 1. Number variation of high stability regions is correlated with gene functions. In this study, I defined high stability regions(HSRs) in coding sequences(CDSs) of Escherichia coli based on the normalized folding free energy. We found that CDSs have a higher number of HSRs compared with the random sequences, which was generated by shuffling sequence order while controlling for base compositions of the sequence. The HSRs tends to locate at the 5' or 3' terminal of CDSs, suggesting different roles of these HSRs in translation regulation. A reduced ribosome speed was detected near the start position of HSR, implying a possibility that HSR can act as an obstacle to drive translational pausing that coordinated protein synthesis. Interestingly, we found that genes with high HSR density are enriched in the processes of translation, protein folding, and cell division. In addition, essential genes exhibit higher HSR density than nonessential genes. Overall, this study presents the previously unappreciated correlation between the number variation of HSR and cellular processes.Part 2. Universally increased m RNA stability downstream of the translation initiation site. In this study, I analyze the structural stability along coding sequences in 27 species using computational method. We found that there is a significant increase in structural stability in the interval of 30-80 nt downstream of the translation start site. We showed the evidence that the increased structural stability is not the by-product of the selection for codon usage bias or amino acid bias in this region. More importantly, we found that the m RNAs encoding secreted proteins have more stable structure in this region compared that in the m RNA encoding non-secreted proteins. The result suggests that m RNA secondary structure in the interval of 30-80 nt downstream of translation start site might participate in the translation regulation which is function to the localization of secreted proteins.Part 3. Deciphering the rules by which dynamics of m RNA secondary structure affect translation efficiency. m RNA secondary structure decreases the elongation rate, as ribosomes must unwind every structure they encounter during translation. Therefore, the strength of m RNA secondary structure is assumed to be reduced in highly translated m RNAs. However, previous studies in vitro reported a positive correlation between m RNA folding strength and protein abundance. The counterintuitive finding suggests that m RNA secondary structure affects translation efficiency in an undetermined manner. Here, I analyzed the folding behavior of m RNA during translation and its effect on translation efficiency in yeast. I simulated translation process based on a novel computational model, taking into account the interactions among ribosomes, codon usage and m RNA secondary structures. The result showed that m RNA secondary structure shortens ribosomal distance through the dynamic change of folding strength. Notably, when adjacent ribosomes are close, m RNA secondary structures between them disappear, and codon usage determines the elongation rate. More importantly, our results showed that the combined effect of m RNA secondary structure and codon usage in highly translated m RNAs causes a short ribosomal distance in structural regions, which in turn eliminates the structures during translation, leading to a high elongation rate. Together, these findings reveal how the dynamics of m RNA secondary structure coupling with codon usage affect translation efficiency.Part4. Genome-wide analysis of selective constraints on high stability regions of m RNA. m RNA is a key component of a complex regulatory network. It accommodates numerous regulatory signals delineated along the protein coding regions in an intricate overlapping manner. A worthy question is how the local structural stability is maintained under the constraint that multiple selective pressures are imposed on m RNA local regions. Here, I performed the first genome-wide study of natural selection operating on high structural stability regions(HSRs) of m RNAs in Escherichia coli. I found that HSR tends to adjust the folded conformation to reduce the harm of mutations, showing a high level of mutational robustness. Moreover, guanine preference in HSR was observed, supporting the hypothesis that the selective constraint for high structural stability may partly account for the high percentage of G content in Escherichia coli genome. Notably, I found a substantially reduced synonymous substitution rate in HSRs compared with that in their adjacent regions. Surprisingly and interestingly, the non-key sites in HSRs, which have slight effect on structural stability, have synonymous substitution rate equivalent to background regions. To explain this result, I identified compensatory mutations in HSRs based on structural stability, and found that a considerable number of synonymous mutations occur to restore the structural stability decreased heavily by the mutations on key sites. Overall, these results suggest a significant role of local structural stability as a selective force operating on m RNA, which furthers our understanding of the constraints imposed on protein-coding RNAs.In summary, in the current work, I performed a systematic analysis on the functions of m RNA secondary structure. The results can provide new insights to the study of the functions and evolution of m RNA secondary structure.