Structure-function relations of RNA molecules involved in gene expression and host defence

This PhD thesis describes the relation of the structure of RNA with its effect on protein expression and the efficacy of CRISPR-Cas12a. Direct control of protein expression by RNA is done via so-called riboswitches, which change their structure depending on the presence of a ligand or external factors like temperature. A theophylline-responsive riboswitch based on the phage T4 td group I intron is described in detail and a method is proposed to obtain riboswitches based on this intron. This method was used to generate a riboswitch library for 3-methylxanthine, a compound closely related to theophylline, and the unrelated citrulline. The second part focuses on the effect of coding and non-coding mRNA on gene translation. Coding regions that are in close proximity of other coding regions have their translation coupled to each other, and the order of the genes appears to be of surprisingly little influence. The next part investigates the effect of the 3’-UTR and the intergenic region. These non-coding elements may have a lot of influence on the translation rate, by a mechanism that is not yet understood. The last part shines a light on the influence of the RNA structure of the CRISPR-Cas12a crRNA. The pre-crRNA forms a characteristic pseudoknot structure that is required for the recognition by Cas12a, which then turns it into crRNA. The low GC content of the pseudoknot allows this structure to be outcompeted by other potential structures, which renders the pre-crRNA unrecognisable. A design strategy was proposed to improve the efficacy of CRISPR-Cas12a