Is there a code behind the code?

Protein synthesis is an essential process for all organisms. Every cell dedicates tremendous amounts of energy and metabolic resources to protein biosynthesis. The central component of this complicated process is a multifunctional complex called ribosome. The ribosome consists of more than 60 different parts and it has to orchestrate more than 80 additional molecules in order to provide fast and accurate protein synthesis. Overall, about 40% of the cellular energy is consumed by this process. The template for the ribosome is the mRNA, which is a transcript of a certain part of the genome that needs to be converted into an amino acid sequence. These mRNAs can also be modified at single nucleotides to regulate gene expression. So far five natural occurring modifications of standard RNA building blocks were found in various eukaryotic organisms: methylated adenosines (m6A and m1A) and cytosines (m5C), inosines (I) as well as pseudouridines (). In a previous study we discovered modifications that, considering current models, should not interfere with the decoding process significantly increased the error rate. One conclusion is, that there are more factors influencing decoding than only the direct interaction between the mRNA and the tRNA. This ignited the idea for this project to use non-natural mRNA modifications to weaken and manipulate this interaction to reveal mechanisms in the background that significantly influence decoding processes. To do so, we introduce nucleotide analogs that can only form a reduced number of hydrogen bonds with the incoming tRNAs (e.g. purine or pyridone). The reduced number of interactions allows several tRNAs to bind to the mRNA codon without being rejected. Consequently, the bound tRNA will be identified and then the factors that impacted the introduction of the particular tRNA can be revealed. It is suggested that possibly the composition of the tRNA pool, tRNA sequences and modifications can interfere to some extend with decoding. By applying modified mRNA codons we will be able to biochemically investigate these influences and potentially reveal new decoding mechanisms behind the codon/anticodon interactions.

Protein synthesis is a central process in every living cell and supplies all organisms with the entity of necessary proteins and enzymes. The center stage for translation is the ribosome, which needs to orchestrate more than 100 different proteins, tRNAs and factors in order to provide fast and accurate protein synthesis. Our aim was to get a better understanding on the decoding process in general and on the decoding of modified mRNAs in particular. We established an experimental system that allowed us to site-specifically introduce modifications into bacterial but also eukaryotic mRNAs. We analyzed the effect of various natural as well as non-natural modifications on translation elongation and thereby could provide a rather detailed picture on the importance of single interactions between the mRNA codon and the tRNA anticodon. In addition, the potential regulatory power of single natural modifications could be determined. Whereas, modifications like 1-methyladenosine inhibited protein synthesis independent of its position, a single 6-methyladenosine or 2'-O methylations were strongly context and position dependent. Other modifications like pseudouridine, or 5-methylcytosine did not alter the decoding efficiency and accuracy. This type of experiments and research was particularly interesting in light of recent developments of mRNA vaccines and mRNA therapeutics. These mRNAs harbor modifications like pseudouridine or 1-methylpseudouridine in order to stabilize the mRNA after injection and reduce unwanted immune responses. Nevertheless, the decoding accuracy and efficiency must not be affected by these modifications. As a detailed understanding of decoding and the interactions within the decoding sites is of importance for further developments in this field a detailed understanding of the decoding process is essential. With this project we were able to contribute an additional piece of knowledge to the complex process of protein synthesis.