MORDOR-Modified mRNAs as Dynamic Operators for Ribosomes

Protein synthesis is an essential process for all organisms. Every cell dedicates tremendous energy and metabolic resources to protein biosynthesis. The central component for this complicated process is a multifunctional particle called ribosome. Ribosomes consists of more than 60 different parts and they have to provide the fast and accurate translation of the genetic information into proteins. A precise and rapid regulation of this translation apparatus is absolutely essential considering that about 40% of the cells energy resources are dedicated to protein synthesis. 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. There are multiple ways how this process of gene expression is regulated. In this project we want to investigate if the modification of the mRNA is a mechanism to regulate the translation machinery. So far four natural occurring modifications of standard RNA building blocks were found in various eukaryotic organisms: methylated adenosines (m6A) and cytosines (m5C), Insosines (I) as well as pseudouridines (. The presence of RNA modifications was reported to be connected with the stability and structural aspects of mRNAs. However, there are contradicting reports how these modified mRNAs affect translation. A systematic investigation of the impact of modifications in the coding sequence of mRNAs was not carried out so far. We established an approach that allows us to systematically introduce these modifications into certain positions the mRNA. By the use of standard molecular biology assays and advanced analytical methods like mass spectrometry we aim to unravel how these modifications influence the translation process. Preliminary data in an E. coli based system were in line with previously published data supporting our experimental approach. This system will now be introduced to incorporate various naturally and also non-naturally occurring modifications in mRNAs. The effects observed will be carefully characterized to get a deeper insight into the role of the modification during translation. We intend to apply this approach not only for prokaryotic but also for eukaryotic translation systems to get an idea about this potential regulatory system in more complex organisms as well. One could assume that different organisms react differently on various modifications. As a long-term goal we also aim to investigate different cell-types or tissues of one organism and determine if modifications are potential cell type specific translation regulators. This will not only allow a deeper insight into the regulatory system of translation but also might open the door for a novel tool for cell targeting or even medical applications.

Protein synthesis is a central process in every living cell providing all organisms with all necessary proteins and enzymes. The main player in this procedure is the ribosome, which orchestrates more than 100 different proteins, RNAs and factors. This elaborate process needs to be exactly fine-tuned and regulated to provide the cells with correctly synthesized crucial proteins at any given time. Over the last years more and more modified nucleotides were identified within the coding sequences of mRNAs, and even the term epitranscriptomics was coined, that implicated a potential functional role of these modifications. Consequently, it was speculated and partly already investigated in different settings, what the function of those modifications can be. They have been linked to the processing, stability, and localization of the modified mRNA. It was also speculated that some modifications might have an effect on protein synthesis in terms of its efficiency and accuracy. Therefore, we modified an mRNA ligation protocol that enabled us to introduce various modifications site-specifically into the reading frame of a reporter mRNA. The translation products were then analyzed for their quantity and quality. The observed effects were very diverse ranging from no effect to a total inhibition of translation elongation. Strikingly, not only the type of modifications was decisive but also the position within the codon. Our results, which were also confirmed by other groups employing different techniques, strongly implicate a regulatory role of mRNA modifications on gene expression in prokaryotic and eukaryotic organisms. Inspired by the effects of mRNA modifications on translation, we also started to manipulate the interaction of mRNAs with either proteins or tRNAs by employing non-natural mRNA nucleotide derivatives. Thereby it was possible to alter the strength of interactions between two binding partners. Consequently, conclusions on their importance for a distinct interaction could be made. Based on structural studies we eliminated several proposed interactions and studied their effects. We could identify the minimal set of interactions between the stop codon and the release factors, responsible for translation termination and also important interactions between the mRNA and tRNA during decoding. Both projects provided biochemical insights into translation termination and also tRNA decoding to a so far unreached level. This established strategy enables a detailed biochemical investigation of published structural studies and proposed interactions.