Periodic Reporting for period 2 - tRNAslation (Deciphering the role of RNA modifications during ribosomal decoding and protein synthesis)
Reporting period: 2022-12-01 to 2024-05-31
All living organisms use information coded in their genomic deoxyribonucleic acid (DNA) to produce proteins, so called enzymes. These molecular working horses are responsible for carrying out fundamental cellular function to reproduce, stay alive and allow them to sense and react to changing environmental conditions. During a process called “transcription” regions that are the blueprints for specific proteins (genes) are activated and give rise to messenger ribonucleic acid (mRNA) molecules. These mRNA molecules contain copies of the respective DNA sequence and in higher organisms are transported out from the nucleus into the cytoplasm. During a process called “translation”, ribosomes and transfer RNA (tRNA) molecules translate sequence information encoded in mRNAs into correctly assembled chains of linked amino acids. These chains fold into specific three-dimensional structures and are then called proteins, which have a specific enzymatic activity depending on their intrinsic properties and structural architecture.
We aim to understand fundamental cellular mechanisms that are highly conserved from yeast to humans and affect protein synthesis at the level of translation. In detail, cells use multi-protein machines to attach small chemical modifications to mRNAs and tRNAs, which guarantee that proteins are produced with the highest precision and at the right speed. In this project, we seek to understand how several of these tRNA modifications enzymes catalyze their reactions in humans and investigate the direct consequences of the modifications for the affected RNA molecules and for the production of cellular proteins during translation.
Mutations in the protein complexes that conduct these modifications are associated with severe neurodegenerative diseases, intellectual disabilities and cancer. After obtaining three-dimensional snapshots of these enzymes, we will be able to better understand the functional consequences of decreased modification levels in affected patient and the molecular reasons for the diseases. This knowledge will ultimately allow us to develop novel diagnostic methods and therapeutic treatment strategies for the benefit of the affected patients.
We aim to understand fundamental cellular mechanisms that are highly conserved from yeast to humans and affect protein synthesis at the level of translation. In detail, cells use multi-protein machines to attach small chemical modifications to mRNAs and tRNAs, which guarantee that proteins are produced with the highest precision and at the right speed. In this project, we seek to understand how several of these tRNA modifications enzymes catalyze their reactions in humans and investigate the direct consequences of the modifications for the affected RNA molecules and for the production of cellular proteins during translation.
Mutations in the protein complexes that conduct these modifications are associated with severe neurodegenerative diseases, intellectual disabilities and cancer. After obtaining three-dimensional snapshots of these enzymes, we will be able to better understand the functional consequences of decreased modification levels in affected patient and the molecular reasons for the diseases. This knowledge will ultimately allow us to develop novel diagnostic methods and therapeutic treatment strategies for the benefit of the affected patients.
We have made substantial progress in characterizing the structures of several human tRNA modification complexes and contributed important new insights into the regulation of translation elongation by RNA modifications. Our results provide deep mechanistic insights into highly dynamic and complex enzymatic process that install various chemical modifications on RNA molecules. In addition, we developed novel methodologies to analyze the effects of individual modifications on the structure, folding and stability of RNAs. Furthermore, we have analyzed the function of various human tRNA modification enzymes carrying patient-derived mutations and provide molecular explanations for their potential to trigger human diseases. Overall, the work performed since the beginning of the project has led to several fundamental discoveries, which will pave the way to generate a more complete picture of protein synthesis in humans in health and disease.
Our results highlight the vast potential of combining cryo-EM with biophysical measurements to analyze the structure-function relationship of tRNAs and other small, folded RNA domains. We reveal novel mechanisms that enable human tRNA modification enzymes to specifically recognize tRNAs and attach small chemical groups at specific sites. Furthermore, we combine structural biology techniques with transcriptome-wide approaches to understand how modifications of rRNAs, tRNAs and mRNAs shape the protein landscape of each cell. We seek to continue our molecular characterization of various tRNA modification pathways and we aim to reconstitute fully modified human tRNAs by the end of the project. Last but not least, we aim to apply our newly developed techniques to a variety of RNA families to not only show the potential of our approaches, but also to discover specific mechanisms that allow RNA molecules to dynamically switch between numerous three-dimensional conformations.