Function and Mechanism of RNA Modification

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Ribosomes are fascinating molecular machines that synthesize all proteins. As such, ribosomes are a prime example of a nanoassembler, a machine capable of constructing a wide range of functional parts on the nanometer scale. The ribosome consists of two subunits each containing large, intricately folded RNAs (ribonucleic acids) and numerous interwoven proteins, raising the question of how all these components come together. It is the long-term goal of my research program to understand ribosome biogenesis as a paradigm for the assembly of complex RNA-protein machines. Ribosome formation begins with the transcription, modification and folding of ribosomal RNA (rRNA). In the past years, I have focused on the molecular mechanism of pseudouridine formation in tRNA as a simple model system for RNA modification. I am now proposing to extend these studies to other tRNA modifications and to rRNA modifications. Using a bacterial model system, we will in particular analyze the interplay of RNA transcription, modification and folding as these processes occur simultaneously and may influence each other during the early stages of ribosome assembly. We apply a combination of biochemical, biophysical and molecular biology techniques asking three main questions:1. How are pseudouridines and other modifications introduced into transfer RNA? Continuing our previous research, I will assess how pseudouridine synthases bind tRNA and locally unfold this small model RNA. We will also address the slow nature of pseudouridylation catalysis by analyzing potential intermediates, an important step towards identifying the catalytic mechanism of these enzymes. Lastly, we will dissect the kinetics of tRNA methylation and the impact thereof on RNA folding as well as the interplay of methylation and pseudouridylation as the most common RNA modifications.2. What is the biologically important function of RNA modification enzymes for the bacterial cell? Here, I ask why the bacterial cell has many RNA modification enzymes if most are not essential, and I suggest that their benefit will be most apparent under stress conditions. If so, how do RNA modifications impact ribosome formation and function under stress conditions? Thereby, we will also identify the most critical rRNA modification enzymes for subsequent in vitro studies. Additionally, we test the hypothesis that RNA modification enzymes act as RNA chaperones contributing to RNA folding.3. How are pseudouridines formed site-specifically in 23S rRNA and how do they contribute to RNA folding and ribosome assembly?I will test the hypothesis that modification enzymes recognize 23S rRNA structure that is folded co-transcriptionally. The establishment of a co-transcriptional 23S rRNA modification system will be the first step towards an in vitro large subunit assembly system, allowing the systematic addition of ribosomal proteins while analyzing RNA modification and folding.My proposed research will provide significant insight (1) into the impact of modification on RNA folding, (2) into the timing and interplay of different RNA modifications, (3) into the cellular function of RNA modification, and (4) into the dependence of RNA folding and modification on transcription. Our studies of short and long bacterial RNAs involved in translation will generate knowledge that will very likely hold true for all organisms as these processes are highly conserved. Ultimately, my research will lead to an experimental system for studying assembly of the large ribosomal subunit in vitro using sophisticated biochemistry experiments. This will also allow manipulation of ribosome assembly with the goal of constructing novel ribosome-based nanoassemblers that will enable the efficient synthesis of novel compounds.