Mechanisms of RNA helicases in ribosome assembly

Ribosomes are the cellular machineries responsible for translating the genetic information encoded by messenger RNA (mRNA) transcripts into proteins. They consist of a small and a large subunit composed of ribosomal RNAs (rRNAs) and ribosomal proteins. The assembly of ribosomes occurs through a highly complex process that takes place in different parts of the cell and requires the coordinated activity of over 200 assembly factors, along with small nucleolar RNAs (snoRNAs). These factors work together to ensure the synthesis of fully functional ribosomes that can produce all necessary proteins in the cell. In this research project, our aim is to investigate the functions of RNA helicases involved in ribosome biogenesis. RNA helicases are a class of enzymes that promote crucial maturation steps during ribosome assembly and are suggested to remodel particularly important or complex regions of the rRNA. However, the precise functions of the 20 RNA helicases involved in the pathway are still largely unknown. These functions may involve not only unwinding and rearranging RNA structures but also separating snoRNAs or protein assembly factors from the rRNA. Additionally, helicases work alongside specific cofactors that regulate their activity and guide them to the right locations and ribosomal precursor particles during the assembly process. To uncover the molecular functions of RNA helicases on evolving ribosomes, we will employ a combination of genetic and sophisticated biochemical methods coupled with structural investigations using cryo-electron microscopy. By selectively mutating specific elements required for helicase function, we will be able to efficiently inhibit ribosome assembly. We will isolate stalled ribosomal precursor particles from cells expressing these helicase mutants and compare them to normal wild-type particles. The purifications of such helicase-bound stalled assembly intermediates will unveil the cellular RNA and protein interaction network of helicases including the specific cofactors that regulate their activities. Furthermore, detailed investigations of the structural changes in the isolated pre-ribosomal particles resulting from helicase inactivity will allow us to determine the functions of RNA helicases on their distinct substrates. In recent years, research on ribosome assembly has gained significance in understanding human diseases since mutations in ribosomal proteins and assembly factors can lead to specific disorders known as ribosomopathies. Moreover, increased ribosome production is observed in cancer cells, which rely on extremely high rates of protein synthesis. Therefore, studying ribosome assembly may provide valuable insights into disease mechanisms and potentially lead to the development of new therapeutic strategies.