Functions of the AAA-ATPase Rix7 in pre-60S assembly

Ribosomes are the molecular machineries inside cells that produce all cellular proteins by translating the genetic information encoded by messenger RNA (mRNA) transcripts into polypeptide chains. Eukaryotic ribosomes consist of a small (40S) subunit, which is responsible for decoding of messenger RNA (mRNA), and a large (60S) subunit, responsible for forming peptide bonds. The assembly of these ribosomal subunits occurs through a highly complex, multi-step process that takes place in different parts of the cell and requires the synchronized activity of over 200 helper molecules, known as maturation factors. These maturation factors work together to safeguard the proper folding of ribosomal RNA (rRNA) and coordinate the correct assembly of ribosomal proteins with rRNA. Among the 200 maturation factors, certain energy-consuming enzymes, including GTPases and ATPases, play crucial roles in driving key assembly steps and ensuring the unidirectional progression of the assembly pathway. In this research project, we aim to investigate the essential functions of Rix7, which belongs to a specific class of ATPases known as AAA (ATPases Associated with a variety of cellular Activities) ATPases. AAA-ATPases utilize the energy derived from ATP hydrolysis to generate mechanical force, which allows them to extract their distinct ligand proteins from macromolecular complexes. Our goal is to uncover how Rix7 interacts with nascent ribosomal 60S precursor subunits and the mechanisms by which it releases its potential maturation factor ligand(s) from these particles. Using the yeast Saccharomyces cerevisiae as a model organism, we will employ a combination of genetic techniques and protein biochemistry assays to study the Rix7-dependent ribosomal maturation and remodelling steps in the test tube. Additionally, with cryo-electron microscopy, we aim to determine the structure of Rix7 when it is bound to ribosomal precursor particles and visualize how the ATPase interacts with its direct maturation factor targets.