Structural and functional analysis of the maturation process leading to the production of the eukaryotic small ribosomal subunit – RIBOPRE40S

To study the structure of precursor particles to the ribosomal subunits, it is first necessary to purify them; purified particles must be sufficiently pure and abundant. In the first part of the project, we have worked on a purification procedure. We use as source of particles yeast cell extracts. Particles are purified from these extracts using a “bait” protein integrated within the precursor particles. The biochemical properties of the purified particles are then assessed and their three-dimensional structure will be modelled using hundreds of electron microscopy images of the particles obtained from samples frozen at very low temperature. The positions within the precursor particle structures of various proteins will be determined by treating these particles with specific antibodies binding to these proteins. Finally, the atomic structure of these proteins will be determined using a method involving X-ray irradiation of protein crystals.

We have determined the three-dimensional structure of a protein enzyme, that plays a key role in the production of the first precursor particle to the small ribosomal subunit, bound to its regulatory protein. We have devised an efficient protocol to purify precursor particles to the small ribosomal subunit and obtained electron microscopy images of these precursor particles. We have identified factors that intervene in the degradation of aberrant precursor particles.

We will now acquire a very large number of electron microscopy images of precursor particles to the small ribosomal subunit at various stages of maturation, that will be used to model their three-dimensional structure. In parallel, we will work on a protocol to purify early precursor particles to the large ribosomal subunit with the aim to determine the structure of these particles using the approaches detailed above.

Scientific articles dealing with the three-dimensional X-ray structure of protein components of precursor particles to the ribosomal subunits are in preparation.

The synthesis of ribosomes is one of the major cellular activities, both quantitatively and qualitatively. The production of eukaryotic ribosomes involves (1) the synthesis in the nucleolus of precursors to the mature ribosomal rRNAs, (2) the proper folding, post-transcriptional modification and processing of these pre-rRNAs to yield the mature rRNAs (3) the stepwise correct association of ribosomal proteins with rRNAs in their precursor context (4) the transport of the maturing pre-ribosomal particles resulting from these associations from the nucleolus to the nucleoplasm and from there through the nuclear pores into the cytoplasm. Close to two hundred non-ribosomal proteins transiently and dynamically interact with pre-ribosomal particles at various stages of maturation. It is widely accepted that these non-ribosomal proteins play crucial roles in the key aspects of ribosome biogenesis listed above, but their precise molecular functions remain elusive. Strikingly, several energy-consuming enzymes feature among these essential non-ribosomal proteins. This suggests that pre-ribosomal particles undergo energy-consuming remodelling events required for their further maturation. In addition, current evidence suggests that correct execution of these remodelling events are monitored by quality control mechanisms or checkpoints. The challenge in the field is to identify the composition and structure at atomic resolution of pre-ribosomal particles at various stages of the maturation process, and how the activities of non-ribosomal and ribosomal proteins drive conversion of a given structure to the subsequent one.
Given the difficulty of the task, we will first concentrate on the maturation of pre-40S particles, the direct precursors to 40S ribosomal subunits. The aim of our proposal is to provide a molecular description of the successive structural transitions that pre-40S pre-ribosomal subunits undergo from their nuclear state all the way to their mature cytoplasmic 40S ribosomal subunit structure. We will also investigate how the cell controls proper execution of pre-40S particle maturation in the cytoplasm.
To achieve these aims, we will proceed as follows. We will investigate the atomic structure of non-ribosomal and some ribosomal protein components of pre-ribosomal pre-40S particles by X-ray crystallography. In parallel, we intend to investigate by chemical/enzymatic probing experiments the 20S pre-rRNA structure and map the 20S pre-rRNA/protein interactions within pre-40S pre-ribosomal particles purified from yeast cells at various stages of maturation. We also want to determine the three-dimensional structure of these purified pre-40S particles using cryo-electron microscopy (cryo-EM). The crude positions of the non-ribosomal protein components and their ribosomal protein partners within the successive pre-ribosomal particles will be determined by immuno-localization using specific antibodies. The biochemical and morphological data from structural probing and immuno-localization experiments will allow a precise fitting of the atomic X-Ray structures of non-ribosomal and ribosomal proteins within the cryo-EM 3D structures of the successive pre-40S particles. Moreover, we will attempt to reconstitute the cytoplasmic steps of pre-40S particle maturation in vitro. The structures of pre-40S particles matured in vitro will be compared with the structures of particles purified from yeast cells. Finally, we will investigate how the No Go decay machinery detects and targets for degradation stalled mRNA-associated pre-40S particles.
The approaches that will be developed and refined during this project using pre-40S pre-ribosomal particles will certainly prove to be of crucial importance to the later study of more complex particles such as the pre-60S and 90S pre-ribosomal particles and more generally to the investigation of complex RNPs undergoing structural transitions.