Shaping the Ribosome - Restructuring Events in 40S Maturation

Ribosomes are the macromolecular machines responsible for protein synthesis in every cell. The production of ribosomes (ribosome biogenesis) is a complex cellular pathway involving multiple assembly factors. These ensure that ribosomes are correctly assembled from their building blocks, the ribosomal RNAs and ribosomal proteins. In the initial stages of ribosome biogenesis, large ribosome precursor particles are formed, which undergo a series of maturation events until the small 40S and the large 60S subunit of mature ribosomes are released. In the course of ribosome biogenesis, precursor particles undergo significant structural re- arrangements. The subject of this proposal is a massive restructuring event within 40S precursor particles that shapes a protrusion of the RNA, the characteristic "beak structure". It was reported previously that formation of the beak structure is connected to the release of two assembly factors, Ltv1 and Enp1, from 40S precursors. The objective of this project is to elucidate the mechanisms of this restructuring event in molecular detail using yeast as a model organism. In preliminary experiments, we discovered that Ltv1 binding to 40S precursors prevents important contacts between ribosomal proteins. Moreover, our results suggest that formation of these contacts is necessary to release Ltv1. In addition, phosphorylation of Ltv1 and Enp1 is necessary for their release, probably because of electrostatic repulsion from their binding sites. Another important pre-requisite for Ltv1 release is the activity of another assembly factor, Rio2. In this project, we aim at unraveling the detailed structural differences between 40S precursors and mature 40S subunits and to clarify which contacts need to be released and which ones need to be newly formed in the course of ribosome biogenesis. Furthermore, mutants, in which the formation of specific contacts is blocked will be investigated with respect to the defects caused by a blocked release. Finally, we will address how the individual events are coordinated to eventually ensure the correct formation of the beak structure. This project is expected to provide not only deep insights into an important maturation step of 40S maturation, but also a better general understanding of restructuring events in ribosome biogenesis and other cellular processes. As all factors investigated in this project are also present in human cells, many of the insights gained in this project will also be applicable to the human ribosome biogenesis pathway.

Ribosomes are extremely important nanomachines responsible for all protein synthesis in each living cell. Their correct functioning is essential for life; therefore, the cell has to put high effort into correctly building these machines. This building process is termed ribosome synthesis. Mistakes in ribosome synthesis can lead to diseases like cancer and bone marrow failure syndroms. Each ribosome is composed of the same building blocks: four ribosomal RNAs and about 80 different ribosomal proteins. The synthesis of ribosomes can be imagined as a mechanical construction work in which many different workers put all of these individual components together in the correct order. Moreover, some of these workers act as culptors that shape the structure of the ribosome. There are more than 200 different workers, termed "ribosome assembly factors", engaged in the production of each single ribosome. Considering that these assembly factors work at physically distant sites, we asked the question how works taking place on different sides of the nascent ribosome are coordinated. To address this question, we analyzed two different important building works taking place on two distant sites of the ribosome. We found that the initial construction works on each of the sides are performed independently from the other side. These independent works however stop at a certain checkpoint. We discovered that a ribosomal protein, Rps20, serves as a communication wire between the two distant building works, transmitting the information about the status of the works to the other side. Only if works have been executed correctly on both sides, Rps20 gives the okay that works can continue. We propose that Rps20 acts as a quality control inspector that only allows the progression to later construction steps once the earlier steps have been executed correctly. Importantly, we were able to re-capitulate these steps with purified ribosome precursors in the test-tube, and were able to block the cascade of maturation events at selected stages. This allowed us to "freeze" the building works at certain steps and then analyze the structure of the intermediates in the ribosome maturation pathway. This helped us to visualize short-lived, so far poorly characterized ribosome synthesis intermediates. The results of this work are expected to lead to a better understanding of how the process of ribosome synthesis works and will consequently also help to find out what is going wrong in diseases caused by defective ribosome synthesis.