Ribosomal RNA maturation and its link to quality control of ribosome assembly – ARNr-QC

Task 1. Studies on the 5S and 23S maturation complexes will be performed using standard structural biology techniques (crystallography, NMR, SAXS). The protein components of the complexes with be produced in E. coli, the RNA components transcribed in vitro. Complexes will be made by incubation of the different components together followed by purification on gel-filtration columns, for crystallography trials and SAXS data collection etc. Task 2. Messenger RNA substrates of RNase P encoding factors that may have an impact on ribosome assembly will be identified both at a global level by RNA sequencing experiments and at a gene specific level by Northern blots probed for mRNAs encoding known assembly proteins. Once potential candidates are identified, we will map cleavage sites both in vivo and in vitro and determine the regulatory mechanisms involved by standard genetic and molecular biology techniques. Task 3. We originally proposed a number of techniques (cross-linking, purification etc) to identify the enzyme involved in 16S rRNA processing. Since we now believe we have the enzyme identified, we will try to determine the exact assembly intermediate that is a substrate for this enzyme by isolating ribosomes blocked at various steps of assembly (using assembly mutants) and then adding the purified enzyme in vitro to see if processing can occur.

1. Identification of preliminary crystallisation conditions and SAXS data for the 5S rRNA/L18/RNase M5 complex.
2. Identification of a potential substrate for RNase P that may explain the 30S ribosomal subunit defect in cells depleted for this enzyme
3. Identification of a candidate enzyme for the 16S rRNA 3' processing reaction

We have identified preliminary crystallography conditions for 5S rRNA/L18/M5 complex that require a significant amount of improvement to hope to be able to resolve the structure. We will soon begin experiments to identify the minimal 23S rRNA fragment for cleavage by Mini-III to begin structural studies of this complex along with co-factor L3. We have identified a potential substrate of RNase P that could explain the 30S assembly defect. Our future experiments will be designed to confirm this effect, to determine wither the effect of RNase P is direct, and if so to map the cleavage site to understand the mechanism. Finally, we believe we have identified the enzyme responsible for 16S rRNA processing. Our future experiments will focus on determining the co-factors involved.

Rae1/YacP, a new endoribonuclease involved in ribsome-dependent mRNA decay in B. subtilis. EMBO J. In press

Characterisation of the quality control link between tRNA processing and ribosome assembly in Bacillus subtilis. 3rd course in Post-transcriptional gene regulation: mechanisms and networks. Curie Institute, Paris. Poster.

Ribosomes, the large ribonucleo-protein complexes that carry out protein synthesis, are essential for life. In rapidly growing cells, the biosynthesis of ribosomes accounts for most of cellular transcription and consumes a major portion of the cell's energy. Mistakes in ribosome assembly are thus understandably not tolerated and cells have active surveillance methods to rapidly degrade misassembled ribosomes and avoid making aberrant proteins. Mutations that increase the likelihood of assembly defects in humans are associated with a number of ‘ribosomopathies’, including cancer, highlighting the importance of accurate ribosome assembly for cellular well-being and how this process is intrinsically linked to other cellular processes, including cell division. In this study, we have uncovered a link between 5’ processing of transfer RNAs and the assembly of the small (30S) ribosomal subunit in the Gram-positive model organism Bacillus subtilis. It would make sense to stop making ribosomes if there is a problem with tRNA synthesis in the cell. We propose to characterise in detail the mechanism underlying this potentially new quality control step in ribosome assembly and determine how it ultimately affects 3’ processing of 16S rRNA. This processing reaction is the only major step in B. subtilis rRNA maturation for which the enzyme is not yet known; identifying it is thus an important goal of this project. We also propose to further our understanding of suspected quality control steps involved in the maturation of the rRNAs in the large (50S) subunit, by studying the roles of specific ribosomal proteins in the processing reactions of 23S and 5S rRNA at the molecular level. Although quality control of ribosome assembly has been well-studied in yeast, this question has not been addressed in depth in bacteria. This project will therefore substantially increase our understanding of this phenomenon and likely have future global implications for medicine in terms of revealing new potential antibiotic targets in evolutionarily related organisms.