Biogenesis, architecture and interactions of RNA

Our Team

The team’s research focuses on understanding RNA maturation, at the molecular level: how RNAs acquire their structure and function during maturation, and how maturation enzymes specifically recognize target nucleotides and catalyze the maturation reactions. We use and develop different methods of structural biology, including NMR spectroscopy, X-ray crystallography and single-particle cryoEM, to tackle these questions. Over the past few years, my team has made significant progress in understanding RNA chemical modifications and RNA processing :

• Maturation of human mitochondrial tRNAs
The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endor- ibonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3′-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3′-cleavage and 3′-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5′ and 3′ tRNA-end processing, methylation and 3′-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3′-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria (PMID 38824131). 

• NMR view of tRNA maturation 
We have developed a methodology using NMR spectroscopy as a tool to observe the maturation of tRNAs in cell extracts. By following the maturation of yeast tRNAPhe with time-resolved NMR measurements, we have showed that some modifications are introduced in a defined sequential order, which is controlled by cross-talks between modification events. In particular, we have uncovered that a strong hierarchy controls the introduction of the T54, Ψ55 and m1A58 modifications in the T-arm of yeast tRNAPhe. Using mass-spectrometry, we have demonstrated in collaboration with S. Kellner-Kaiser that the modification circuits identified in yeast extracts with NMR spectroscopy also impact the tRNA modification process in living cells. This NMR-based methodology is currently being further exploited in our team to investigate different aspects of tRNA maturation (PMID 31358763, PMID 37650648). 

• rRNA processing
We have solved the cryoEM structures of RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNAs. Mini-III binds both cleavage sites of the double-stranded 23S rRNA substrate helix, whereas M5 initially binds the 3’-cleavage site of the 5S rRNA substrate helix. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing (PMID 32991829, PMID 33541205).