Yu Qiao
Visiting PhD student in Carrie Tisné’s lab, Laboratoire d’expression génétique microbienne (UMR8261 CNRS/université de Paris), Team “Biogenèse, architecture et interactions des ARN”

Title:
Functional and structural study of mRNA N⁶-methyladenosine (m⁶A) modification and its methyltransferase in Tetrahymena

Abstract:

Marta Kwapisz
MCD-CBI-Université de Toulouse, Team: “RNA Biology of Archaea”

Title:
The RNA Degradation Machineries in Hyperthermophile Archaea

“RNArchaea group (M. Ansart, J. Pham, M. Tassoni, M. Batista, R. Capeyrou, B. Clouet d'Orval, M. Bouvier) in collaboration with OncoRib team (P. Espirito-Santo, D. Rinaldi, C. Plisson-Chastang), CBI (Centre de Biologie Intégrative), Toulouse, France”

Abstract:
Our study model are the hyperthermophile archaea Thermococcus barophilus and Pyrococcus abyssi from Thermococcales order. Their ability to grow at temperatures above 100°C provides unique opportunity to address the stability of RNA, DNA and proteins at extremely high temperatures. Archaea are ubiquitous in all ecosystems (oceans, soil, microbiota) but serve as exemplary models for the study of life in extreme conditions. Their unique, intermediate status between bacteria and eukaryotes offers insights into the evolution of both molecular and genetic processes and central metabolic pathways. We have identified key players of RNA degradation machinery in Thermococcales: the RNA exoribonucleases aRNaseJ (5'-3') and the RNA Exosome (3'-5'), as well as RNA Helicases - ASH-Ski2 & Lhr2. The objective of our research is to gain insight into the mechanisms by which these factors are specifically recruited to regulate the RNA life cycle at the post-transcriptional level. The network of molecular interactions established in Thermococcales by affinity pull-down experiments suggests the existence of interactions between 5' and 3' degradation machineries, as well as with the ribosome and helicases. Our recent research has focused on the comprehensive characterization of the RNA exosomes. The RNA exosome in archaea is constituted by nine protein subunits that assemble to form a barrel-like structure, with a cap at its apex. The barrel is composed of a trimer of Rrp41/Rrp42 dimers. The phosphorolytic activity of the complex is carried out by the three Rrp41 subunits. The cap is comprised of a trimer of Rrp4 and Csl4, the in vivo stoichiometry of which is currently unknown. This trimeric complex serves to facilitate the recognition of RNA substrates and their subsequent insertion into the lumen of the catalytic barrel, where the RNA degradation occurs. This 3'-5' degradation machinery is orthologous to nuclear and cytoplasmic RNA exosomes in eukaryotes. We aim at characterizing the RNA exosome composition in vivo, its protein partners and substrates. In this purpose, we have performed co-immunoprecipitations of the RNA exosome of T. barophilus followed by LC/MS analysis. All exosome subunits and the exosome's known in vitro partners, ASH-Ski2 and aRNaseJ, were successfully recovered. LC/MS analysis identified over 30 proteins that were enriched in the sample. In order to characterize the different variants of exosome in vivo, we have reconstituted in vitro the RNA exosome with two different homogeneous caps (Rrp4 and Csl4). We have obtained cryo-electron microscopy (Cryo-EM) images of RNA exosomes at 3.4A resolution. These models serve as a basis for the subsequent in vivo characterization of the RNA exosome variants. I will present the progress of this work.