Group leader: Nicolas Bologna
Interplay of DCL- and AGO- RNA complexes during RNA silencing in Arabidopsis thaliana.
Small RNAs (sRNAs) are key regulators of gene expression. They are essential during development, stress responses, to preserve genome integrity, and for general immunity to viruses. sRNA are generated from long double-strand RNA (dsRNA) processed by DICER-LIKE (DCL) proteins and then are loaded into ARGONAUTE (AGO) proteins to fulfil their biological function. Eukaryotic organisms have evolved a large variety of pathways over the DICER–AGO consortium, which most likely underpins important aspects of their phenotypic plasticity. The Arabidopsis thaliana genome encoded four different DICER-LIKE (DCL) and ten ARGOUNATES (AGO) proteins. Although Arabidopsis nuclear DCL proteins play a central role in plant sRNA biogenesis, their dsRNA substrates have never been directly isolated and compared. Recently, we have produced catalytically inactive versions of DCL1, 2, 3 and 4 using point mutations introduced in the RNase III domains of the proteins. These Dicer catalytically inactive proteins, never engineered before in any other eukaryotic organism, bound effectively to their interacting RNAs but not release them. We already performed immunoprecipitation assays followed by RNA sequencing, showing and increased amount of nuclear RNA substrate complexes and providing a whole batch of targets and new putative functions. Furthermore, in the past years, research in the RNA silencing field has focused on steady-state subcellular localization and the functions of the main effector of RNA pathways. While DCL proteins are known to localize in either in the nucleus or in the cytoplasm, AGO proteins are known to shuttle between the nucleus and cytoplasm compartments. However, transient localizations have remained unknown in all eukaryotic organisms. This restricts our comprehension of AGO proteins’ specific subcellular functions and the intracellular sRNA/AGO complexes. As part of a preliminary project, we have developed a prototype of a computational tool that improves the scoring of true nuclear localization signals and nuclear export signals, reducing false positive rates. This information allowed to generate a collection of AGO proteins impaired in their nuclear import or export, which allowed us to uncouple cytoplasmic and nuclear AGO/sRNA complexes and their respective functions. By a combination of computational and experimental approaches, the proposed project attempts to identify and characterise novel dsRNA/DCL and sRNA/AGO complexes and decipher their interplay among the different sRNA pathways.
Bologna NG, Iselin R, Abriata LA, Sarazin A, Pumplin N, Jay F, Grentzinger T, Dal Peraro M, Voinnet O. Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway. Mol Cell. 2018 Feb 15;69(4):709-719.e5. doi: 10.1016/j.molcel.2018.01.007. Epub 2018 Feb 1. PMID: 29398448.
Potential collaborations with other research groups
Max Planck Institute for Plant Breeding Research (MPIPZ)
Potential collaborations with associated partners