Résumé
The abundant and universally conserved Signal Recognition Particle (SRP) is essential for targeting most transmembrane and secretory proteins to the endoplasmic reticulum (ER). In mammals, SRP consists of a single RNA molecule, the 7SL, and of six proteins: SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72. Remarkably, SRP assembly largely occurs in the nucleolus where the initial steps of ribosome synthesis take place. Why SRP assembles in the nucleolus and whether its biogenesis is somehow coordinated with that of ribosomal subunits is totally unknown. In fact, only very few SRP assembly factors have been identified so far. Given the fundamental roles played by SRP in cells, our aim is to determine how it is assembled, and how its biogenesis is regulated in the nucleolus. We are particularly interested to identify novel assembly factors involved in this process. To carry out our studies, we have constructed stable human cell lines expressing GFP-tagged full-length SRP proteins. Our immunofluorescence analysis revealed unexpected new interesting features of SRP assembly. Indeed, we have observed that nuclear GFP-SRP19 and GFP-SRP72 concentrate in Cajal Bodies (CBs) in addition to nucleoli, which suggests the existence of a previously undocumented step of SRP assembly in CBs. Moreover, our quantitative proteomic studies using affinity purification combined with stable isotope labeling (SILAC) to identify the partners associated with GFP-SRP proteins interestingly revealed the presence of nucleolar proteins known to be involved in ribosome biogenesis and/or nucleolar structure. Our data suggest that the biogenesis of the ribosome and the SRP may share common assembly factors. We are currently exploring this hypothesis and the potential role of some of these identified factors in SRP biogenesis. Furthermore, by probing the requirement for an intact nucleolar structure in SRP biogenesis, we found that disrupting this biomolecular condensate by interfering with its function, strongly alters the distribution and localization of GFP-SRP19 and GFP-SRP72.Altogether, our results are starting to illuminate the importance of a structurally and functionally intact nucleolus on SRP biogenesis.