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 is assembled in the nucleolus and whether its biogenesis is somehow coordinated with that of ribosomal subunits is totally unknown. Despite the fundamental roles played by SRP in cells, its biogenesis mechanism is still unclear, as well as the assembly factors involved. Using stable human cell lines expressing GFP-tagged full-length SRP proteins, we previously found that disrupting the nucleolus by interfering with its function strongly alters the distribution and localization of GFP-SRP proteins. Thus, an intact nucleolar structure seems to be required for SRP biogenesis. Moreover, 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. We showed that GFP-tagged mutated SRP proteins, which have lost their capability to interact with other SRP components, accumulate in the nucleolus of the cells. The mutated protein interactomes differ from the WT ones. Interestingly, some nucleolar factors areparticularly enriched in complexes associated with mutated SRP proteins, while others are less present. We showed that the knockdown of some of these nucleolar proteins led to reduced levels of 7SL RNA in cells. Our data suggest that the biogenesis of the ribosome and the SRP may share common assembly factors.