In vivo labelling of functional ribosomes reveals spatial regulation during starvation in Podospora anserina

Here, we report the successful tagging of functional ribosomal particles with a S7-GFP chimaeric protein, making it possible to observe in vivo ribosome dynamics in the filamentous fungus Podospora anserina. Microscopic observations revealed a novel kind of ribosomal protein regulation during the passage between cell growth and stationary phases, with a transient accumulation of ribosomal proteins and/or ribosome subunits in the nucleus, possibly the nucleolus, being observed at the beginning of stationary phase.Nuclear sequestration can be another level of ribosomal protein regulation in eukaryotic cells.This may contribute to the regulation of cell growth and division.Because translation utilizes a large proportion of the cell energy, its components are tightly regulated, especially ribosomes. Indeed, as cells are depleted of nutrients, a reduction in ribosome function occurs. In E. coli, ribosomes are converted by dimerization through the action of the RMF protein into 100S particles that are unable to perform translation [1]. In eukaryotes, ribosomal proteins are down regulated. This occurs through transcriptional regulation in yeast [2] and mostly through translational regulation in mammals and Dictyostelium [3]. To date, in vivo observation of a tagged and functional ribosomal particle during various cellular growth phases has not been reported (despite the fact that the large ribosomal subunit has recently been successfully tagged with GFP [4]), so there has been no evidence of whether spatial regulation can also be involved in the regulation of ribosomal proteins. Here, we report a spatial regulation of a small subunit ribosomal protein upon entrance into stationary phase.In order to observe the ribosomes in vivo, the S7 small subunit ribosomal protein of P. anserina was tagged with GFP. S7 belongs to the E. coli S4 family and is encoded by the su12 gene [5]. To this end, the GFP coding sequence was inserted in frame in the place of the su12 stop codon to yi