In vivo labelling of functional ribosomes reveals spatial regulation during starvation in Podospora anserina
© Lalucque and Silar; licensee BioMed Central Ltd. 2000
Received: 30 August 2000
Accepted: 16 November 2000
Published: 16 November 2000
To date, in eukaryotes, ribosomal protein expression is known to be regulated at the transcriptional and/or translational levels. But other forms of regulation may be possible.
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 . In eukaryotes, ribosomal proteins are down regulated. This occurs through transcriptional regulation in yeast  and mostly through translational regulation in mammals and Dictyostelium . 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 ), 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.
Results and discussion
We report here for the first time a nuclear sequestration of a ribosomal protein during a transient period at the onset of stationary phase. A plausible explanation is that this kind of regulation permits a rapid production of ribosomes if nutrients are encountered before a more pronounced stationary phase is entered. However, recent data [9, 10] show that release of cdc14, sequestered in the nucleolus, is involved in the proper exit from mitosis. Because ribosomes might regulate cell cycle progression , it is possible that sequestering ribosome in the nucleus is an additional level of regulation involved in ensuring a correct cell cycle arrest.
Materials and Methods
DNA manipulation were made according to . To construct the su12-GFP gene, the su12 coding sequence was amplified from plasmid psu12-S3  by PCR with oligos 3382 (5'-ACTATAGGGCGAATTGG-3') and su12-3' (5'-CGGGATCCCGAAAACATACCTGATCACGCAGAG-3'). This yielded a PCR product, in which the su12 stop codon is replaced by a Bam HI site allowing for the fusion with the GFP coding sequence. To this end, the PCR product was digested by Eco RI and Bam HI and cloned into pEGFP-1 (Clontech) at the corresponding sites. The sequence of the complete su12 coding sequence along with the junction with the GFP coding sequence revealed that no mutation had occur in the su12 coding sequence during plasmid construction. Podospora S strain was co-transformed using the method of  by this plasmid and pBC-hygro vector . Transformants resistant to hygromycin were selected and examined for GFP fluorescence and resistance to paromomycin. Several such transformants were obtained. Two were subjected to genetic analysis through a cross with wild type.
We would like to thank all members of the laboratory for useful discussion and Denise Zickler and Françoise James for helping with the microscope. This work was supported by ARC grant n° 5388.
- Wada A: Growth phase coupled modulation of Escherichia coli ribosomes. Genes to Cells. 1998, 3: 203-208. 10.1046/j.1365-2443.1998.00187.x.View ArticlePubMedGoogle Scholar
- Warner JR: The economics of ribosome biosynthesis in yeast. Trends Biochem Sci. 1999, 24: 437-440. 10.1016/S0968-0004(99)01460-7.View ArticlePubMedGoogle Scholar
- Meyuhas O, Avni D, Shama S: Translational control of ribosomal protein mRNA in Eukaryotes. In Translational control Edited by Hersey JWB, Mathews MB, Sonenberg N Cold Spring harbor Laboratory Press. 1996, 363-388.Google Scholar
- Hurt E, Hannus S, Schmelzl B, Lau D, Tollervey D, Simos G: A novel in vivo assay reveals inhibition of ribosomal nuclear export in Ran-cycle and nucleoporin mutants. J Cell Biol. 1999, 144: 389-401. 10.1083/jcb.144.3.389.PubMed CentralView ArticlePubMedGoogle Scholar
- Silar P, Koll F, Rossignol M: Cytosolic ribosomal mutations that abolish accumulation of circular intron in the mitochondria without preventing Senescence of Podospora anserina. Genetics. 1997, 145: 697-705.PubMed CentralPubMedGoogle Scholar
- Dequard-Chablat M: Different alterations of the ribosomal protein S7 lead to opposite effects on translational fidelity in the fungus Podospora anserina. J Biol Chem. 1986, 261: 4117-4121.PubMedGoogle Scholar
- Blanchard SC, Fourmy D, Eason RG, Puglisi JD: rRNA chemical groups required for aminoglycosid binding. Biochemistry. 1998, 37: 7716-7724. 10.1021/bi973125y.View ArticlePubMedGoogle Scholar
- Palmer E, Wilhem JM: Mistranslation in a eukaryotic organism. Cell. 1978, 13: 329-334.View ArticlePubMedGoogle Scholar
- Shou W, Seol JH, Shevchenko A, Baskerville C, Moazed D, Cheng ZWS, Jang J, Shevchenko A, Charbonneau H, Deshaies RJ: Exit from mitosis is triggered by Tem1-dependent release of the protein phosphatase Cdc14 from Nucleolar RENT complex. Cell. 1999, 97: 233-244.View ArticlePubMedGoogle Scholar
- Visitin R, Hwang ES, Amon A: Cfi1 prevents premature exit from mitosis by anchoring Cdc14 phosphatase in the nucleolus. Nature. 1999, 398: 818-823. 10.1038/19775.View ArticleGoogle Scholar
- Thomas G: An encore for ribosome biogenesis in the control of cell proliferation. Nature Cell Biol. 2000, 2: E71-E72. 10.1038/35010581.View ArticlePubMedGoogle Scholar
- Rizet G: Les phénomènes de barrages chez Podospora anserina. I Analyse génétique des barrages entre souches S et s. Rev Cytol Biol Veget. 1952, 13: 51-92.Google Scholar
- Esser K: Podospora anserina. In Handbook of Genetics Edited by King RC, vol. 1 New York: Plenum. 1974, 531-551.Google Scholar
- Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Strul (eds): K: Current Protocols in Molecular Biology. 1987Google Scholar
- Brygoo Y, Debuchy R: Transformation by integration in Podospora anserina. I. Methodology and phenomenology. Mol Gen Genet. 1985, 200: 128-131.View ArticleGoogle Scholar
- Silar P: Two new easy to use vectors for transformations. Fung Genet Newslett. 1995, 42: 73-Google Scholar
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